100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect total number of EdU + ( B ) ( S - phase ) in hair matrix . Unpaired t - performed using N of 9 - 12 HFs from 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.


C ) 100 nM paclitaxel treatment ( 24h ) significantly ( p = < 0 . 0001 [ * * * * ] ) increased number of mitotic phospho - histone H3 ( pH3 ) + in hair matrix . Welch ' s t - performed using N of 9 HFs from 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

C) 100 nM paclitaxel treatment (24h) significantly (p= <0.0001 [****]) increased the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of 9 HFs from 3 patients.


D ) 100 nM docetaxel treatment ( 24h ) significantly ( p = 0 . 0004 [ * * * ] ) increases number of pH3 + in hair matrix . Unpaired t - performed using N of 8 - 9 HFs from 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) 100 nM docetaxel treatment (24h) significantly (p = 0.0004 [***]) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8-9 HFs from 3 patients.


E ) Representative immunofluorescence highlight effects of 24h 100 nM taxane treatment on ( i ) Ki - 67 expression [ paclitaxel ] ; ( ii ) EdU incorporation pH3 immunoreactivity [ paclitaxel ] ; ( iii ) pH3 immunoreactivity [ docetaxel ] . 20 µm scale . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20 µm scale. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


presence of micronucleated in hair matrix in paclitaxel ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U performed using N of 12 - 13 HFs ( paclitaxel ) from 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The presence of micronucleated cells in the hair matrix in paclitaxel (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 12-13 HFs (paclitaxel) from 3 patients.


presence of micronucleated in hair matrix in docetaxel treated ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U performed using N of 8 HFs ( docetaxel ) from 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The presence of micronucleated cells in the hair matrix in docetaxel treated (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 8 HFs (docetaxel) from 3 patients.


C ) Hoechst 33342 of healthy nuclei comprising hair matrix ( lined ) dermal papilla in untreated ( vehicle ) human HFs . 20 µm scale .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix (lined) and dermal papilla in untreated (vehicle) human HFs. 20 µm scale.


D ) Paclitaxel treatment ( 100 nM , 24h ) induces formation of micronucleated bodies , as visualised by Hoechst 33342 ( arrows ) , localising to proliferative region of hair matrix . i - 20 µm scale ; ii - 10 µm scale .

D) Paclitaxel treatment (100 nM, 24h) induces the formation of micronucleated bodies, as visualised by Hoechst 33342 staining (arrows), localising to the proliferative region of the hair matrix. i - 20 µm scale; ii - 10 µm scale.


E ) 100 nM docetaxel treatment was also seen to promote formation of micronucleated bodies ( arrows ) . 10 µm scale . Error bars are standard error .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10 µm scale. Error bars are standard error.


A ) Nascent synthesis , as detected by ethynyl uridine ( EU ) incorporation , is blocked within clusters of nuclei in hair matrix ( arrows ) following paclitaxel treatment ( 100 nM , 24h ) . 20 µm scale .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24h). 20 µm scale.


B ) Quantitative highlights a significant ( p = < 0 . 0001 [ * * * * ] ) decrease in number of EU + nuclei following 24h paclitaxel treatment . Welch ' s t - performed using N of 11 - 12 hair follicles ( HFs ) from 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Quantitative analysis highlights a significant (p =<0.0001 [****]) decrease in the number of EU+ nuclei following 24h paclitaxel treatment. Welch's t-test performed using N of 11-12 hair follicles (HFs) from 3 patients.


C ) Representative dual stain highlights how EU incorporation in hair matrix is blocked within pH3 + population that accumulates in response to paclitaxel treatment 10 µm scale .

C) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3+ cell population that accumulates in response to paclitaxel treatment 10 µm scale.


D ) caspase 3 expression in hair matrix following 24h paclitaxel treatment . 20 µm scale .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O

D) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment. 20 µm scale.


E ) 100 nM paclitaxel treatment significantly ( p = 0 . 0016 [ exact ] [ * * ] ) increases number of caspase 3 + in hair matrix after 24h . Mann Whitney U performed using N of 16 - 18 HFs from 5 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) 100 nM paclitaxel treatment significantly (p = 0.0016 [exact] [**]) increases the number of cleaved caspase 3+ cells in the hair matrix after 24h. Mann Whitney U test performed using N of 16-18 HFs from 5 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


Ai ) Paclitaxel treatment promotes mitotic arrest ( orange arrows / pH3 + ) apoptosis ( arrows / caspase 3 + ) , within K15 + bulge K15 + proximal bulb outer root sheath ( pbORS ) / progenitor compartments of human hair follicle ( HF ) . 20 µm scale . Aii ) High magnification montage demonstrating mitotic arrest ( orange arrow ) apoptosis / caspase 3 positivity ( arrow ) within K15 + bulge . 10 µm scale . pbORS - proximal bulb outer root sheath .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

Ai) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20 µm scale. Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase 3 positivity (white arrow) within the K15+ bulge. 10 µm scale. pbORS - proximal bulb outer root sheath.


B ) Representative immunofluorescence of heightened caspase 3 immunoreactivity within K15 + bulge following extended paclitaxel organ culture experiments 50 µm scale . EC - extended cultures .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

B) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments 50 µm scale. EC - extended cultures.


C ) Graph showing significantly ( p = 0 . 029 [ * * ] ) increased K15 / caspase 3 double + within bulge following extended paclitaxel HF organ cultures . Welch ' s t - performed using N of 8 - 9 HFs from 3 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Graph showing significantly (p = 0.029 [**]) increased K15/caspase 3 double+ cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


D ) K15 + of human HF bulge express Ki - 67 during extended organ culture experiments . Paclitaxel treatment did not significantly affect number of bulge K15 / Ki - 67 double + . Unpaired t - performed using N of 8 - 9 HFs from 3 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double+ cells. Unpaired t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


E ) Representative double immunofluorescence of elevated γH2A . X immunoreactivity within K15 + bulge following extended paclitaxel organ culture experiments ( see methods ) . 50 µm scale . EC - extended cultures .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

E) Representative double immunofluorescence images of elevated γH2A.X immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures.


F ) γH2A . X showing a significant ( p = 0 . 065 [ * * ] ) increase in number of with double - strand breaks in K15 + bulge following extended organ culture experiments . Mann Whitney U performed using N of 5 - 6 HFs from 2 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) γH2A.X analysis showing a significant (p = 0.065 [**]) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann Whitney U test performed using N of 5-6 HFs from 2 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


B ) Palbociclib - only palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki - 67 expression in hair matrix beyond that seen with just 24h treatment Data also confirms that Ki - 67 expression is unaffected by paclitaxel treatment performed using N of 6 - 9 HFs per condition from 3 . Ordinary one - way ANOVA with multiple comparisons performed . Adjusted p values = 0 . 0001 [ * * * ] 0 . 0001 [ * * * * ] respectively . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Palbociclib-only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki-67 expression in the hair matrix beyond that seen with just 24h treatment Data also confirms that Ki-67 expression is unaffected by paclitaxel treatment Analysis performed using N of 6-9 HFs per condition from 3 patients. Ordinary one-way ANOVA with multiple comparisons performed. Adjusted p values = 0.0001[***] and 0.0001 [****] respectively. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


C ) Data confirming that paclitaxel treatment significantly increases ( adjusted p value = 0 . 0001 [ * * * ] ) number of pH3 + in hair matrix This effect was not observed when paclitaxel treated hair follicles were pre - co - incubated with palbociclib . performed using N of 6 - 9 HFs per condition from 3 Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Data confirming that paclitaxel treatment significantly increases (adjusted p value = 0.0001[***]) the number of pH3+ cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre- and co-incubated with palbociclib. Analysis performed using N of 6-9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


D ) caspase 3 in hair follicles from a donor that showed apoptotic sensitivity to paclitaxel treatment ( adjusted p value = 0 . 0056 [ * * ] ) . Paclitaxel treated hair follicles pre - co - incubated with palbociclib do not show enhanced caspase 3 expression , contrasting with paclitaxel - only treatment . Ordinary one - way ANOVA with multiple comparisons performed using N of 4 - 5 HFs per condition . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment (adjusted p value = 0.0056 [**]). Paclitaxel treated hair follicles pre- and co-incubated with palbociclib do not show enhanced cleaved caspase 3 expression, contrasting with paclitaxel-only treatment. Ordinary one-way ANOVA with multiple comparisons performed using N of 4-5 HFs per condition. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


E ) Immunofluorescence data representing how palbociclib / or paclitaxel affect ( i ) Ki - 67 expression , ( ii ) pH3 immunoreactivity ( iii ) caspase 3 expression . 20 µm scale .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O

E) Immunofluorescence data representing how palbociclib and/or paclitaxel affect (i) Ki-67 expression, (ii) pH3 immunoreactivity and (iii) cleaved caspase 3 expression. 20 µm scale.


B ) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle . synthesis in hair matrix resumed in palbociclib - only palbociclib plus paclitaxel dual treated conditions following washout Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 13 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard error .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib-only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one-way ANOVA with multiple comparisons performed using N of 8-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


C ) A trending increase in number of caspase 3 + was seen in hair matrix of HFs dual treated with paclitaxel palbociclib following drug washout , but this was not significant , whereas paclitaxel - only treatment significantly increased number of caspase 3 + in hair matrix ( adjusted p value = 0 . 0003 [ * * * ] ) . Palbociclib - only treatment did not increase number of caspase 3 + in hair matrix following drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard error .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) A trending increase in the number of cleaved caspase 3+ cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel-only treatment significantly increased the number of cleaved caspase 3+ cells in the hair matrix (adjusted p value = 0.0003 [***]). Palbociclib-only treatment did not increase the number of cleaved caspase 3+ cells in the hair matrix following drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


D ) HFs treated with paclitaxel alone showed a significant ( adjusted p value = 0 . 0316 [ * ] ) increase in number of pH3 + in matrix after washout . A significant ( adjusted p value = 0 . 0278 [ * ] ) increase in number pH3 + was seen in HFs dual treated with both paclitaxel palbociclib following drug washout . Palbociclib treatment alone showed no significant change in number of pH3 + hair matrix keratinocytes after drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard error .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) HFs treated with paclitaxel alone showed a significant (adjusted p value = 0.0316 [*]) increase in the number of pH3+ cells in the matrix after washout. A significant (adjusted p value = 0.0278 [*]) increase in the number pH3+ cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3+ hair matrix keratinocytes after drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


E ) Representative of EdU pH3 / caspase 3 in paclitaxel - only palbociclib plus paclitaxel dual treated HFs following drug washout . Where synthesis has resumed following reversal of G1 arrest , paclitaxel cytotoxicity is emergent . HS - hair shaft ; " wo " - washout . 20 µm scale .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Representative fluorescence images of EdU and pH3/cleaved caspase 3 staining in paclitaxel-only and palbociclib plus paclitaxel dual treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent. HS - hair shaft; "wo" - washout. 20 µm scale.


B ) Paclitaxel treatment did not affect number of EdU + within HF outer root sheath ( ORS ) following washout . In contrast , palbociclib treated HFs lacked EdU incorporation following drug washout ( adjusted p value = 0 . 0001 [ * * * ] ) . number of EdU + was significantly decreased in outer root sheath ( ORS ) of HFs dual treated with both paclitaxel palbociclib ( adjusted p value = 0 . 0021 [ * * ] ) . Ordinary one - way ANOVA with multiple comparisons performed using N of 12 - 13 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Paclitaxel treatment did not affect the number of EdU+ cells within the HF outer root sheath (ORS) following washout. In contrast, palbociclib treated HFs lacked EdU incorporation following drug washout (adjusted p value = 0.0001 [***]). The number of EdU+ cells was significantly decreased in the outer root sheath (ORS) of HFs dual treated with both paclitaxel and palbociclib (adjusted p value = 0.0021 [**]). Ordinary one-way ANOVA with multiple comparisons performed using N of 12-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


C ) HFs treated with paclitaxel showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in number of caspase 3 + in ORS following drug washout . Palbociclib - only palbociclib plus paclitaxel dual treated HFs showed no change in number of caspase 3 + ORS . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) HFs treated with paclitaxel showed a significant (adjusted p value = 0.0001 [****]) increase in the number of cleaved caspase 3+ cells in the ORS following drug washout. Palbociclib-only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3+ ORS cells. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


D ) Paclitaxel - only treated HFs showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in number of pH3 + ORS following drug washout . Palbociclib treatment alone had no significant effect on number of pH3 + ORS . There was a trending , yet non - significant , increase in number of pH3 + ORS in HFs dual treated with palbociclib plus paclitaxel . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Paclitaxel-only treated HFs showed a significant (adjusted p value = 0.0001 [****]) increase in the number of pH3+ ORS cells following drug washout. Palbociclib treatment alone had no significant effect on the number of pH3+ ORS cells. There was a trending, yet non-significant, increase in the number of pH3+ ORS cells in HFs dual treated with palbociclib plus paclitaxel. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


E ) Representative image of ORS EdU incorporation in palbociclib plus paclitaxel treated HFs following drug washout , whereby synthesis has not resumed . ; wo - washout . 50 µm scale ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout, whereby DNA synthesis has not resumed. ; wo - washout. 50 µm scale;


F ) Representative of pH3 / caspase 3 within ORS of paclitaxel - only palbociclib plus paclitaxel treated HFs following drug washout . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 50 µm

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Representative images of pH3/caspase 3 staining within the ORS of paclitaxel-only and palbociclib plus paclitaxel treated HFs following drug washout. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 50 µm scale


Immunofluorescence illustrating how palbociclib blocks paclitaxel - induced accumulation of caspase 3 + ( G ) in K15 + bulge proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of cleaved caspase 3+ (G) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.


Immunofluorescence illustrating how palbociclib blocks paclitaxel - induced accumulation of pH3 + ( H ) in K15 + bulge proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of pH3+ (H) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.


( A - C ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS ( Earle ' s Balanced Salt Solution ) for 30 min . Total membranes ( mem ) from lysed were collected incubated in a lipidation reaction with cytosols prepared from starved HEK293T . Reactions contained indicated concentrations of PI3K inhibitor ( PI3KI ) 3 - methyladenine ( 3 - MA ) ( B ) or FYVE ( C ) . A diagram of experimental scheme is shown in ( A ) . RPN1 , Ribophorin

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR

(A-C) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS (Earle's Balanced Salt Solution) for 30 min. Total membranes (mem) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells. Reactions contained the indicated concentrations of PI3K inhibitor (PI3KI) 3-methyladenine (3-MA) (B) or FYVE protein (C). A diagram of the experimental scheme is shown in (A). RPN1, Ribophorin 1


( D , E ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS in absence or presence of 20 nM wortmannin ( Wtm ) or 10 mM 3 - methyladenine ( 3 - MA ) for 30 min . Membranes from each treated sample were collected subjected to a differential centrifugation to separate 3K × g , 25K × g 100K × g pellet fractions followed by a lipidation as above ( E ) . A diagram is shown in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS in the absence or presence of 20 nM wortmannin (Wtm) or 10 mM 3-methyladenine (3-MA) for 30 min. Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K ×g, 25K ×g and 100K ×g pellet fractions followed by a lipidation assay as above (E). A diagram is shown in (D).


( F , G ) Atg5 KO MEFs were starved for 30 min . Membranes in 25K × g 100K × g pellets from a differential centrifugation were collected as described above . A similar lipidation was performed in presence of indicated concentrations ( Conc in G ) of 3 - MA , wortmannin ( F ) FYVE as well as a PI3P binding - deficient FYVE mutant ( C / S ) ( G ) . of lipidation activity is shown as of LC3 - II to LC3 - I ( II / I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O

(F, G) Atg5 KO MEFs were starved for 30 min. Membranes in the 25K ×g and 100K ×g pellets from a differential centrifugation were collected as described above. A similar lipidation assay was performed in the presence of indicated concentrations (Conc in G) of 3-MA, wortmannin (F) and FYVE protein as well as a PI3P binding-deficient FYVE mutant protein (C/S) (G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


( B ) A small vesicle generation as above was performed with indicated conditions . Slowly - sedimenting vesicles were collected to determine activity in LC3 lipidation reaction . 3 - MA , 5 mM ; Wortmannin , 20 nM ; S , starved Atg5 KO MEF cytosol ; N , untreated Atg5 KO MEF cytosol ; − / AP , in absence of ATP regeneration GTP in presence of apyrase ( AP ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

(B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP).


( D ) A small vesicle generation followed by a lipidation reaction was performed with 3K , P F1 - 4 fractions ( upper four panels : Small vesicle ) . SDS - PAGE immunoblot with indicated antibodies were carried out to probe organelle enrichment of each fraction ( lower five panels : Fraction input ) . TFR , transferrin

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR

(D) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K, P and F1-4 fractions (upper four panels: Small vesicle). SDS-PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction (lower five panels: Fraction input). TFR, transferrin receptor


( E ) A small vesicle generation as shown in ( A ) was performed in presence of indicated or drugs . Slowly - sedimenting vesicles were collected incubated in a lipidation reaction in presence of GST , H79G ( Sar1A ( H79G ) , 0 . 7 μM ) , wortmannin ( 20 nM ) , or BFA ( brefeldin A , 0 . 5 μg / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml).


( F ) A small vesicle generation as shown in ( A ) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown in absence or presence of Sar1A ( H79G ) or 3 - MA followed by a lipidation reaction .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction.


( G ) Atg5 KO MEFs were starved for 30 min . Differential centrifugation as shown in Figure 1D was performed to collect 25K × g 100K × g pellet fractions . LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 ( H79G ) . of lipidation activity is shown as of LC3 - II to LC3 - I ( II / I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O

(G) Atg5 KO MEFs were starved for 30 min. Differential centrifugation as shown in Figure 1D was performed to collect the 25K ×g and 100K ×g pellet fractions. The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 (H79G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


( A ) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min . were harvested ERGIC membranes were isolated by pooling fractions 3 4 ( F2 in Figure 2C , D ) of OptiPrep gradient in three - step fractionation approach followed by immunoblot to examine amount of indicated markers on ERGIC membranes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min. Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 (F2 in Figure 2C,D) of the OptiPrep gradient in the three-step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes.


( B ) Atg5 KO MEFs were transfected with a plasmid encoding an HA - tagged SEC31A . After transfection ( 24 hr ) , were treated as shown in ( A ) . Immunofluorescence was performed with anti - ERGIC53 anti - HA antibodies were examined by confocal microscopy . Bar : 10 μM ( C ) of SEC31A overlapped with ERGIC53 shown in ( B ) using Pearson ' s correlation coefficient ( PCC ) . Error bars represent standard deviations from ∼ 20 in three independent experiments . p values were calculated by T - .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Atg5 KO MEFs were transfected with a plasmid encoding an HA-tagged SEC31A. After transfection (24 hr), the cells were treated as shown in (A). Immunofluorescence was performed with anti-ERGIC53 and anti-HA antibodies and the cells were examined by confocal microscopy. Bar: 10 μM (C) Quantification of SEC31A overlapped with ERGIC53 shown in (B) using the Pearson's correlation coefficient (PCC). Error bars represent standard deviations from ∼20 cells in three independent experiments. p values were calculated by T-test.


A - D ) Confocal image of cryostat sections from adult CX3CR1 + / GFP ; Sfrp1 + / βgal ( A , B ) ; Sfrp1 + / βgal ( C ) CX3CR1 + / GFP ; Sfrp1 - / - ( D ) mouse brains three days after intra - cortical infusion of saline or LPS . Sections were immunostained for βgal ( magenta , green in C ) Iba1 ( green in A ; red in C ) or Sfrp1 ( magenta ) GFP ( green , B D ) , GFAP ( cyan in A ; B , D , red in C ) . Arrowheads indicate βgal / GFAP ( A ) Sfrp1 / GFP co - localization ( B ) . No Sfrp1 is detected in null mice independently of treatment ( two bottom lines ) . Scale bar 25μm . E ) ELISA determination of Sfrp1 in brain extracts from 3 months - old wt Sfrp1 - / - mice . WT mice were injected either with saline or LPS . Three days after injection region around injected side ( 10 mm3 cortical cube ) was isolated SFRP1 content compared with that present in similar region of non - injected or Sfrp1 - / - mice used as negative control ( n = 5 mice for each group ) . Error bars represent Standard Error . Statistical significance : ns P > 0 . 5 * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni multiple comparisons .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D) Confocal image analysis of cryostat sections from adult CX3CR1+/GFP;Sfrp1+/βgal (A,B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1-/- (D) and mouse brains three days after intra-cortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A;B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar 25μm. E) ELISA determination of Sfrp1 levels in brain extracts from 3 months-old wt and Sfrp1-/- mice. WT mice were injected either with saline or LPS. Three days after injection the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1-/- mice used as negative control (n=5 mice for each group). Error bars represent Standard Error. Statistical significance: ns P>0.5 ****P<0.0001; One-way ANOVA followed by Bonferroni multiple comparisons test.


A ) Coronal cryostat sections of LV - IRES - Gfp or LV - Sfrp1 - IRES - Gfp infected brains 1 - month post - infusion , immunostained for SFRP1 ( magenta ) Iba1 ( green ) GFAP ( cyan ) , or TauP ( red ) . Lv , lateral ventricle . Scale bar 100μm . B ) graph shows normalised level of GFAP , Iba1 CD45 immunoreactivity ( IR ) occupied by TauP signal ( n = 24 acquisitions , dots ; N = 3 mice , black dots , for each group ) , normalized to LV - IRES - Gfp infected brains . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 by two - sided Student ' s t - .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Coronal cryostat sections of LV-IRES-Gfp or LV-Sfrp1-IRES-Gfp infected brains 1-month post-infusion, immunostained for SFRP1 (magenta) Iba1 (green) GFAP (cyan), or TauP (red). Lv, lateral ventricle. Scale bar 100μm. B) The graph shows the normalised level of GFAP, Iba1 and CD45 immunoreactivity (IR) and the area occupied by TauP signal (n=24 acquisitions, white dots; N=3 mice, black dots, for each group), normalized to LV-IRES-Gfp infected brains. Error bars represent Standard Error. Statistical significance: *P<0.05; **P<0.01; ****P<0.0001 by two-sided Student's t-test.


C , D ) Coronal sections from wt Sfrp1 - / - mouse brains three days after infusion of saline or LPS , immunostained for GFAP ( cyan , C ) or CD45 ( magenta , D ) . are high power views of somatosensory cortex ( lower power view in Fig EV1D ) . Scale bar 60μm . E , F ) graphs show normalised of immunoreactivity ( IR ) for GFAP ( E ) CD45 ( F , P = 0 , 006 ) present in cortical sections ( n = 24 acquisitions dots ; from N = 3 , black dots , per group ) from wt Sfrp1 - / - infused with saline or LPS . Bars represent Standard Error . Statistical significance calculated per biological replicas is indicated in green that on number of acquisitions in black . * * or # # P < 0 . 01 ; * * * or # # # P < 0 . 001 ; * * * * or # # # # P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons . * # indicate significance between genotypes treatments , respectively .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D) Coronal sections from wt and Sfrp1-/- mouse brains three days after infusion of saline or LPS, immunostained for GFAP (cyan, C) or CD45 (magenta, D). The images are high power views of the somatosensory cortex (lower power view in Fig EV1D). Scale bar 60μm. E, F) The graphs show the normalised levels of immunoreactivity (IR) for GFAP (E) and CD45 (F, P=0,006) present in cortical sections (n=24 acquisitions white dots; from N=3 animals, black dots, per group) from wt and Sfrp1-/- animals infused with saline or LPS. Bars represent Standard Error. Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black. ** or ## P<0.01; *** or ### P<0.001; **** or #### P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. * and # indicate significance between genotypes and treatments, respectively.


C ) Wt Sfrp1 - / - mice immunized with MOG sacrificed 16 days post immunization . Cryostat sections of thoracic spinal cords were stained with antibodies against CD4 , Iba1 , GFAP MBP . show region dorsal fasciculus . arrowheads point to disruption of pia surface in wt . Scale bar 100μm . D ) of CD4 + infiltrated lymphocytes , Iba1 normalised immunoreactivity , MBP normalised immunoreactivity MBP immune - reactive in dorsal fasciculus of spinal cord sections from wt Sfrp1 - / - mice 16 days after immunization ( n = 16 acquisitions , dots ; from N = 4 , black dots , per genotype ) . Error bars represent Standard Error . Statistic refers to number of acquisitions .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Wt and Sfrp1-/- mice immunized with MOG and sacrificed 16 days post immunization. Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4, Iba1, GFAP and MBP. Images show the region dorsal fasciculus. White arrowheads point to the disruption of the pia surface in wt. Scale bar 100μm. D) Quantification of CD4+ infiltrated lymphocytes, Iba1 normalised immunoreactivity, MBP normalised immunoreactivity and MBP immune-reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1-/- mice 16 days after immunization (n=16 acquisitions, white dots; from N=4 animals, black dots, per genotype). Error bars represent Standard Error. Statistic refers to number of acquisitions.


D ) ELISA determination of SFRP1 present in media of microglia ( n = 4 cultures per genotype ) , astrocytes ( n = 4 cultures per genotype ) or mixed astrocytes microglial ( 2 : 1 , n = 7 cultures per genotype ) cultures derived from wt or Sfrp1 - / - pups exposed for 24h to saline or LPS ( 1μg / ml ) . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) ELISA determination of SFRP1 levels present in the media of microglia (n=4 cultures per genotype), astrocytes (n=4 cultures per genotype) or mixed astrocytes and microglial (2:1, n=7 cultures per genotype) cultures derived from wt or Sfrp1-/- pups exposed for 24h to saline or LPS (1μg/ml). Error bars represent Standard Error. Statistical significance: *P<0.05; ***P<0.001; ****P<0.0001; One-way ANOVA followed by Bonferroni Test.


Secretory profile of cytokines present in medium of microglia , astrocytes or mixed astrocytes microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - genotype ) . content of IL1β , IL10 was determined by Multiplex ELISA . Values are represented in absolute . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IL1β, IL10 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.


F ) Secretory profile of cytokines present in medium of microglia , astrocytes or mixed astrocytes microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - genotype ) . content of IFNγ , TNFα , IL4 , IL6 was determined by Multiplex ELISA . Values are represented in absolute . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IFNγ, TNFα, IL4, IL6 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.


B , C ) Volcano plots of differential gene expression from CX3CR1GFP / + ( B ) or CX3CR1GFP / + ; Sfrp1 - / - ( C ) microglial in response to LPS . Data are represented as Log2 Fold Change vs - Log10 adjusted p - value by Wald corrected for multiple comparisons by Benjamini Hochberg method . Blue vertical lines represent an increase of 75 400 % in expression respectively . Green horizontal lines represent a 0 . 05 or 0 . 01 adjusted p - value of statistical significance respectively . Genes with an expression change higher than 75 % an adjusted p - value lower than 0 . 05 are highlighted in red ( + FC ) blue ( - FC ) . Genes with an expression change higher than 400 % an adjusted p - value lower than 0 . 01 are coloured in red ( + FC ) blue ( - FC ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C) Volcano plots of differential gene expression from CX3CR1GFP/+ (B) or CX3CR1GFP/+;Sfrp1-/- (C) microglial cell in response to LPS. Data are represented as Log2 Fold Change vs -Log10 adjusted p-value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. Blue vertical lines represent an increase of 75 and 400% in the expression levels respectively. Green horizontal lines represent a 0.05 or 0.01 adjusted p-value of statistical significance respectively. Genes with an expression change higher than 75% and an adjusted p-value lower than 0.05 are highlighted in dark red (+FC) and dark blue (-FC). Genes with an expression change higher than 400% and an adjusted p-value lower than 0.01 are coloured in light red (+FC) and light blue (-FC).


B ) Integrative Genomics Viewer transcription profile of represented genes in microglial isolated for saline LPS treated CX3CR1GFP / + CX3CR1GFP / + ; Sfrp1 - / - mouse brains . Scale bar : 2 Kbp .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O

B) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains. Scale bar: 2 Kbp.


A . localization of NSUN3 was analysed in HEK293 stably expressing NSUN3 - GFP ( green ) . NSUN3 - GFP with a Mitotracker ( red ) are shown separately in an overlay with DAPI to indicate nuclei . scale bar represents 5 m .

O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A. The localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3-GFP (green). NSUN3-GFP and staining with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.


B . To analyse submitochondrial localization of NSUN3 , human mitochondria were isolated either left untreated , swollen in hypotonic buffer ( Mitoplasts ) or solubilized by sonication ( Sonic . ) before treatment with different amounts of Proteinase K ( PK ) where indicated , followed by SDS - PAGE Western blotting using antibodies against human TIM44 , TIM23 , TOM70 or FLAG - tagged NSUN3 . Note that TIM44 localises to matrix , while N - terminus of TIM23 localises to intermembrane space TOM70 is largely exposed on mitochondrial surface . asterisk indicates a cross - reaction of TOM70 antibody .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O

B. To analyse submitochondrial localization of NSUN3, human mitochondria were isolated and either left untreated, swollen in hypotonic buffer (Mitoplasts) or solubilized by sonication (Sonic.) before treatment with different amounts of Proteinase K (PK) where indicated, followed by SDS-PAGE and Western blotting using antibodies against human TIM44, TIM23, TOM70 or FLAG-tagged NSUN3. Note that TIM44 localises to the matrix, while the N-terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface. The asterisk indicates a cross-reaction of the TOM70 antibody.


B . HEK293 expressing NSUN3 - HisPrcFLAG ( NSUN3 ) or HisPrcFLAG - tag alone ( FLAG ) were either not crosslinked ( - ) , UV crosslinked ( UV ) or treated with 5 - azacytidine ( 5 - AzaC ) . - complexes were affinity purified bound was trimmed , end - labelled with 32P phosphate ligated to linkers . - complexes were separated by SDS - PAGE , transferred to nitrocellulose exposed to an X - ray film .

O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG-tag alone (FLAG) were either not crosslinked (-), UV crosslinked (UV) or treated with 5-azacytidine (5-AzaC). The protein-RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and exposed to an X-ray film.


G . 5 - AzaC crosslinking was performed associated with wildtype NSUN3 , catalytically inactive NSUN3 mutant ( C265A ) or FLAG tag alone was isolated as described in ( B ) . analysed by northern blot using probes against mt - tRNAMet , mt - tRNAPro mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on left eluates ( 50 % ) on right .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. 5-AzaC crosslinking was performed and RNA associated with wildtype NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA analysed by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


A . In vitro methylation reactions were performed using recombinant His14 - MBP - NSUN3 ( NSUN3 ) or catalytically inactive mutant His14 - MBP - NSUN3 - C265A ( C265A ) , 3H - labelled S - adenosyl methionine as a methyl group donor in vitro transcribed mt - tRNAMet , mt - tRNAPro mt - tRNAGlu . was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) to indicate inputs exposed to an X - ray film to analyse methylation ( 3H - Me ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosyl methionine as a methyl group donor and in vitro transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me).


B . distribution of Illumina reads along mt - tRNAMet obtained from CRAC experiments with NSUN3 after UV ( grey ) or 5 - AzaC crosslinking ( grey ) is given as reads per million mapped reads . position of anticodon is indicated by a bar .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC crosslinking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar.


D . In vitro methylation assays were performed as described in ( A ) with His14 - MBP - NSUN3 in vitro transcribed wildtype mt - tRNAMet cytidine mutants of anticodon loop region indicated in ( C ) . Two exposure times of X - ray films are shown : 16 hours ( short ) , 3 days ( long ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long).


E . In vitro methylation of in vitro transcribed mt - tRNAMet chemically synthesized mt - tRNAMet containing an m5C modification at wobble position . experiment were performed as described in ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. In vitro methylation assay of in vitro transcribed mt-tRNAMet and chemically synthesized mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A).


B . In vitro methylation assays using [ 3H - methyl ] - labelled S - adenosyl methionine , in vitro transcripts of mt - tRNAMet mutants described in ( A ) recombinant His14 - MBP - NSUN3 . was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) , dried exposed to an X - ray film to detect methylated transcripts ( 3H - Me ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. In vitro methylation assays using [3H-methyl]-labelled S-adenosyl methionine, the in vitro transcripts of the mt-tRNAMet mutants described in (A) and recombinant His14-MBP-NSUN3. RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr), dried and exposed to an X-ray film to detect methylated transcripts (3H-Me).


C . In vitro methylation using chemically synthesised ASL . experiment were performed as described in ( B ) .

C. In vitro methylation assay using chemically synthesised ASL. The experiment and analysis were performed as described in (B).


A . localization of ABH1 was analysed by immunofluorescence in HEK293 . ABH1 ( green ) localisation mitochondria stained with a Mitotracker ( red ) are shown separately in an overlay with DAPI to indicate nuclei . scale bar represents 5 m .

O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. The localization of ABH1 was analysed by immunofluorescence in HEK293 cells. ABH1 (green) localisation and mitochondria stained with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.


B . HEK293 expressing ABH1 - HisPrcFLAG ( ABH1 ) or HisPrcFLAG - tag alone ( FLAG ) were UV crosslinked ( UV ) - complexes were affinity purified . Co - precipitated was isolated by northern blot using probes against mt - tRNAMet , mt - tRNAPro mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on left eluates ( 50 % ) on right .

O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HEK293 cells expressing ABH1-HisPrcFLAG (ABH1) or the HisPrcFLAG-tag alone (FLAG) were UV crosslinked (UV) and protein-RNA complexes were affinity purified. Co-precipitated RNA was isolated and analysis by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


A . In vitro transcribed mt - tRNAMet was methylated at C34 using recombinant NSUN3 3H - labelled S - adenosyl methionine as a methyl group donor . Radiolabelled mt - tRNAMet was re - extracted then subjected to oxidation assays without ( - ) , with maltose binding ( MBP ) , with dioxygenase FTO or using wildtype ( ABH1 ) or mutant ( R338A , D233A ) His14 - MBP - ABH1 . Besides ABH1 controls lacking a - ketoglutarate ( KG ) or Fe2 + ions , samples contained a - ketoglutarate Fe2 + ions . After oxidation , was precipitated tritium released upon oxidation of radiolabelled mt - tRNAMet was quantified in supernatant per minute ( CPM ) are shown for experiments performed in triplicate with error bars indicating ± SD ( upper panel ) . Pelleted was separated on a denaturing polyacrylamide gel exposed to an X - ray film to analyse tritium retained ( 3H - Me ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. In vitro transcribed mt-tRNAMet was methylated at C34 using recombinant NSUN3 and 3H-labelled S-adenosyl methionine as a methyl group donor. Radiolabelled mt-tRNAMet was re-extracted and then subjected to oxidation assays without protein (-), with maltose binding protein (MBP), with the dioxygenase FTO or using wildtype (ABH1) or mutant (R338A, D233A) His14-MBP-ABH1. Besides ABH1 controls lacking a-ketoglutarate (KG) or Fe2+ ions, all samples contained a-ketoglutarate and Fe2+ ions. After oxidation, RNA was precipitated and tritium released upon oxidation of radiolabelled mt-tRNAMet was quantified in the supernatant and counts per minute (CPM) are shown for experiments performed in triplicate with error bars indicating ± SD (upper panel). Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X-ray film to analyse the tritium retained (3H-Me).


B . Synthetic anticodon loop ( ASL ) was radioactively labelled subjected to oxidation assays that were performed analysed as described in ( A ) using no ( - ) , MBP or wildtype His14 - MBP - ABH1 ( ABH1 ) . Experiments were performed in triplicate with error bars indicating ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O

B. Synthetic anticodon stem loop (ASL) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in (A) using no protein (-), MBP or wildtype His14-MBP-ABH1 (ABH1). Experiments were performed in triplicate with error bars indicating ± SD.


C . Anion exchange HPLC was performed on synthetic m5C containing ASL ( 20nt ) before after oxidation by ABH1 . small shift in retention time indicates formation of f5C modified . ABH1 oxidation product was then treated with NaBH4 to generate hm5C - modified . three samples were analysed by ESI - MS molecular ( m . w . ) is indicated on HPLC trace . Only f5C - containing was labelled efficiently with 1 - ethyl - 2 , 3 , 3 - trimethylindoleninium - 5 - sulfonate ( TMI ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Anion exchange HPLC analysis was performed on synthetic m5C containing ASL (20nt) before and after oxidation by ABH1. The small shift in retention time indicates formation of f5C modified RNA. The ABH1 oxidation product was then treated with NaBH4 to generate hm5C-modified RNA. All three samples were analysed by ESI-MS and the molecular weight (m.w.) is indicated on the HPLC trace. Only the f5C-containing RNA was labelled efficiently with 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate (TMI).


D . different retention times of m5C , hm5C f5C - modified ASL were confirmed by co - injection of samples shown in ( C ) . HPLC was performed as in C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. The different retention times of m5C, hm5C and f5C-modified ASL RNA were confirmed by co-injection of samples shown in (C). HPLC was performed as in C.


A . MTIF2 - dependent reading of initiation codons AUG , AUA or AUU in P site of ribosome by unmodified ( unmod . ) or C34 - modified [ 14C ] Met - tRNAMet . Binding was determined by nitrocellulose filtration [ 14C ] Met - tRNAMet retrieved on was quantified by scintillation counting . Binding in absence of ribosomes ( no 70S ) or mRNA ( no mRNA ) served as controls . Data from three independent experiments are presented with error bars indicating ± . statistical significance of results was analysed by T - is indicated by asterisks in graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. MTIF2-dependent reading of initiation codons AUG, AUA or AUU in the P site of the ribosome by unmodified (unmod.) or C34-modified [14C]Met-tRNAMet. Binding was determined by nitrocellulose filtration and [14C]Met-tRNAMet retrieved on the membrane was quantified by scintillation counting. Binding in the absence of ribosomes (no 70S) or mRNA (no mRNA) served as controls. Data from three independent experiments are presented with error bars indicating ± SEM. The statistical significance of the results was analysed by T-test and is indicated by the asterisks in the graph.


B . TUFM - dependent recognition of A site codons during elongation . Data from three independent experiments are presented with error bars indicating ± statistical as in ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. TUFM-dependent recognition of A site codons during elongation. Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in (A).


A . HeLa were transfected with two different siRNAs against NSUN3 ( siNSUN3 _ 1 , siNSUN3 _ 2 ) , ABH1 ( siABH1 _ 1 , siABH1 _ 2 ) or with nontarget ( siNT ) siRNA knockdown efficiency was analysed by quantitative PCR . relative abundance of NSUN3 or ABH1 mRNA was normalized to GAPDH . Data are presented as mean ± SD .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

A. HeLa cells were transfected with two different siRNAs against NSUN3 (siNSUN3_1, siNSUN3_2), ABH1 (siABH1_1, siABH1_2) or with nontarget (siNT) siRNA and the knockdown efficiency was analysed by quantitative PCR. The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels. Data are presented as mean ± SD.


B . Chemically synthesised f5C modified mt - tRNAMet total from wild - type ( WT ) or those transfected with siRNAs as in ( A ) were treated with TMI to specifically label f5C residues . Primer extension , using a radiolabelled antisense primer , was performed under limited dNTP conditions . Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder RNAs were detected using a phosphorimager .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Chemically synthesised f5C modified mt-tRNAMet and total RNA from wild-type (WT) cells or those transfected with siRNAs as in (A) were treated with TMI to specifically label f5C residues. Primer extension, using a radiolabelled antisense primer, was performed under limited dNTP conditions. Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager.


C . Primer extension reactions were performed on total from transfected with siRNAs as described in B . Stops corresponding to position C34 in mt - tRNAMet were quantified in three independent experiments results are shown graphically as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B. Stops corresponding to position C34 in mt-tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD.


D . from wild - type HeLa treated with siRNAs against NSUN3 or ABH1 ( as in A ) was either first reduced with NaBH4 or directly treated with bisulfite . After deamination desulfonation , mt - tRNAMetRNAs were reverse transcribed , amplified , cloned sequenced . proportions of thymine ( grey ) generated by bisulfite - conversion or non - converted cytosine ( black ) at position 34 of mt - tRNAMet are shown . Note that for sequences from non - reduced samples , thymine can originate from unmodified or f5C - containing mt - tRNAMet , while in reduced samples it originates from unmodified cytosine .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. RNA from wild-type HeLa cells and cells treated with siRNAs against NSUN3 or ABH1 (as in A) was either first reduced with NaBH4 or directly treated with bisulfite. After deamination and desulfonation, mt-tRNAMetRNAs were reverse transcribed, amplified, cloned and sequenced. The proportions of thymine (grey) generated by bisulfite-conversion or non-converted cytosine (black) at position 34 of mt-tRNAMet are shown. Note that for sequences from non-reduced samples, thymine can originate from unmodified or f5C-containing mt-tRNAMet, while in reduced samples it originates from unmodified cytosine.


A . HeLa were treated with non - target siRNAs ( siNT ) or those targeting NSUN3 ( siNSUN3 _ 1 or siNSUN3 _ 2 ) or ABH1 ( siABH1 _ 1 or siABH1 _ 2 ) for 72 h before labelling of mitochondrial translation products with [ 35S ] methionine . samples were separated by SDS - PAGE then transferred to a . Labelled were detected using a phosphorimager of tubulin were determined by western blotting using an antibody against endogenous for normalisation . B . Mitochondrially - translated that could be clearly detected were quantified in three independent experiments results are shown graphically as mean ± SD .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. HeLa cells were treated with non-target siRNAs (siNT) or those targeting NSUN3 (siNSUN3_1 or siNSUN3_2) or ABH1 (siABH1_1 or siABH1_2) for 72 h before labelling of mitochondrial translation products with [35S]methionine. Protein samples were separated by SDS-PAGE then transferred to a membrane. Labelled proteins were detected using a phosphorimager and the levels of tubulin were determined by western blotting using an antibody against the endogenous protein for normalisation.B. Mitochondrially-translated proteins that could be clearly detected were quantified in three independent experiments and the results are shown graphically as mean ± SD.


SnRK2 OST1 activates ABFs for expression of a luciferase reporter ( LUC ) in yeast . Luciferase activity is given on a Log10 scale as relative of emission per second ( rcps ) normalized to density . OST1 - mediated induction factor is indicated above columns . In absence of ABFs , LUC activity was 3 x 103 rcps .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

The SnRK2 OST1 activates ABFs for expression of a luciferase reporter (LUC) in yeast. Luciferase activity is given on a Log10 scale as relative counts of light emission per second (rcps) normalized to the cell density. The OST1-mediated induction factor is indicated above the columns. In the absence of ABFs, the LUC activity was 3 x 103 rcps.


Combining ABA signaling components ABF2 , OST1 , PP2C ABI1 , RCAR11 provides ABA - controlled LUC expression . reporter is under control of a minimal promoter that contains ABA - responsive cis elements ( ABREs ) . ABA signaling involves RCAR receptor that inhibits ABA coreceptor ( PP2C ) in presence of ABA allows activation of kinase SnRK2 . SnRK2 phosphorylates activates key transcription factor ABF , which targets promoters containing ABRE . Reporter activity was determined 16 h after exogenous ABA ( 0 . 1 mM ) administration referred to samples expressing ABF2 alone .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

Combining the ABA signaling components ABF2, OST1, the PP2C ABI1, and RCAR11 provides ABA-controlled LUC expression. The reporter is under the control of a minimal promoter that contains ABA-responsive cis elements (ABREs). Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor (PP2C) in the presence of ABA and allows the activation of the protein kinase SnRK2. SnRK2 phosphorylates and activates the key transcription factor ABF, which targets promoters containing ABRE. Reporter activity was determined 16 h after exogenous ABA (0.1 mM) administration and referred to samples expressing ABF2 alone.


In yeast , OST1 - ABF2 - mediated reporter activity was inhibited by ABI1 ( ‑ RCAR ) . subsequent recovery of response was determined by co - expressed ABA receptors RCAR1 - RCAR14 belonging to three subfamilies , in presence or absence of 30 µM ABA . ABA response is expressed relative to response obtained by replacing ABI1 with catalytically inactive ABI1D177A . Regulation of an ABA - responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1 - effector in combination with 5 µg ABA receptor ( ± 10 µM ABA ) per sample containing 105 protoplasts .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1-effector in combination with 5 µg ABA receptor DNA (± 10 µM ABA) per sample containing 105 protoplasts.


In yeast , OST1 - ABF2 - mediated reporter activity was inhibited by ABI1 ( ‑ RCAR ) . subsequent recovery of response was determined by co - expressed ABA receptors RCAR1 - RCAR14 belonging to three subfamilies , in presence or absence of 30 µM ABA . ABA response is expressed relative to response obtained by replacing ABI1 with catalytically inactive ABI1D177A . Regulation of an ABA - responsive LUC reporter in Arabidopsis protoplasts by ectopic

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression


ABA - dependent regulation of PP2Cs ABI1 ( A , D , G ) , ABI2 ( B , E , H ) , HAB1 ( C , F , I ) by RCAR12 RCAR13 were analyzed in yeast ( A - C ) , Arabidopsis protoplasts ( D - F ) , in vitro ( G - I ) . A - C ) in yeast was conducted as mentioned in Fig . 2A except that other PP2C - RCAR combinations were used ABA concentrations varied as indicated . D - F ) of protoplasts was performed in ABA - deficient background aba2 - 1 in presence of different exogenous ABA concentrations . ABA response was approximately tenfold inhibited by ectopic expression of respective PP2Cs ( Tischer et al , 2017 ) . induction of reporter gene is related to protoplasts without ectopic RCAR expression , in absence of exogenous ABA . G - I ) in vitro was performed by using 50 nM PP2C 100 nM RCAR at different ABA concentrations . Maximum inhibition of PP2C activity by ABA was set to 100 % . Data information : In ( A - C ) each data point represents mean ± s . d . ; n = 9 biological replicates derived from three independent yeast transformants . In ( D - F ) data represent mean ± s . d . of three independent transfections . In ( G - I ) data represent mean ± s . d . of three replicates .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I). A-C) The analysis in yeast was conducted as mentioned in Fig. 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated. D-F) Analysis of protoplasts was performed in the ABA-deficient background aba2-1 in the presence of different exogenous ABA concentrations. The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs (Tischer et al, 2017). The induction of the reporter gene is related to protoplasts without ectopic RCAR expression, and in the absence of exogenous ABA. G -I) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations. Maximum inhibition of PP2C activity by ABA was set to 100%. Data information: In (A-C) each data point represents the mean ± s.d.; n = 9 biological replicates derived from three independent yeast transformants. In (D-F) data represent the mean ± s.d. of three independent transfections. In (G-I) data represent the mean ± s.d. of three replicates.


ABA - dependent regulation of PP2Cs ABI1 ( A , D , G ) , ABI2 ( B , E , H ) , HAB1 ( C , F , I ) by RCAR12 RCAR13 were analyzed in yeast ( A - C ) , Arabidopsis protoplasts ( D - F ) , in vitro ( G - I ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I).


Expression of SnRK2s in yeast revealed ABF2 - mediated transactivation by SnRK2 . 1 , SnRK2 . 4 , SnRK2 . 5 , SnRK2 . 10 in addition to OST1 . Inset : Immuno - detection of FLAG - tagged SnRK2s expressed in yeast . data in ( B ) are normalized to control samples expressing ABF2 but no SnRK2

O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

Expression of the SnRK2s in yeast revealed ABF2-mediated transactivation by SnRK2.1, SnRK2.4, SnRK2.5, and SnRK2.10 in addition to OST1. Inset: Immuno-detection of FLAG-tagged SnRK2s expressed in yeast. The data in (B) are normalized to control samples expressing ABF2 but no SnRK2


Expression of SnRK2s in yeast revealed ABF2 - mediated transactivation by SnRK2 . 1 , SnRK2 . 4 , SnRK2 . 5 , SnRK2 . 10 in addition to OST1 . Inset : Immuno - detection of FLAG - tagged SnRK2s expressed in yeast . data in ( B ) are normalized to control samples expressing ABF2 but no

O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O


active subgroup I members together with SnRK2 . 2 SnRK2 . 3 were analyzed for differential activation of ABFs . OST1 data shown in Fig . 1A is included for better comparison . induction level of ABF4 relative to empty vector control ( ev ) is indicated .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

The active subgroup I members together with SnRK2.2 and SnRK2.3 were analyzed for differential activation of ABFs. The OST1 data shown in Fig. 1A is included for better comparison. The induction level of ABF4 relative to the empty vector control (ev) is indicated.


of phosphorylation status in activation loop of SnRK2s expressed in yeast . Phosphorylation occurred at serine 175 or threonine 176 , at threonine 179 . relative abundance is given as of signal of phosphorylated versus non - phosphorylated peptide for OST1 , SnRK2 . 3 , SnRK2 . 4 in mass spectrometric . numbering of phosphosites corresponds to OST1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast. Phosphorylation occurred at serine 175 or threonine 176, and at threonine 179. The relative abundance is given as the ratio of signal intensity of phosphorylated versus non-phosphorylated peptide for OST1, SnRK2.3, and SnRK2.4 in the mass spectrometric analysis. The numbering of phosphosites corresponds to OST1.


Inhibition of OST1 SnRK2 . 4 by clade A PP2Cs . data in ( G ) to expressing inactive ABI1D177A instead of ABI1 .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O

Inhibition of OST1 and SnRK2.4 by clade A PP2Cs. The data in (G) to cells expressing inactive ABI1D177A instead of ABI1.


A , B ) ABF2 - mediated reporter induction by different SnRK2s was assessed in presence of receptor RCAR11 ABI1 , varying exogenous ABA concentrations . Data information : ABA that provides half maximum response recovery is indicated by dashed vertical lines . represent Replicates were normalized to controls without SnRK2 in ( A , B )

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B) ABF2-mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1, and varying exogenous ABA concentrations. Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. represent Replicates were normalized to controls without SnRK2 in (A, B)


C ) Maximum ABA response recovery of samples shown in ( A , B ) relative to reporter activity of 0 . 1 mM ABA - treated yeast expressing SnRK2s but no ABI1 . Data information : Replicates were normalized to controls without ABI1 RCAR for each SnRK2 in ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

C) Maximum ABA response recovery of samples shown in (A,B) relative to the reporter activity of 0.1 mM ABA-treated yeast cells expressing SnRK2s but no ABI1. Data information: Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in (C).


D - F ) of ABA response mediated by OST1 SnRK2 . 4 using receptors RCAR1 ( D ) , RCAR8 ( E ) , RCAR14 ( F ) . Data information : ABA that provides half maximum response recovery is indicated by dashed vertical lines . data in ( D - F ) are mean ± s . e . m ; n = 12 , biological replicates from four independent yeast transformants for OST1 + RCAR1 / RCAR8 , SnRK2 . 4 + RCAR8 .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O

D-F) Analysis of the ABA response mediated by OST1 and SnRK2.4 using the receptors RCAR1 (D), RCAR8 (E), and RCAR14 (F). Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. The data in (D-F) are the mean ± s.e.m; n = 12, biological replicates from four independent yeast transformants for OST1 + RCAR1/RCAR8, and SnRK2.4 + RCAR8.


FRET - FLIM of GFP : OST1 or GFP : SnRK2 . 4 effect of ABI1 fused to mCherry fluorophore in presence of RCAR1 ABA . GFP lifetimes were determined after transient expression of Arabidopsis in leaves of Nicotiana benthamiana . Representative false colour of GFP : SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2 . 4 in combination with mCherry : ABI1 RCAR1 without exogenous ABA . bar marks 20 µm nucleus ( n ) cytosol ( c ) are indicated .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

FRET-FLIM analysis of GFP:OST1 or GFP:SnRK2.4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA. GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana.   Representative false colour images of GFP:SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2.4 in combination with mCherry:ABI1 and RCAR1 without exogenous ABA. The bar marks 20 µm and the nucleus (n) and cytosol (c) are indicated.


of SnRK2 - ABI1 interaction as mentioned in ( A ) but in presence of RCAR14 different exogenous ABA . RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1 . Representative GFP lifetime false colour image of shown in C ) 2h after 30 µM exogenous ABA administration . Details are as described in ( B ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of SnRK2-ABI1 interaction as mentioned in (A) but in the presence of RCAR14 and different exogenous ABA levels. RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1.   Representative GFP lifetime false colour image of the analysis shown in C) 2h after 30 µM exogenous ABA administration. Details are as described in (B).


of SnRK2 - ABI1 interaction as mentioned in ( A ) but in presence of RCAR14 different exogenous ABA . RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

Analysis of SnRK2-ABI1 interaction as mentioned in (A) but in the presence of RCAR14 and different exogenous ABA levels. RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1.


A ) ( left ) t - sne plot from scRNAseq data of human organoids cluster assignment of colonic populations . Red color corresponds to undifferentiated , green corresponds to surface colonocytes ( enterocytes ) . t - sne plot for selected genes from single RNAseq of human colon epithelial revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 AQP8 , which also express IFN - γ receptor IFNGR2 . B ) Immunofluorescence for ACE2 ( red ) KRT20 ( green ) of human colon tissue section indicating ACE2 expression in surface colonocytes . Scale bar : 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

A) (left) t-sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations. Red color corresponds to undifferentiated cells, light green corresponds to surface colonocytes (enterocytes). t-sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8, which also express the IFN-γ receptor IFNGR2. B) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of human colon tissue section indicating ACE2 expression in surface colonocytes. Scale bar: 50 µm.


C ) Human colon organoids cultured in full medium ( FM ) condition ( upper panel ) or FM treated with IFN - γ ( lower panel ) . Left : bright field of 7 - day cultured human colon organoids , scale bar : 100 µm . Right : Immunofluorescence for KRT20 ( green ; note increased expression upon IFN - γ treatment ) . Scale bar : 25 µm .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

C) Human colon organoids cultured in full medium (FM) condition (upper panel) or FM treated with IFN-γ (lower panel). Left: bright field images of 7-day cultured human colon organoids, scale bar: 100 µm. Right: Immunofluorescence for KRT20 (green; note increased expression upon IFN-γ treatment). Scale bar: 25 µm.


D ) Human colon organoids cultured in basic medium ( upper panel ) or additionally treated with IFN - γ ( lower panel ) . Left : bright field of organoids that were cultured for 4 days in basic medium in addition for 3 days with IFN - γ ( lower panel ) scale bar : 100 µm . Middle : Immunofluorescence labelling for KRT20 ( green ) Right : Immunofluorescence labelling for E - cadherin ( green ) to visualize epithelial boundaries ; insets with higher magnification indicate elongation of epithelial that are grown in basic medium treated with IFN - γ . Scale bar : 25 µm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

D) Human colon organoids cultured in basic medium (upper panel) or additionally treated with IFN-γ (lower panel). Left: bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN-γ (lower panel) scale bar: 100 µm. Middle: Immunofluorescence labelling for KRT20 (green) Right: Immunofluorescence labelling for E-cadherin (green) to visualize epithelial cell boundaries; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN-γ. Scale bar: 25 µm.


Comparison of E ) KRT20 , F ) MUC2 , G ) CHGA , H ) LGR5 mRNA expression in organoids grown in FM or in basic medium either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of E) KRT20, F) MUC2, G) CHGA, H) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


ACE2 mRNA expression in organoids grown in FM or in basic medium either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


J ) Immunofluorescence for ACE2 ( red ) KRT20 ( green ) of organoids grown in full medium ( upper ) or basic medium ( lower ) additionally treated with IFN - γ ( right ) , indicating expression of ACE2 in differentiated enterocytes . arrows point to ACE2 . Scale bar : 10

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O

J) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of organoids grown in full medium (upper) or basic medium (lower) and additionally treated with IFN-γ (right), indicating expression of ACE2 in differentiated enterocytes. White arrows point to ACE2. Scale bar: 10 µm


A ) Immunofluorescence for N - ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) basic medium + IFN - γ ( lower right ) . Yellow arrows point to N - positive . Scale bar : 25 µm .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

A) Immunofluorescence for N-protein (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Yellow arrows point to N-protein positive cells. Scale bar: 25 µm.