mRNA ( top ) ( bottom ) of indicated genes in transfected with indicated siRNAs . n = 8 . Error bars are s . e . m . P - values computed two - sided t - . * * P ≤ 0 . 01 * * * * P ≤ 0 . 0001 .

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mRNA (top) and protein (bottom) levels of the indicated genes in cells transfected with the indicated siRNAs. n=8. Error bars are s.e.m. P-values computed two-sided t-test. ** P≤0.01 **** P≤0.0001.


Differences in Nuc / Cyto ( log2 ) ratios for tiles containing SIRLOIN element ( up to 6 mismatches ) other NucLibB tiles ( only WT tiles were considered ) . Boxplots are as in Figure 1C . P - values computed using two - sided Wilcoxon rank - sum .

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Differences in the Nuc/Cyto (log2) ratios for tiles containing the SIRLOIN element (up to 6 mismatches) and other NucLibB tiles (only WT sequence tiles were considered). Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test.


IP / Input ( top ) changes in Nuc / Cyto ( log2 ) ratios upon siRNA targeting of indicated factor ( top ) for WT NucLibB tiles with indicated number of indicated motifs . Number of tiles in each group is indicated in parentheses . * - P < 0 . 05 * * P < 0 . 005 * * * P < 0 . 0005 for comparing group with indicated number of matches of motif with tiles containing a lower motif . Boxplots are as in Figure 1C . P - values computed using two - sided Wilcoxon rank - sum . * - P < 0 . 05 , * * - P < 0 . 005 , * * * - P < 0 . 0005 .

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IP/Input (top) and changes in Nuc/Cyto (log2) ratios upon siRNA targeting of the indicated factor (top) for WT NucLibB tiles with the indicated number of the indicated motifs. Number of tiles in each group is indicated in parentheses. *- P<0.05 ** P<0.005 *** P<0.0005 for comparing the group with the indicated number of matches of the motif with all tiles containing a lower motif count. Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test. * - P<0.05, ** - P<0.005, *** - P<0.0005.


( A - D ) Color fundus SW - AF examinations on four of β - thalassemia subject to chelation therapy by DFO for at least 16 years . Fundus , including RPE mottling depigmentation in macula ( Case Ⅰ ) ( Gelman et al , 2014 ) , choroidal sclerosis in perimacular areas ( Case Ⅱ ) , peripapillary ( Case Ⅲ Ⅳ ) , subretinal pigmentation ( Case Ⅳ ) were evaluated . RPE lesions including concentric distribution of stippled hyper - autofluorescence at macula ( Case Ⅰ ) , large areas of hypo - autofluorescent regions at parapapillary or perimacular ( Case Ⅱ III ) , extensive RPE loss in both eyes ( Case Ⅳ ) were observed by SW - AF .

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(A - D) Color fundus photographs and SW-AF examinations on four patients of β-thalassemia subject to chelation therapy by DFO for at least 16 years. Fundus phenotypes, including RPE mottling and depigmentation in the macula (Case Ⅰ) (Gelman et al, 2014), choroidal sclerosis in the perimacular areas (Case Ⅱ), peripapillary (Case Ⅲ and Ⅳ), and subretinal pigmentation (Case Ⅳ) were evaluated. RPE lesions including concentric distribution of stippled hyper-autofluorescence at the macula (Case Ⅰ), large areas of hypo-autofluorescent regions at parapapillary or perimacular area (Case Ⅱ and III), and extensive RPE loss in both eyes (Case Ⅳ) were observed by SW-AF.


( A - C ) Adult C57BL / 6J mice ( four months old ) were intraperitoneally injected with DFO at 100 mg / kg three times a week for three months in a row . SW - AF were acquired longitudinally at one , two three months post injection .

(A - C) Adult C57BL/6J mice (four months old) were intraperitoneally injected with DFO at 100 mg/kg three times a week for three months in a row. SW-AF images were acquired longitudinally at one, two and three months post injection.


( I , J ) Flat mount RPE was harvested from mice treated with DFO for five months stained by TUNEL to detect . Hoechst was used for . Untreated mice of similar were used as healthy controls . Flat mount were captured by 10 × confocal microscopy stitched together .

(I, J) Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death. Hoechst was used for nuclear staining. Untreated mice of similar age were used as the healthy controls. Flat mount images were captured by 10× confocal microscopy and stitched together.


( K - N ) RPE was dissected from mice injected with DFO for three nine months respectively stained with phalloidin . - matched untreated mice were included as controls . were taken at 40 × magnification .

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(K - N) The RPE was dissected from the mice injected with DFO for three and nine months respectively and stained with phalloidin. Age-matched untreated mice were included as the controls. The images were taken at 40× magnification.


( A - D ) microscopic examination of iRPE 24 48 hours post treatment . Scale bar : 50 µm .

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(A - D) Light microscopic examination of the iRPE cells 24 and 48 hours post treatment. Scale bar: 50 µm.


( E - H ) iRPE were stained with ZO - 1 24 48 hours post treatment . Untreated iRPE were used as control .

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(E - H) iRPE cells were stained with ZO-1 24 and 48 hours post treatment. Untreated iRPE cells were used as the control.


( I - L ) among iRPE population was examined by TUNEL 24 hours 48 hours post treatment . Untreated iRPE were included as control . were counterstained with Hoechst .

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(I - L) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment. Untreated iRPE cells were included as the control. The cells were counterstained with Hoechst.


( A ) iRPE treated with DFO were collected lysed to measure expression of HIF1α HIF2α 24 48 hours after treatment . iRPE supplemented with 0 . 1 % DMSO in culture media were used as control . Actin was used as loading control . HIF1α ( post - translationally modified ) was predicted to be ~ 100 kDa ; HIF2α ( post translationally modified ) was predicted to be ~ 120 kDa . ( B ) iRPE treated with DFO were lysed fractionated by centrifugation to measure expression of HIF1α HIF2α in nuclei 24 48 hours after treatment . Untreated iRPE were used as normal control . Histone H3 was used as loading control . (

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(A) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the control. Actin was used as the loading control. HIF1α (post-translationally modified) was predicted to be ~100 kDa; HIF2α (post translationally modified) was predicted to be ~ 120 kDa. (B) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment. Untreated iRPE cells were used as the normal control. Histone H3 was used as the loading control. (


( A ) Ferrous iron was measured . statistics are analyzed by one - way ANOVA with Tukey . results are presented as mean ± S . E . M . , n = 3 iRPE lines for each group . * p < 0 . 05 .

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(A) Ferrous iron was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05.


( B ) transcripts of ferroptosis - associated markers were determined by qPCR using iRPE as template . extract from untreated iRPE was included as control . level of each transcript was normalized to ACTB . statistics are analyzed by paired student ' s t - for each target gene . results are presented as mean ± S . E . M . , n = 3 iRPE lines for each group . * p < 0 . 05 ; * * p < 0 . 01 . Round dots : untreated iRPE ; square dots : DFO - treated iRPE .

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(B) The transcripts of ferroptosis-associated markers were determined by qPCR using iRPE cells as the template. The RNA extract from untreated iRPE was included as the control. The level of each transcript was normalized to ACTB. The statistics are analyzed by ratio paired student's t-test for each target gene. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05; ** p < 0.01. Round dots: untreated iRPE; square dots: DFO-treated iRPE.


( C ) level of ROS was measured . statistics are analyzed by one - way ANOVA with Tukey . results are presented as mean ± S . E . M . , n = 6 iRPE lines for each group . * p < 0 . 05 , * * p < 0 . 01 . DHE : Dihydroethidium .

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(C) The level of ROS was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 6 iRPE lines for each group. * p < 0.05, ** p < 0.01. DHE: Dihydroethidium.


( D ) Mitochondrial respiration of iRPE with DFO treatment was determined by seahorse extracellular flux . OCR was determined over a course of time . Each data point is shown as mean ± S . E . M . , n = 4 iRPE lines for each group . OCR : oxygen consumption rate ; FCCP : carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ; Rot : rotenone ; AA : antimycin A .

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(D) Mitochondrial respiration of the iRPE cells with DFO treatment was determined by seahorse extracellular flux assay. OCR was determined over a course of time. Each data point is shown as mean ± S.E.M., n = 4 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.


( A - C ) microscopy of morphological protection of AKG against DFO toxicity on iRPE . were treated with DFO at 100 mM for 48 hours ( B ) . AKG was supplemented at 10 mM simultaneously ( C ) . control group was supplemented with 0 . 1 % DMSO ( A ) . Scale bar : 100 µm . ( D - F ) DFO - treated iRPE with or without AKG supplementation were subject to phalloidin ( E F ) . iRPE treated with 0 . 1 % DMSO - containing media were included as controls ( D ) . were captured at 40 × magnification . Scale bar : 30 µm . ( G - I ) Detection of in DFO - treated iRPE with or without AKG supplementation by TUNEL . Hoechst was used for . were captured at 40 × magnification . Scale bar : 30 µm . (

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(A - C) Light microscopy of morphological protection of AKG against DFO toxicity on iRPE. The cells were treated with DFO at 100 mM for 48 hours (B). AKG was supplemented at 10 mM simultaneously (C). The control group was supplemented with 0.1% DMSO (A). Scale bar: 100 µm. (D - F) The DFO-treated iRPE cells with or without AKG supplementation were subject to phalloidin staining (E and F). The iRPE cells treated with 0.1% DMSO-containing media were included as the controls (D). The images were captured at 40× magnification. Scale bar: 30 µm. (G - I) Detection of cell death in the DFO-treated iRPE cells with or without AKG supplementation by TUNEL staining. Hoechst was used for nuclear staining. The images were captured at 40× magnification. Scale bar: 30 µm. (J


( J - L ) SW - AF was performed longitudinally on mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months .

(J - L) SW-AF was performed longitudinally on the mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months.


( A ) Mitochondrial respiration of DFO - treated ( 100 mM ) iRPE with AKG supplementation ( 10 mM ) for 24 hours was determined by seahorse extracellular flux . OCR was measured over a course of time . Each dot is shown as mean ± S . E . M . , n = 5 iRPE lines for each group . OCR : oxygen consumption rate ; FCCP : carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ; Rot : rotenone ; AA : antimycin A .

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(A) Mitochondrial respiration of the DFO-treated (100 mM) iRPE cells with AKG supplementation (10 mM) for 24 hours was determined by seahorse extracellular flux assay. OCR was measured over a course of time. Each dot is shown as mean ± S.E.M., n = 5 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.


( G ) DFO - treated ( 100 mM ) iRPE supplemented with AKG ( 10 mM ) for 48 hours were lysed for immunoblotting against HIF2α . iRPE supplemented with 0 . 1 % DMSO in culture media were used as controls . HIF2α ( post translationally modified ) was predicted to be ~ 120 kDa . Actin was included as loading control . ( H ) RPE lysate from one - year - old mice subject to 10 - month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α . RPE of - matched untreated mice was included as control . HIF2α ( degraded ) was predicted to be 40 - 80 kDa . Actin was used as loading control .

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(G) The DFO-treated (100 mM) iRPE cells supplemented with AKG (10 mM) for 48 hours were lysed for immunoblotting against HIF2α. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the controls. HIF2α (post translationally modified) was predicted to be ~ 120 kDa. Actin was included as the loading control. (H) RPE lysate from the one-year-old mice subject to 10-month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α. The RPE of the age-matched untreated mice was included as the control. HIF2α (degraded) was predicted to be 40 - 80 kDa. Actin was used as the loading control.


Macrophages produce little to no inflammatory cytokines when incubated with either ( a ) whole iRBC for 24 h . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance Each symbol represents value obtained for from an independent donor in an independent experiment . ( a ) n = 4 ; Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared with

Macrophages produce little to no inflammatory cytokines when incubated with either (a) whole iRBC for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


Macrophages produce little to no inflammatory cytokines when incubated with either ( b ) fresh iRBCL for 24 h . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance Each symbol represents value obtained for from an independent donor in an independent experiment . ( b ) n = 5 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared with

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Macrophages produce little to no inflammatory cytokines when incubated with either (b) fresh iRBCL for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (b) n=5 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


( c ) Macrophages were pre - incubated with IFNγ ( 1000U / ml ) for 18 h before iRBCL stimulation . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance Each symbol represents value obtained for from an independent donor in an independent experiment . ( c ) n = 3 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared with

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(c) Macrophages were pre-incubated with IFNγ (1000U/ml) for 18 h before iRBCL stimulation. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=3 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


( d ) Phase - contrast microscopy of macrophages 24 h after addition of RBC , iRBC or LPS . hemozoin crystals can be observed within macrophages . Bar is 30 µm .

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(d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm.


( e ) Macrophages were incubated with iRBCL 24 h before addition of LPS . Paired t - was performed to determine statistical significance . Each symbol represents value obtained for from an independent donor in an independent experiment . ( e ) n = 3 . Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared RBCL ( e ) .

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(e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared RBCL (e).


Cytokine secretion of macrophages incubated with indicated stimuli for 24 h . RBCL , iRBCL , XO were added to macrophages for 24 h . Each symbol represents value obtained for from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL ( a ) n = 7 One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. RBCL, iRBCL, XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL (a) n=7 One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with indicated stimuli for 24 h . , XO , Hypoxanthine ( Hyx ) were added to macrophages for 24 h . Each symbol represents value obtained for from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO, Hypoxanthine (Hyx) were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with indicated stimuli for 24 h . XO added to macrophages for 24 h . Febuxostat ( Feb ) was pre - incubated for 30 minutes with XO before addition to macrophages . Each symbol represents value obtained for from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. XO added to macrophages for 24 h. Febuxostat (Feb) was pre-incubated for 30 minutes with XO before addition to macrophages. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with indicated stimuli for 24 h . , XO anti - oxidants N - acetyl - L - cysteine ( NAC ) 1 - Thioglycerol ( 1 - TG ) , heat inactivated ( HI ) XO were added to macrophages for 24 h . Each symbol represents value obtained for from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO the anti-oxidants N-acetyl-L-cysteine (NAC) and 1-Thioglycerol (1-TG), and heat inactivated (HI) XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of PBMC ( e ) incubated with indicated stimuli for 24 h . RBC , iRBC , RBCL , iRBCL , XO Each symbol represents value obtained for from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL n = 3 . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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Cytokine secretion of PBMC (e) incubated with the indicated stimuli for 24 h. RBC, iRBC, RBCL, iRBCL, XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


( a ) XO was incubated with macrophages for indicated times , when wells were washed twice media was replaced . Supernatants were collected for cytokine determinations 24 h later ( n = 3 ) . Kruskal - Wallis statistical method was used for statistical . Blue asterisks indicate significance when values were compared with time 0 ( * p < 0 . 05 ) .

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(a) XO was incubated with macrophages for the indicated times, when wells were washed twice and media was replaced. Supernatants were collected for cytokine determinations 24 h later (n=3). Kruskal-Wallis statistical method was used for statistical analysis. Blue asterisks indicate significance when values were compared with time 0 (*p<0.05).


( b ) Diagram shows experimental set up with macrophages in bottom of well XO added either in dialysis cassette ( DC , blue ) or well ( black ) . XO activity panels show that enzyme did not leak through DC . Secretion of IL6 TNF was measured 24 h after addition of XO in each compartment . A representative result of two independent experiments is shown .

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(b) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette (DC, blue) or the well (black). XO activity panels show that the enzyme did not leak through the DC membrane. Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment. A representative result of two independent experiments is shown.


( a ) Plasma of TNF IL8 from with uncomplicated ( black circles , n = 14 ) or cerebral ( red circles , n = 9 ) malaria correlate with of XO - produced ROS detected in each sample . ROS is expressed as fold change over plasma from healthy controls . Uncomplicated malaria , gray circles , cerebral malaria , red circles . Linear regression ( a ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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 O O O O O O O O O O O O O O O O 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) Plasma levels of TNF and IL8 from patients with uncomplicated (black circles, n=14) or cerebral (red circles, n=9) malaria correlate with levels of XO-produced ROS detected in each sample. ROS is expressed as fold change over plasma from healthy controls. Uncomplicated malaria, gray circles, cerebral malaria, red circles. Linear regression (a) were performed to determine statistical significance (*p<0.05, **p<0.01).


( b ) of XO activity in malaria correlate with disease severity UM ( uncomplicated malaria ) , CM ( cerebral malaria ) . Mann - Whitney ( b ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

(b) Levels of XO activity in malaria patients correlate with disease severity UM (uncomplicated malaria), CM (cerebral malaria). Mann-Whitney test (b) were performed to determine statistical significance (*p<0.05, **p<0.01).


( c ) Plasma from malaria induce macrophages to secrete cytokines , which are inhibited by a XO specific inhibitor . Plasma from a healthy control ( HP ) from three : P1 ( with cerebral malaria ) , P2 P3 ( both with uncomplicated malaria ) was pre - incubated for 30 min with febuxostat ( Feb ) or alone at 37ºC before addition to macrophages at 1 : 2 dilution in media for 30 min . were washed cytokine secretion measured in triplicates after 24 h of incubation . unpaired t - ( c ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

(c) Plasma from malaria patients induce macrophages to secrete cytokines, which are inhibited by a XO specific inhibitor. Plasma from a healthy control (HP) and from three patients: P1 (with cerebral malaria), P2 and P3 (both with uncomplicated malaria) was pre-incubated for 30 min with febuxostat (Feb) or alone at 37ºC before addition to macrophages at 1:2 dilution in media for 30 min. Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation. unpaired t-tests (c) were performed to determine statistical significance (*p<0.05, **p<0.01).


Cytokine secretion by macrophages after 24 h incubation with indicated stimuli . ( b ) IL1β after addition of increasing of RBCL or iRBCL ( 1 : 2 , 1 : 4 1 : 8 macrophage : iRBCL ) . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) . Each symbol represents value obtained for from an independent donor in an independent experiment . ( a ) n = 4 ; ( b ) n = 2 Insets show paired samples by donor in two different conditions .

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 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

Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli. (b) IL1β levels after addition of increasing concentration of RBCL or iRBCL (1:2, 1:4 and 1:8 macrophage:iRBCL). One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=2 Insets show paired samples by donor in two different conditions.


chemokine secretion by macrophages after 24 h incubation with indicated stimuli . One - way ANOVA with Tukey for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) . Each symbol represents value obtained for from an independent donor in an independent experiment . ( c ) n = 4 . Insets show paired samples by donor in two different conditions .

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

chemokine secretion by macrophages after 24 h incubation with the indicated stimuli. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=4. Insets show paired samples by donor in two different conditions.


( a ) Gene expression of macrophages incubated with indicated stimuli expressed as fold change over RBCL . Dotted red indicates 2 - fold increase in expression .

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(a) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL. Dotted red line indicates 2-fold increase in RNA expression.


( b ) Western Blot of proIL1β . Relative abundance is average of 3 independent experiments with different donors .

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(b) Western Blot analysis of proIL1β. Relative abundance is the average of 3 independent experiments with different donors.


( c ) FLICA of activated caspase - 1 . Results are representative of 2 independent experiments with different donors .

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

(c) FLICA analysis of activated caspase-1. Results are representative of 2 independent experiments with different donors.


( d ) Two distinct siRNAs against NLRP3 an irrelevant control siRNA were utilized to knock down by lipofectamine transfection in macrophages that were later incubated with iRBCL , or RBCL as control , with or without XO , before of secreted IL1β . Results are representative of 2 independent experiments with different donors .

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(d) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL, or RBCL as control, with or without XO, before quantification of secreted IL1β. Results are representative of 2 independent experiments with different donors.


( B , C ) of anti - HEL IgG in serum as measured by ELISA at indicated days post immunization with HEL - OVA / CFA following adoptive transfer of BHEL without CD4OT - II ( no T control ) or with control CD4OT - II or NEF - expressing CD4OT - II . ( B ) NEF WT of HIV - 1SF2 ( C ) - derived NEF variants . Shown are relative of HEL IgG production with mean of IgG in mice receiving control between days 12 - 14 post immunization arbitrarily set to 1 . Each dot represents one . Bars represent mean values with SD from 2 - 10 mice analyzed from 3 ( B ) 2 ( C ) independent experiments . Statistical significance was assessed by Mann Whitney U . * * * P ≤ 0 . 001 , * P ≤ 0 .

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-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-CONTROLLED_VAR 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 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

(B, C) Levels of anti-HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL-OVA/CFA following adoptive transfer of BHEL cells without CD4OT-II cells (no T cell control) or with control CD4OT-II cells or NEF-expressing CD4OT-II cells. (B) NEF WT of HIV-1SF2 (C) patient-derived NEF variants. Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1. Each dot represents one animal. Bars represent mean values with SD from 2-10 mice analyzed from 3 (B) and 2 (C) independent experiments. Statistical significance was assessed by Mann Whitney U test. ***P ≤0.001, *P ≤0.05


( E ) Representative confocal of p - tyrosine ( p - Tyr ) polarization at B - T contacts after 1 - 2h of co - culture . conjugates were stained with anti - pTyr antibody . Scale bar , 5µm . top panels display only p - Tyr signal in grey , bottom panel depicts p - Tyr ( red ) , BHEL ( blue ) CD4OT - II ( green ) . ( F ) of p - Tyr accumulation at B - T interface as shown in E . Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment .

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(E) Representative confocal micrographs of p-tyrosine (p-Tyr) polarization at B-T cell contacts after 1-2h of co-culture. Cell conjugates were stained with anti-pTyr antibody. Scale bar, 5µm. The top panels display only the p-Tyr signal in grey, the bottom panel depicts p-Tyr (red), BHEL cells (blue) and CD4OT-II cells (green). (F) Quantification of p-Tyr accumulation at the B-T interface as shown in E. Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment.


( G ) NEF prevents induction of F - actin remodeling in BHEL during IS . BHEL ( blue ) CD4OT - II ( green ) were placed on PLL - coated slides subjected to live - time - lapse imaging . Shown are representative still from indicated time points post contact formation ( see Movies EV1 EV2 ) . Red signals indicate actin polymerization specifically in BHEL . Scale bars , 5µm . Filled empty arrowheads indicate examples of accumulation of polarized actin or p - Tyr signal at B - T interface during IS formation or lack thereof , respectively . Asterisks mark pairs of interacting T B . ( H ) of B actin polymerization as shown in G with frequency of actin polymerization in B co - cultured with control T arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment .

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(G) NEF prevents induction of F-actin remodeling in BHEL during IS. BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated slides and subjected to live-cell time-lapse imaging. Shown are representative still images from the indicated time points post contact formation (see Movies EV1 and EV2). Red signals indicate actin polymerization specifically in BHEL cells. Scale bars, 5µm. Filled and empty arrowheads indicate examples of accumulation of polarized actin or p-Tyr signal at B-T interface during IS formation or lack thereof, respectively. Asterisks mark pairs of interacting T and B cell. (H) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment.


( L ) Relative B proliferation ( quantified by gating on last 3 - 4 generations ) is plotted as mean values with SD from four independent experiments . Control T + Ag were arbitrarily set to 1 .

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(L) Relative B cell proliferation (quantified by gating on the last 3-4 generations) is plotted as mean values with SD from four independent experiments. Control T cells +Ag were arbitrarily set to 1.


( B ) Production of anti - HEL IgG at indicated day post immunization with HEL - OVA / CFA following adoptive transfer of BHEL CD4OT - II expressing NEF or various NEF mutants . Shown are relative of HEL IgG production with median of IgG in mice receiving control on day 14 post immunization arbitrarily set to 1 . Each dot represents one . Shown are mean values with SD from 5 - 12 mice from 4 experiments .

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(B) Production of anti-HEL IgG at the indicated day post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and CD4OT-II cells expressing NEF or various NEF mutants. Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post immunization arbitrarily set to 1. Each dot represents one animal. Shown are mean values with SD from 5-12 mice from 4 experiments.


( D ) of B actin polymerization as shown in C with frequency of actin polymerization in B co - cultured with control T arbitrarily set to 100 % . Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition .

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(D) Quantification of B cell actin polymerization as shown in C with frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition.


( H ) Relative surface expression of CD4 , CD28 , OX40 , ICOS , CD45 CD54 on T expressing NEF WT or indicated NEF mutants . of surface expression was performed using flow cytometry 3 - 7 d post transduction of T . Depicted are mean values with SD from 3 - 6 independent experiments , with control T arbitrarily set to 100 % as indicated by dashed .

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(H) Relative surface expression of CD4, CD28, OX40, ICOS, CD45 and CD54 on T cells expressing NEF WT or the indicated NEF mutants. Analysis of surface expression was performed using flow cytometry 3-7 d post transduction of T cells. Depicted are mean values with SD from 3-6 independent experiments, with control T cells arbitrarily set to 100% as indicated by the dashed line.


( I ) Representative confocal of subcellular distribution of IL - 4 in CD4OT - II expressing indicated engaged in an IS with BHEL . B T were placed on PLL - coated cover glasses for 60 - 90min , fixed stained for intracellular IL - 4 . Filled empty arrowheads indicate presence or absence of IL - 4 accumulation at IS , respectively . Scale bars , 5µm . ( J ) of IL - 4 accumulation at IS as depicted in I . Shown are mean values with SD from 3 independent experiments relative to control set to 100 % .

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(I) Representative confocal images of the subcellular distribution of IL-4 in CD4OT-II cells expressing the indicated proteins engaged in an IS with BHEL cells. B cells and T cells were placed on PLL-coated cover glasses for 60-90min, fixed and stained for intracellular IL-4. Filled and empty arrowheads indicate presence or absence of IL-4 accumulation at the IS, respectively. Scale bars, 5µm. (J) Quantification of IL-4 accumulation at the IS as depicted in I. Shown are mean values with SD from 3 independent experiments relative to control cells set to 100%.


( A ) Representative cross - sections of draining LNs ( dLNs ) from mice 7d post immunization with HEL - OVA / CFA following adoptive transfer of BHEL transduced CD4OT - II . dLN were cryosectioned stained with fluorophore - conjugated antibodies against GL - 7 to reveal GCs ( green ) , CD21 / 35 to stain for follicular dendritic ( FDC , red ) Hoechst to stain for nuclei ( blue ) . Scale bar , 250µm . ( B , C ) of number of GCs relative to B follicles ( B ) of GC ( C ) on confocal as shown in A . Shown are mean values with SD from dLNs from three mice in two independent experiment . Each dot represents one LN ( 3 LNs / mouse ) in B one GC 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 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 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 O O O O O O O O O O

(A) Representative cross-sections of draining LNs (dLNs) from mice 7d post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and transduced CD4OT-II cells. dLN were cryosectioned and stained with fluorophore-conjugated antibodies against GL-7 to reveal GCs (green), CD21/35 to stain for follicular dendritic cells (FDC, red) and Hoechst to stain for cell nuclei (blue). Scale bar, 250µm. (B, C) Quantification of the number of GCs relative to B cell follicles (B) and size of GC (C) on confocal micrographs as shown in A. Shown are mean values with SD from dLNs from three mice in two independent experiment. Each dot represents one LN (3 LNs/mouse) in B and one GC in C.


( A ) Still of Movies EV3 ( Control , upper panel ) EV4 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL ( green ) mTomato CD4OT - II ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) , respectively . High endothelial venules ( HEVs ) ( ) were labeled by intravenous injection of Alexa660 - labeled Meca - 79 antibody collagen ( blue ) is visualized by second harmonic generation ( SHG ) . Yellow orange arrows indicate stable transient ISs , respectively . Numbering of ISs corresponds to that in respective Supplemental movies . Scale bar 20µm . ( B ) of duration of interactions as indicated by arrows in A . Touching T B were manually using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Data is derived from 5 to 6 movies , acquired from 4 mice in 2 independent experiments . Percentages on top indicate frequency of interactions that lasted 20min or longer .

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 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 O O O O

(A) Still images of the Movies EV3 (Control, upper panel) and EV4 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel), respectively. High endothelial venules (HEVs) (white) were labeled by intravenous injection of Alexa660-labeled Meca-79 antibody and collagen (blue) is visualized by second harmonic generation (SHG). Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (B) Quantification of the duration of interactions as indicated by the arrows in A. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Data is derived from 5 to 6 movies, acquired from 4 mice in 2 independent experiments. Percentages on top indicate frequency of interactions that lasted 20min or longer.


( C ) Still of Movies EV5 ( Control , upper panel ) EV6 ( NEF , lower panel ) , obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL - OVA / CFA , following adoptive transfer of GFP BHEL ( green ) mTomato CD4OT - II ( red ) expressing empty vector ( Control ; upper panel ) or NEF WT ( lower panel ) . Follicular dendritic ( FDCs ) ( ) were labeled by subcutaneous injection of Alexa647 - labeled anti - CD21 / CD35 antibody collagen ( blue ) is visualized by SHG . Yellow orange arrows indicate stable transient ISs , respectively . Numbering of ISs corresponds to that in respective Supplemental movies . Scale bar 20µm . ( D ) of duration of interactions as indicated by arrows in C . Touching T B were manually using virtual reality ( InViewR , Arivis ) . Each dot represents one T - B interaction pair . Red bar shows median . Shown is one representative dataset out of two , derived from 5 to 8 movies , acquired from 4 mice . Percentages on top indicate frequency of interactions that lasted 20min or longer .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Still images of the Movies EV5 (Control, upper panel) and EV6 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel). Follicular dendritic cells (FDCs) (white) were labeled by subcutaneous injection of Alexa647-labeled anti-CD21/CD35 antibody and collagen (blue) is visualized by SHG. Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (D) Quantification of the duration of interactions as indicated by the arrows in C. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Shown is one representative dataset out of two, derived from 5 to 8 movies, acquired from 4 mice. Percentages on top indicate frequency of interactions that lasted 20min or longer.


( B ) replication as assessed by of RT activity in culture supernatant . Shown are mean values with SD from 6 donors .

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(B) Virus replication as assessed by quantification of RT activity in the cell culture supernatant. Shown are mean values with SD from 6 donors.


( C ) Production of IgG antibodies against TT from infected blocks at indicated time point . Each dot represents one donor , bar indicates mean value . Tonsillar blocks were infected with indicated for 24h . After washing off , blocks were stimulated with TT antigen for 24h . Samples were collected at different time intervals to estimate viral replication antibody production .

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(C) Production of IgG antibodies against TT from infected blocks at indicated time point. Each dot represents one donor, bar indicates mean value. Tonsillar blocks were infected with indicated virus for 24h. After washing off the virus, blocks were stimulated with TT antigen for 24h. Samples were collected at different time intervals to estimate viral replication and antibody production.


Distribution of RT shifts calculated with - ΔL + ΔE method described in Appendix Figure S1 . Different transgenes in a mid - late - replicating region are compared : IL2R reporter gene under control of βA - globin promoter ( βA pro ) containing an inactive ( i , N = 10 ) or an active origin ( ii , N = 8 ) flanked by two copies of FIV ( 2xFIV ) , blasticidin resistance gene ( BsR ) under control of β - actin promoter ( β - act pro ) ( iii , N = 7 ) or a combination of these two transgenes ( iv , N = 8 ) . Blue black triangles represent reactive loxP sites recombined inactive loxP sites respectively . Rectangle edges correspond to 0 . 25 0 . 75 quartiles , thick black lines represent median , triangles represent mean whiskers extend to smallest largest - ΔL + ΔE values . Statistical was performed with Wilcoxon nonparametric two - ( ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

Distribution of RT shifts calculated with the -ΔL+ΔE method described in Appendix Figure S1. Different transgenes in a mid-late-replicating region are compared: the IL2R reporter gene under the control of the βA-globin promoter (βA pro) containing an inactive (i, N=10) or an active origin (ii, N=8) flanked by two copies of FIV( 2xFIV), the blasticidin resistance gene (BsR) under the control of the β-actin promoter (β-act pro) (iii, N=7) or a combination of these two transgenes (iv, N=8). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (ns, not significant; **p<0.01; ***p<0.001).


A . UCSC genome browser visualization of mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 71 , 000 , 000 - 74 , 100 , 000 bp , 3 . 1 Mb ; galGal5 ) . RT weighted average ( WA ) values for wt βA - globin + β - actin lines are shown . Early - replicated regions ( E ) are represented in orange late replicating regions ( L ) in blue . Annotated genes ( Ref Seq genes ) are represented below . mid - late insertion site is indicated with a red arrow dotted .

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

A. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:71,000,000-74,100,000 bp, 3.1 Mb; galGal5). RT weighted average (WA) values for the wt and the βA-globin+β-actin cell lines are shown. Early-replicated regions (E) are represented in orange and late replicating regions (L) in blue. Annotated genes (Ref Seq genes) are represented below. The mid-late insertion site is indicated with a red arrow and dotted line.


B . UCSC genome browser visualization of mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 72 , 450 , 000 - 72 , 650 , 000 bp ; 200 kb ; galGal5 ) . Tracks of nascent strands ( NS ) enrichments in four S phase fractions were represented separately ( S1 to S4 ) for wt 2x ( βA - globin + β - actin ) lines . NS enriched depleted regions for each fraction are represented in purple blue respectively . Single reads form SNS aligned track of replication origins ( Ori peaks ) determined in ( Massip et al , 2019 ) are reported in between . three mid - late insertion sites 1 , 2 3 are indicated with red arrows . Annotated genes CpG Islands are shown below . Initiation zones ( IZ ) termination zones ( TZ ) are reported .

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

B. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:72,450,000-72,650,000 bp; 200 kb; galGal5). Tracks of nascent strands (NS) enrichments in the four S phase fractions were represented separately (S1 to S4) for the wt and the 2x(βA-globin+β-actin) cell lines. NS enriched and depleted regions for each fraction are represented in purple and blue respectively. Single reads form SNS aligned and track of replication origins (Ori peaks) determined in (Massip et al, 2019) are reported in between. The three mid-late insertion sites 1, 2 and 3 are indicated with red arrows. Annotated genes and CpG Islands are shown below. Initiation zones (IZ) and termination zones (TZ) are reported.


A . Distribution of - ΔL + ΔE values for clonal lines containing one advanced replicon ( βA - globin + β - actin ) inserted at site 1 ( i , N = 8 ) or 3 ( iii , N = 3 ) or at both sites on same chromosome with one GFP reporter construct composed of GFP reporter gene under control of βA - globin promoter / origin linked to a 1 . 6 kb fragment of human chromosome 7 ( h . K7 ) inserted at site 2 , either on same chromosome as 1 3 ( 1 + 2 + 3 ) , or on other chromosome ( 1 + 3 ) ( ii , N = 5 ) . Blue black triangles represent reactive loxP sites recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to 0 . 25 0 . 75 quartiles , thick black lines represent median , triangles represent mean whiskers extend to smallest largest - ΔL + ΔE values . Statistical was performed with Wilcoxon nonparametric two - ( * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of -ΔL+ΔE values for clonal cell lines containing one advanced replicon (βA-globin+β-actin) inserted at site 1 (i, N=8) or 3 (iii, N=3) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA-globin promoter/origin linked to a 1.6 kb fragment of human chromosome 7 (h.K7) inserted at site 2, either on the same chromosome as 1 and 3 (1+2+3), or on the other chromosome (1+3) (ii, N=5). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


C . Interaction profiles of allele 1 + 3 / 2 with chr1 for combined Hi - C libraries : - corrected interactions , i . e . observed / expected ( OE ) , binned at 500 kb with corresponding compartment ( EV1 ) tracks . Red arrows represent loci that show preferred interactions with allele 1 + 3 that coincide with A compartment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Interaction profiles of allele 1+3/2 with chr1 for combined Hi-C libraries: distance-corrected interactions, i.e. observed/expected (OE), binned at 500 kb with corresponding compartment ( EV1) tracks. Red arrows represent the loci that show preferred interactions with allele 1+3 and that coincide with A compartment.


D . OE interactions of allele 1 + 3 ( orange rectangles ) allele 2 ( blue rectangles ) averaged over A B compartments for p q - arms at 100kb bin . A random compartment status permutation was made then we calculated one - sided p - value for observed value of ' difference allele 1 + 3 / 2 A - B ' . Values corresponding to OE interactions of allele 1 + 3 allele 2 averaged over shuffled A ( red circles , N = 1000 ) B compartments ( blue circles , N = 1000 ) for p q - arms . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. OE interactions of allele 1+3 (orange rectangles) and allele 2 (blue rectangles) averaged over A and B compartments for p and q-arms at 100kb bin size. A random compartment status permutation test was made and then we calculated the one-sided p-value for the observed value of 'difference allele 1+3 / 2 A - B'. Values corresponding to OE interactions of allele 1+3 and allele 2 averaged over shuffled A (red circles, N=1000) and B compartments (blue circles, N=1000) for p and q-arms. ns, not significant; **p<0.01;***p<0.001.


E . Scatterplot of density of interactions between allele 1 + 3 allele 2 with chromosomes larger than 5 , 000 , 000 bp .

O 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. Scatterplot of density of interactions between allele 1+3 and allele 2 with chromosomes larger than 5,000,000 bp.


A . Distribution of - ΔL + ΔE values for clonal lines containing one β - actin construct at site 1 ( i , N = 7 ) or at site 1 3 ( ii , N = 5 ) , on same chromosome . Data information : Blue black triangles represent reactive loxP sites recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to 0 . 25 0 . 75 quartiles , thick black lines represent median , triangles represent mean whiskers extend to smallest largest - ΔL + ΔE values . Statistical was performed with Wilcoxon nonparametric two - ( * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of -ΔL+ΔE values for clonal cell lines containing one β-actin construct at site 1 (i, N=7) or at site 1 and 3 (ii, N=5), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


B . Distribution of - ΔL + ΔE values for clonal lines containing one βA - globin construct at site 1 ( i , N = 8 ) or at site 1 3 ( ii , N = 6 ) , on same chromosome . Data information : Blue black triangles represent reactive loxP sites recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to 0 . 25 0 . 75 quartiles , thick black lines represent median , triangles represent mean whiskers extend to smallest largest - ΔL + ΔE values . Statistical was performed with Wilcoxon nonparametric two - ( * * p < 0 . 01 ) .

B. Distribution of -ΔL+ΔE values for clonal cell lines containing one βA-globin construct at site 1 (i, N=8) or at site 1 and 3 (ii, N=6), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


C . After 1 , 8 , 24 48 h of 4 - hydroxytamoxifen treatment , genomic was extracted quantified by semi - quantitative PCR ( see Appendix Figure S6 ) . Error bars correspond to standard deviation for PCR duplicates . percentages of with large 1 + 3 loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) or β - actin ( 1 + 3 ) excision at each time point are shown for different lines .

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

C. After 1, 8, 24 and 48 h of 4-hydroxytamoxifen treatment, genomic DNA was extracted and quantified by semi-quantitative PCR (see Appendix Figure S6). Error bars correspond to the standard deviation for PCR duplicates. The percentages of cells with the large 1+3 and loxP_LE (1) + βA-globin+β-actin (3) or β-actin (1+3) excision at each time point are shown for different cell lines.


A , B . Clones 1 + 2 + 3 , 1 + 3 ( A ) loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) ( B ) were used to assess accessibility within transgenes . positions of amplicons used for in each shifting construct individually ( thick black lines ; 5 ' 2xFIV - 1 or - 3 , β - actin pro - 1 or - 3 , BsR gene , PuroR gene , LoxP site ) , in both shifting constructs ( βA pro 1 2 , IL2R gene , 3 ' 2xFIV1 - 3 ) or in GFP reporter construct ( βA - GFP , GFP gene , h . K7 ) are shown . insertion sites on wt chromosome ( insertion sites 1 , 2 3 ) a genomic region located 5 kb downstream from insertion site ( insertion site + 5kb ) were used as controls for targeted genomic region . endogenous active MED14 promoter was analyzed as a control . Blue black triangles represent reactive loxP sites recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Quantifications by real - time qPCR of total ( input ) extracted from two 1 + 2 + 3 , 1 + 3 loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) clonal lines , after digestion with micrococcal nuclease ( MNAse , 2 . 5 U , 10 U , 40 U , 160 U / mL ) selection were shown . Error bars indicate standard deviation for qPCR triplicates , for two independent clones . Data are presented as total input relative to condensed genomic regions ( cond1 cond2 ) used for normalization .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Clones 1+2+3, 1+3 (A) and loxP_LE (1) + βA-globin+β-actin (3) (B) were used to assess chromatin accessibility within the transgenes. The positions of the amplicons used for quantification in each shifting construct individually (thick black lines; 5' 2xFIV-1 or -3, β-actin pro-1 or -3, BsR gene, PuroR gene, LoxP site), in both shifting constructs (βA pro 1 and 2, IL2R gene, 3' 2xFIV1-3) or in the GFP reporter construct (βA-GFP, GFP gene, h.K7) are shown. The insertion sites on the wt chromosome (insertion sites 1, 2 and 3) and a genomic region located 5 kb downstream from the insertion site (insertion site +5kb) were used as controls for the targeted genomic region. The endogenous active MED14 promoter was analyzed as a control. Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Quantifications by real-time qPCR of total chromatin (input) extracted from two 1+2+3, 1+3 and loxP_LE (1)+ βA-globin+β-actin (3) clonal cell lines, after digestion with micrococcal nuclease (MNAse, 2.5 U, 10 U, 40 U, 160 U/mL) and size selection were shown. Error bars indicate the standard deviation for qPCR triplicates, for two independent clones. Data are presented as total chromatin input relative to the condensed genomic regions (cond1 and cond2) used for the normalization.


A , B . UCSC genome browser visualization of two parts of chromosome 1 containing insertion sites ( genomic positions : chr1 : 69 , 000 , 000 - 74 , 200 , 000 ; 5 . 2 Mb ( A ) chr1 : 175 , 300 , 000 - 180 , 500 , 000 ; 5 . 2 Mb ( B ) ; galGal5 ) . RT weighted average ( WA ) values obtained after sorting into four S - phase fractions , BrdU - labeled nascent strands ( NS ) immunoprecipitation sequencing are shown . Early - replicated regions are represented in orange late - replicating regions in blue . Below , track of eigenvector 1 values corresponding to A / B compartments at 100 kb resolution after Hi - C in wt , track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) annotated genes are reported . three sites chosen for insertion are indicated with red arrows dotted lines ( mid - late , late 1 ( A ) late 2 ( B ) insertion ) . At bottom , an enlargement of late insertion sites ( chr1 : 69 , 500 , 000 - 71 , 500 , 000 ( A ) , chr1 : 177 , 000 , 000 - 179 , 000 , 000 ( B ) ; galGal5 ) , displaying genes , mRNAs ESTs for chicken other species together with track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) is shown . Tracks of RefSeq genes are represented in blue , tracks of Ensembl genes in red , tracks of AUGUSTUS ab initio gene predictions TransMap alignements in green tracks of mRNAs ESTs in black . Insertion sites are indicated with red dotted lines black dotted lines determine borders of enlarged regions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites (genomic positions: chr1:69,000,000-74,200,000; 5.2 Mb (A) and chr1: 175,300,000-180,500,000; 5.2 Mb (B); galGal5). RT weighted average (WA) values obtained after cells sorting into four S-phase fractions, BrdU pulse-labeled nascent strands (NS) immunoprecipitation and sequencing are shown. Early-replicated regions are represented in orange and late-replicating regions in blue. Below, track of eigenvector 1 values corresponding to A/B compartments at 100 kb resolution after Hi-C analysis in wt cells, track of replication origins determined in (Massip et al, 2019) (Ori peaks) and annotated genes are reported. The three sites chosen for insertion are indicated with red arrows and dotted lines (mid-late, late 1 (A) and late 2 (B) insertion). At the bottom, an enlargement of late insertion sites (chr1: 69,500,000-71,500,000 (A), chr1: 177,000,000-179,000,000 (B); galGal5), displaying genes, mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in (Massip et al, 2019) (Ori peaks) is shown. Tracks of RefSeq genes are represented in blue, tracks of Ensembl genes in red, tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black. Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions.


A , B . RT profiles of chromosomal alleles following targeted integration of a βA - globin + β - actin construct into two late - replicating loci , 1 ( A ) 2 ( B ) in two or three clonal lines respectively . Nascent strands ( NS ) were quantified by real - time qPCR in four S phase fractions . Specific primer pairs determine RT profile for modified allele ( With ) , wt allele ( Without ) both alleles ( Both ) . endogenous β - globin locus was analyzed as an early - replicated control . modified ( Mod ) wild - type ( wt ) alleles are shown . Difference - ΔL + ΔE values calculated at target site following transgene integration are indicated ( see Appendix Figure S1 ) . Error bars correspond to standard deviation for qPCR duplicates . black vertical bars represent precise insertion position .

O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT profiles of chromosomal alleles following the targeted integration of a βA-globin+β-actin construct into two late-replicating loci, 1 (A) and 2 (B) in two or three clonal cell lines respectively. Nascent strands (NS) were quantified by real-time qPCR in four S phase fractions. Specific primer pairs determine the RT profile for the modified allele (With), the wt allele (Without) and both alleles (Both). The endogenous β-globin locus was analyzed as an early-replicated control. The modified (Mod) and wild-type (wt) alleles are shown. Difference -ΔL+ΔE values calculated at the target site following transgene integration are indicated (see Appendix Figure S1). Error bars correspond to the standard deviation for qPCR duplicates. The black vertical bars represent the precise insertion position.


B , C Both mRNA of RBM47 were elevated in SeV - infected ( B ) or IFN - α - stimulated ( C ) 293T , HFF , HUVEC , THP - 1 . were infected with SeV ( MOI = 1 ) or stimulated with IFN - α for 6 or 12 h , expression of RBM47 mRNA ( at 6 12 h post treatment ) ( at 6 12 h post treatment ) were tested . PCR results are represented as means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

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

B, C Both mRNA and protein levels of RBM47 were elevated in SeV-infected (B) or IFN-α-stimulated (C) 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with SeV (MOI = 1) or stimulated with IFN-α for 6 or 12 h, and the expression of RBM47 mRNA (at 6 and 12 h post treatment) and protein (at 6 and 12 h post treatment) were tested. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


C , D of viral genome , , progeny of VSV ( C ) or HSV - 1 ( D ) were quantified in - infected 293T with or without RBM47 ectopic expression at indicated time points . viral were tested 12 h post VSV infection 24 h post HSV - 1 infection . PCR viral titer data are represented as means ± SD of n = 3 biological replicates . Data information : data shown are representative of n = 3 independent experiments . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

O O O O O O 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

C, D Levels of viral genome, protein, and progeny of VSV (C) or HSV-1 (D) were quantified in virus-infected 293T cells with or without RBM47 ectopic expression at the indicated time points. The viral protein levels were tested 12 h post VSV infection and 24 h post HSV-1 infection.The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments.**P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


D , E mRNA expression of IFIT1 , ISG15 , Cig5 in SeV - infected 293T overexpressing RBM47 ( D ) or RBM47 knocked down ( E ) . were infected with SeV for 24 h , gene expression was determined using qRT - PCR . PCR results are represented as means ± SD of n = 3 biological replicates . Data information : data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

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

D, E mRNA expression of IFIT1, ISG15, and Cig5 in SeV-infected 293T cells overexpressing RBM47 (D) or RBM47 knocked down cells (E). Cells were infected with SeV for 24 h, and gene expression was determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


F expression of IFIT1 , ISG15 , Cig5 in RBM47 - overexpressing or control 293T stimulated by IFN - α for 12 h . PCR results are represented as means ± SD of n = 3 biological replicates . Data information : data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The expression of IFIT1, ISG15, and Cig5 in RBM47-overexpressing or control 293T cells stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


G expression of IFIT1 , ISG15 , Cig5 in WT RBM47 + / - peritoneal macrophages stimulated by IFN - α for 12 h . PCR results are represented as means ± SD of n = 3 biological replicates . Data information : data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The expression of IFIT1, ISG15, and Cig5 in WT and RBM47+/- peritoneal macrophages stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


B IFNAR1 mRNA stability after DRB treatment . 293T were transfected with RBM47 - Flag or empty vectors treated with DRB ( 50 μg / mL ) . Total was harvested at 0 , 15 , 30 , 45 , 60 min , IFNAR1 mRNA were measured using qRT - PCR normalized to β - actin . PCR results are represented as means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

O B-MEASURED_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 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

B IFNAR1 mRNA stability after DRB treatment. 293T cells were transfected with RBM47-Flag or empty vectors and treated with DRB (50 μg/mL). Total RNA was harvested at 0, 15, 30, 45, and 60 min, and IFNAR1 mRNA levels were measured using qRT-PCR and normalized to β-actin. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


A mRNA of IFNAR1 , but not IFNAR2 or STAT1 , was stabilized by RBM47 ectopic expression in 293T with or without IFN - α stimulation . were treated with IFN - α or PBS for 6 h , relative gene expression were determined using qRT - PCR . PCR results are represented as means ± SD of n = 3 biological replicates . Data information : data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) . data shown are representative of three independent experiments .

O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O 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 mRNA of IFNAR1, but not IFNAR2 or STAT1, was stabilized by RBM47 ectopic expression in 293T cells with or without IFN-α stimulation. Cells were treated with IFN-α or PBS for 6 h, and the relative gene expression levels were determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.


F , G of IFNAR1 phosphorylation of TYK2 , STAT1 , STAT2 in RBM47 - overexpressing ( F ) or knockdown ( G ) 293T . were treated with PBS or IFN - α for 6 h , relative band densities were analyzed using ImageJ software normalized to GAPDH . Data information : data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) . data shown are representative of three independent experiments .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O 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, G Protein levels of IFNAR1 and phosphorylation levels of TYK2, STAT1, and STAT2 in RBM47-overexpressing (F) or knockdown (G) 293T cells. Cells were treated with PBS or IFN-α for 6 h, and the relative band densities were analyzed using ImageJ software and normalized to GAPDH. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.


G RBM47 + / + RBM47 + / - mice were infected with HSV - 1 ( 1 × 107 pfu ) via caudal vein injection . Viral genome progeny titers were tested using blood samples from infected mice . Data are represented as means ± SD of n = 6 biological replicates . H qRT - PCR of IFNAR1 Cig5 mRNA in blood samples from HSV - 1 - infected mice . PCR results are represented as means ± SD of n = 6 biological replicates . Data information : data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

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 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 O O O O O O O O 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 RBM47+/+ and RBM47+/- mice were infected with HSV-1 (1× 107 pfu) via caudal vein injection. Viral genome DNA and progeny titers were tested using blood samples from infected mice. Data are represented as the means ± SD of n = 6 biological replicates. H qRT-PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV-1-infected mice. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


D mRNA of VSV RBM47 in blood samples of IFNAR1 deficient ( IFNAR1 - / - ) mice ( n = 6 per group ) infected with VSV . Mice were infected with shmRBM47 or control lentivirus for 7 days then challenged with VSV for another 24 h . Each dot represents one mouse . PCR results are represented as means ± SD of n = 6 biological replicates . Data information : data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( Student ' s t - ) .

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

D The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient (IFNAR1-/-) mice (n = 6 per group) infected with VSV. Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h. Each dot represents one mouse. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


A ) Upper panels : Representative single z - planes from 3D - SIM image stacks of DAPI - stained nuclei of AC16 kept at 37 ° C or exposed to different cold temperatures for 24h . Lower panels : DAPI signal segmented into interchromatin regions according to relative . Scale bar : 5 μm . Arrows mark concentrated at periphery ( P ) around nucleoli ( N ) . B ) Boxplots of of volume to volume for exposed to different conditions . Median ( central ) , interquartile range ( IQR , box edges ) , most extreme data point no more than 1 . 5x IQR from box edges ( whiskers ) . Number of nuclei : 37 ° C ( 31 ) , 28 ° C 24h ( 26 ) , 18 ° C 5h ( 19 ) , 18 ° C 24h ( 22 ) , 18 ° C 24h then 37 ° C 2h ( 25 ) , 8 ° C 24h ( 23 ) . C ) Left central panels : representative single z - planes from 3D - SIM image stacks of DAPI - stained nuclei of kept at 37 ° C or exposed to 18 ° C for 24h showing spatial distribution of immunofluorescence ( IF ) signal of hnRNPC ( green ) pore complexes ( NPCs , red ) . Right panel : DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in z - plane shown . Scale bar whole nuclei : 5 μm . Scale bar enlarged section : 1 μm . D ) Bar graph of mean NPC centroid to nearest hnRNPC centroid for each condition . Number of distances : 37 ° C ( 112511 ) , 28 ° C 24h ( 94521 ) , 18 ° C 5h ( 72340 ) , 18 ° C 24h ( 74998 ) , 18 ° C 24h then 37 ° C 2h ( 92896 ) , 8 ° C 24h ( 80570 ) .

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

A) Upper panels: Representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of AC16 cells kept at 37°C or exposed to different cold temperatures for 24h. Lower panels: DAPI signal segmented into chromatin and interchromatin regions according to relative intensity. Scale bar: 5 μm. Arrows mark chromatin concentrated at the nuclear periphery (P) and around nucleoli (N). B) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions. Median (central line), interquartile range (IQR, box edges), most extreme data point no more than 1.5x IQR from box edges (whiskers). Number of nuclei: 37°C (31), 28°C 24h (26), 18°C 5h (19), 18°C 24h (22), 18°C 24h then 37°C 2h (25), 8°C 24h (23). C) Left and central panels: representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of cells kept at 37°C or exposed to 18°C for 24h showing the spatial distribution of the immunofluorescence (IF) signal of hnRNPC (green) and nuclear pore complexes (NPCs, red). Right panel: DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z-plane shown. Scale bar whole nuclei: 5 μm. Scale bar enlarged section: 1 μm. D) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition. Number of distances: 37°C (112511), 28°C 24h (94521), 18°C 5h (72340), 18°C 24h (74998), 18°C 24h then 37°C 2h (92896), 8°C 24h (80570).


A ) Number of genes showing significant ( adjusted p < 0 . 05 , Wald ) up or downregulation in or upon transfer of AC16 from 37 ° C to different cold temperatures for 24h .

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A) Number of genes showing significant (adjusted p < 0.05, Wald test) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37°C to different cold temperatures for 24h.


B ) Relationship between log2 fold change in level in nucleus that in cytoplasm for genes upon transfer of AC16 from 37 ° C to different cold temperatures for 24h . Points are color - coded from low to high density ( black < blue < red < yellow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. Points are color-coded from low to high density (black < blue < red < yellow).


C ) Relationship between log2 fold change in level for genes upon transfer of AC16 from 37 ° C to 28 ° C or 18 ° C ( top ) , 28 ° C or 8 ° C ( middle ) 18 ° C or 8 ° C ( bottom ) for 24h in both nucleus cytoplasm . Points are shaded from low to high density ( grey < black ) . Genes that show significant differential expression at a specific or at both temperatures are highlighted with colored circles or triangles , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37°C to 28°C or 18°C (top), 28°C or 8°C (middle) and 18°C or 8°C (bottom) for 24h in both the nucleus and cytoplasm. Points are shaded from low to high density (grey < black). Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles, respectively.


A ) Boxplots of standardized at time points during transfer of AC16 from 37 ° C to 18 ° C for 24h then back to 37 ° C for 24h for group of 1285 genes showing significant upregulation in at 18 ° C for 24h time point . Range ( whiskers ) , interquartile range ( IQR , box edges ) , median + / - IQR / [ square root ( number of genes ) ] ( notch ) median ( central ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18°C for 24h time point. Range (whiskers), interquartile range (IQR, box edges), median +/-IQR/[square root(number of genes)] (notch) and median (central line).


B ) Log2 fold change in ( black , left axis ) ( red , right axis ) level of circadian clock negative limb genes at time points during transfer of AC16 from 37 ° C to 18 ° C for 24h then back to 37 ° C for 24h relative to kept at 37 ° C . Error bars show standard error .

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B) Log2 fold change in cytoplasmic (black line, left axis) and nuclear (red line, right axis) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h relative to cells kept at 37°C. Error bars show the standard error.


C ) full - - Seq data from AC16 kept at 37 ° C or exposed to 18 ° C for 24h for REV - ERBα CRY2 . Data for each track have been normalized to show per million aligned ( CPM ) for each nucleotide . Number of 3 ' end - seq sample replicates : 37 ° C ( C : 6 , N : 7 ) , 18 ° C 5h ( 4 ) , 10h ( 4 ) , 24h ( 6 ) , 18 ° C 24h then 37 ° C 2h ( 4 ) , 5h ( 2 ) , 10h ( 2 ) , 24h ( 2 ) . Number of ( C ) ( N ) samples are same for each condition except 37 °

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 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 O O O O O O O O O O O O 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) Nuclear and cytoplasmic full-length RNA-Seq data from AC16 cells kept at 37°C or exposed to 18°C for 24h for REV-ERBα and CRY2. Data for each track have been normalized to show counts per million aligned counts (CPM) for each nucleotide. Number of 3' end RNA-seq sample replicates: 37°C (C: 6, N: 7), 18°C 5h (4), 10h (4), 24h (6), 18°C 24h then 37°C 2h (4), 5h (2), 10h (2), 24h (2). Number of cytoplasmic (C) and nuclear (N) samples are the same for each condition except 37°C