Fis1 knockdown prevents DFP - induced mitophagic autolysosomes . Nsi or Fis1i together with mito - pHfluorin were transfected into HeLa in absence or presence of DFP ( 24 h ) , which were analysed using mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or indicating occurrences of mitophagy marked by arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Blue : Fis1 ; Scale bar , 10 μm ; n = 25 per condition from three experiments ; * * * p < 0 . 001 ; unpaired - t ) .

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

Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).


Flag - Fis1 is deSUMOylated by SENP3 . SENP3 knockdown enhances Fis1 SUMOylation . Nsi or SENP3i was transfected into HEK293 expressing Flag - Fis1 , His - SUMO - 2 Ubc9 for 48 h . His - pulldown lysate samples were detected by immunoblotting for Flag , SENP3 or β - actin . Values are presented as mean ± ( n = 3 , biological replicates ; * p < 0 . 05 ; paired - t ) .

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

Flag-Fis1 is deSUMOylated by SENP3. SENP3 knockdown enhances Fis1 SUMOylation. Nsi or SENP3i was transfected into HEK293 cells expressing Flag-Fis1, His-SUMO-2 and Ubc9 for 48 h. His-pulldown and lysate samples were detected by immunoblotting for Flag, SENP3 or β-actin. Values are presented as mean ± SEM (n=3, biological replicates; * p<0.05; paired-t test).


SUMOylation status of Fis1 influences its mitochondrial localization . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG was expressed in HeLa for 48 h . Whole lysates mitochondrial fractions were prepared blotted as indicated ( upper panel ) . Histograms ( right panel ) show normalized of Flag - Fis1 WT or Flag - Fis1 K149R mutant associated with or mitochondrial fraction ( Value are presented as mean ± are normalized to control value ; n = 7 , biological replicates ; * , p < 0 . 05 ; paired t - ) .

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

The SUMOylation status of Fis1 influences its mitochondrial localization. Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG was expressed in HeLa cells for 48 h. Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated (the upper panel). Histograms (the right panel) show normalized levels of Flag-Fis1 WT or Flag-Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction (Value are presented as mean ± SEM and are normalized to the control value; n=7, biological replicates; *, p<0.05; paired t-test).


SUMOylation status of Fis1 may influence its association with Endoplasmic Reticulum ( ER ) . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG along with GFP - Cb5 as an ER marker were expressed in HeLa for 48 h . Immunocytochemistry was then performed ( Green : ER labelled by GFP - Cb5 ; Blue : different Flag - Fis1 forms ; Scale bar , 10 μm )

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

The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum (ER). Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG along with GFP-Cb5 as an ER marker were expressed in HeLa cells for 48 h. Immunocytochemistry was then performed (Green: ER labelled by GFP-Cb5; Blue: different Flag-Fis1 forms; Scale bar, 10 μm)


Expressing a SUMOylation - deficient Fis1 mutant reverses effect of SENP3 knockdown on DFP - induced LC3 - II induction . pcDNA3 , Flag - Fis1 WT or Flag - Fis1 K149R mutant was transfected into HeLa in which SENP3 was depleted using siRNA for 48 h ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ( I ) ; siRNA , 20nM ) , were treated with DFP for a further 24 h ( DFP , 1mM ) . Lysate samples were blotted as indicated .

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

Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated.


SENP3 depletion does not abolish mitophagic detected in HeLa expressing SUMOylation deficient CFP - Fis1 K149R in presence of DFP . CFP - Fis1 WT or CFP - Fis1 K149R mutant were transfected into HeLa in which SENP3 was depleted using siRNA for 48 h , were treated with DFP for a further 24 h ( Red : mCherry - A or indicating occurrences of mitophagy marked by arrows ; Green : SEP - A ; Cyan / blue : CFP - Fis1 ; Magenta , SENP3 ; Scale bar , 10 μm ) ; Values are presented as mean ± ( n = 21 per condition from two experiments ; * * * * p < 0 . 0001 ; unpaired t - ) . Knockdown of SENP3 was further confirmed by immunoblotting ( lower right panel ) .

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

SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).


B . BrdU - sensitized U - 2 - OS stably expressing GFP - NLS - CDKL5 were pre - incubated with DMSO ( mock ) , olaparib ( 5 μM ) , talazoparib ( 50 nM ) or PDD00017273 ( 0 . 3 μM ) for 1 h prior to micro - irradiation live - imaging at indicated times post - irradiation . One of three independent experiments is shown . Scale bar is 10

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B. BrdU-sensitized U-2-OS cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273 (0.3 μM) for 1 h prior to micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μm


E . BrdU - sensitized parental or PARP1Δ / Δ , PARP2Δ / Δ , PARP1 / 2Δ / Δ U - 2 - OS transiently expressing GFP NLS - CDKL5 were subjected to 355 nm micro - irradiation followed by time lapse imaging . One of two independent experiments is shown . Scale bar is 10

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E. BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ, PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μm


G . subjected to workflow in F . were detergent - extracted fixed before with anti - GFP or fibrillarin ( nucleoli ) . Scale bar is 10 μm H . of detergent - insoluble GFP - NLS - CDKL5 signal ( minus nucleolar signal ) . mean ± SD from three biological experiments is shown . Statistical significance was assessed by one - way - ANOVA - . Asterisks * * indicate P - value of < 0 . 01 ; ns - not significant .

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 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

G. Cells subjected to the workflow in F. were detergent-extracted and fixed before staining with anti-GFP or fibrillarin (nucleoli). Scale bar is 10 μm H. Quantification of the detergent-insoluble GFP-NLS-CDKL5 signal (minus nucleolar signal). The mean ± SD from three biological experiments is shown. Statistical significance was assessed by one-way-ANOVA-test. Asterisks ** indicate P-value of <0.01; ns - not significant.


B . BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) stably expressing GFP - NLS , GFP - NLS - CDKL5 deletion mutants shown in A , or full ( FL ) GFP - NLS - CDKL5 were subjected to micro - irradiation ( 355 nm ) time lapse imaging . Three independent experiments were performed , one representative experiment is shown . Scale bar is 10

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

B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL) GFP-NLS-CDKL5 were subjected to line micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μm


D . Same as in B except that GFP - NLS tagged CDKL5 fragments indicated were examined . Scale bar is 10

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D. Same as in B except that the GFP-NLS tagged CDKL5 fragments indicated were examined. Scale bar is 10 μm


F . Recombinant fragments of CDKL5 fused to GST ( 1 . 2 , 2 . 5 , 5 , 10 µg ) , or GST , were dot - blotted on nitrocellulose then incubated with synthetic PAR . PAR binding was detected by far western blotting . APLF was used as positive control . One of three independent experiments is

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

F. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on nitrocellulose membrane and then incubated with synthetic PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown


( G - H ) . U - 2 - OS ( Flp - In T - Rex ) stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min . Extracts were subjected to immunoprecipitation with antibodies against CDKL5 ( G ) or PAR ( H ) ( or non - specific IgG as control ) . Precipitates , input lysates , were analyzed by western blotting using indicated antibodies . One of two independent experiments is

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 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

(G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting using the indicated antibodies. One of two independent experiments is shown


A . ( Top ) BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) stably expressing GFP - NLS - CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro - irradiation ( 405 nm ) . ( Bottom ) Quantitation of spot intensities . Data represent mean ± of two independent experiments ; > 50 micro - irradiated per point . Scale bar is 10

O O O 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

A. (Top) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. Scale bar is 10 μm


BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) stably expressing GFP - NLS - CDKL5 , mCherry - XRCC1 or mCherry - FAN1 were pre - incubated with indicated transcription inhibitors prior to micro - irradiation ( 355 nm ) time lapse imaging . One of three independent experiments is shown . Scale bar is 10

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

BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μm


E . Same as B - D . except that BrdU - sensitized stably expressing GFP - NLS - CDKL5 were also pre - incubated with olaparib as control . were subjected to micro - irradiation , fixed then subjected to indirect immunofluorescence using antibodies against GFP , PAR γH2AX . Scale bar is 10

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E. Same as B-D. except that BrdU-sensitized cells stably expressing GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μm


F . Stable lines were permeabilized incubated with RNase A or PBS before irradiation imaging . Scale bar is 10

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F. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm


B . Boxplots showing VSN - normalised of phospho - peptides corresponding to EP400 pSer729 , ELOA pSer311and TTDN1 pSer40 from experiment in A . central band of boxplot indicates median value , while hinges represent first third quartile ( bottom top of boxplot , respectively ) . whiskers extend to largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times interquartile range from their respective hinge . In cases data was derived from 3 biological replicates .

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

B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates.


C . Boxplots of VSN - adjusted TMT reporter ion intensities for peptides for each TMT label in case of FLAG - EP400 , FLAG - ELOA FLAG - TTDN1 from experiment in A . central band of boxplot indicates median value , while hinges represent first third quartile ( bottom top of boxplot , respectively ) . whiskers extend to largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times interquartile range from their respective hinge . Datapoints further removed than whiskers are plotted individually . experiment was conducted using three biological replicates within each respective group , each TMT channel represents a single biological replicate .

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

C. Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400, FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT channel represents a single biological replicate.


A . HEK293 were co - transfected with CDKL5 ( wild type " WT " or kinase - dead " KD " K42R mutant ) fused to an NLS , FLAG - ELOA ( wild type " WT " or a S311A mutant " SA " ) . Anti - FLAG precipitates or extracts were probed with antibodies indicated . One of three independent experiments is shown . B . Same as A . showing a range of pathogenic ( red ) benign ( blue ) CDKL5 variants .

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 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 O O

A. HEK293 cells were co-transfected with CDKL5 (wild type "WT" or kinase-dead "KD" K42R mutant) fused to an NLS, and FLAG-ELOA (wild type "WT" or a S311A mutant "SA"). Anti-FLAG precipitates or cell extracts were probed with the antibodies indicated. One of three independent experiments is shown. B. Same as A. showing a range of pathogenic (red) and benign (blue) CDKL5 variants.


( C - E ) . Wild type ( WT ) , CDKL5 - disrupted ( CDKL5Δ / Δ ) or siRNA - transfected were subjected to indirect immunofluorescence with indicated antibodies at tracks . of ELOA - pSer311 signal at tracks is shown . Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated ( n ) . For simplicity , only intensities greater than zero are shown . Statistical significance was assessed by one - way - ANOVA - or unpaired t with Welch ' s correction . Asterisks * * * * indicate P - values of < 0 . 0001 . Scale bar is 10

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

(C-E). Wild type (WT), CDKL5-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm


D . ( Left ) of transcription in U - 2 - OS 263 IFII reporter from experiment in B . > 150 were analysed per condition per experiment . mean ± SD from four independent experiments is shown . ( Right ) Quantitative RT - PCR of YFP - MS2 mRNA in U - 2 - OS 263 reporter . Data represent mean ± SD in different biological replicates as indicated ( n ) . Statistical significance for data was assessed by two - way - ANOVA - , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 ; ns - not significant .

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

D. (Left) Quantification of transcription in U-2-OS 263 IFII reporter cells from experiment in B. >150 cells were analysed per condition per experiment. The mean ± SD from four independent experiments is shown. (Right) Quantitative RT-PCR analysis of YFP-MS2 mRNA in U-2-OS 263 reporter cells. Data represent mean ± SD in different biological replicates as indicated (n). Statistical significance for all the data was assessed by two-way-ANOVA-test, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant.


B , C . Quantitative PCR with reverse transcription ( qRT - PCR ) of SLCO5a1 ( B ) RYR2 expression ( C ) ( left panels ) U - 2 - OS HA - ER - I - PpoI depleted of CDKL5 transiently transfected with FLAG - tagged CDKL5 wild type ( WT ) or a K42R - mutated kinase dead ( KD ) mutant , or empty vector , at times indicated after inducing I - PpoI . mean ± SD from two qPCR replicates of two independent experiments is shown . Statistical significance for data was assessed by two - way - ANOVA - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 ; ns - not significant . I - PpoI - mediated cutting efficiency in relevant gene is shown in right -

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantitative PCR with reverse transcription (qRT-PCR) analysis of SLCO5a1 (B) and RYR2 expression levels (C) (left panels) U-2-OS HA-ER-I-PpoI cells depleted of CDKL5 transiently transfected with FLAG-tagged CDKL5 wild type (WT) or a K42R-mutated kinase dead (KD) mutant, or empty vector, at the times indicated after inducing I-PpoI. The mean ± SD from two qPCR replicates of two independent experiments is shown. Statistical significance for all the data was assessed by two-way-ANOVA-test *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant. I-PpoI-mediated cutting efficiency in the relevant gene is shown in the right-hand panel


A . summary of Aft1 induction experiment showing genes that change with | log2 ( fold - change ) | > 1 . 5 .

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A. Heatmap summary of Aft1 induction experiment showing all genes that change with |log2(fold-change)| > 1.5.


( D ) NIH 3T3 transfected by equal amounts of plasmid fused with gfp ( without ATG ) to from 5 ' cap of pri - miR - 31 to ATG site or which mutated to ATT of sORF . were selected by G418 for 2 days . of GFP expression by confocal microscopy or flow cytometry , scale bar 100 μm .

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(D) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp (without ATG) to the sequence from 5' cap of pri-miR-31 to ATG site or which mutated to ATT of the sORF. Cells were selected by G418 for 2 days. Analysis of the GFP expression by confocal microscopy or flow cytometry, scale bar 100 μm.


( E ) Immunoblot of GFP miPEP31 in NIH 3T3 transfected by pEGFP - N1 - miPEP31 or pEGFP - N1 , red arrow indicate predicted of miPEP31 - GFP band .

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(E) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP-N1-miPEP31 or pEGFP-N1, the red arrow indicate predicted size of miPEP31-GFP band.


( F ) Flow - fish to detect localization of pri - miR - 31 . location of pri - miR - 31 was analyzed by confocal microscopy . Scale bar 5 μm .

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(F) Flow-fish to detect the localization of pri-miR-31. The location of pri-miR-31 was analyzed by confocal microscopy. Scale bar 5 μm.


( G ) Immunoblot of multiple mouse tissues with a specific antibody against miPEP31 . Values are expressed as fold changes relative to controls normalized to GAPDH , red arrow indicate predicted of miPEP31 .

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(G) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31. Values are expressed as fold changes relative to controls and normalized to GAPDH, the red arrow indicate predicted size of miPEP31.


( H ) Immunoblot of spleen , lymph node thymus from WT or miR - 31 - / - mice with miPEP31 antibody , red arrow indicate predicted of miPEP31 .

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(H) Immunoblot analysis of spleen, lymph node and thymus from WT or miR-31-/- mice with miPEP31 antibody, the red arrow indicate predicted size of miPEP31.


( A ) CD4 + T were activated with anti - CD3 / 28 treated with different of FAM - miPEP31 for 24 hours , fluorescein was detected by Flow Cytometry .

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(A) CD4+ T cells were activated with anti-CD3/28 and treated with different concentration of FAM-miPEP31 for 24 hours, the fluorescein was detected by Flow Cytometry.


( B ) Confocal Microscopy was used to analyze of FAM - miPEP31 in anti - CD3 / 28 activated CD4 + T treated with 10 μM FAM - miPEP31 . Scale bar 5 μm .

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(B) Confocal Microscopy was used to analyze the fluorescence of FAM-miPEP31 in anti-CD3/28 activated CD4+ T cells treated with 10 μM FAM-miPEP31. Scale bar 5 μm.


Anti - CD3 / 28 activated CD4 + T were cultured with 10 μM miPEP31 for 24 hours . ( C ) Western Blotting of Cytochrome C Histone H2A in total lysates lysates .

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Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (C) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates.


Anti - CD3 / 28 activated CD4 + T were cultured with 10 μM miPEP31 for 24 hours . ( D ) ELISA to detect of miPEP31 in of total extracts in CD4 + T Treg treated with or without 10 μM miPEP31 . Data in ( D ) are presented as mean ± of four biological replicates . ns , not significant ( CD4 + T versus Treg ) , unpaired two - Student ' s t - . .

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Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (D) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4+ T and Treg cells treated with or without 10 μM miPEP31. Data in (D) are presented as mean ± SEM of four biological replicates. ns, not significant (CD4+ T versus Treg), unpaired two-tailed Student's t-test..


( E ) Naïve CD4 + T were isolated from WT C57BL / 6 mice , activated by anti - CD3 / 28 for 48 hours . Subsequently , FAM - labeled miPEP31 was added . Imaging Flow Cytometry of FAM labeled miPEP31 in CD4 + T . Scale bar 7 μm .

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(E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, FAM-labeled miPEP31 was added. Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4+ T cells. Scale bar 7 μm.


( F , G ) C57BL / 6 mice were injected intravenously with 100 μg FAM - labeled miPEP31 . Splenocytes were isolated stained for CD4 DAPI . Imaging Flow Cytometry of miPEP31 in CD4 + T ( F ) non - CD4 splenocytes ( G ) , scale bar 10 μm .

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(F, G) C57BL/6 mice were injected intravenously with 100 μg FAM-labeled miPEP31. Splenocytes were isolated and stained for CD4 and DAPI. Imaging Flow Cytometry analysis of miPEP31 in CD4+ T cells (F) and non-CD4 splenocytes (G) , scale bar 10 μm.


( D , E ) Naïve CD4 + T were isolated from WT C57BL / 6 mice , activated by anti - CD3 / 28 for 48 hours . Subsequently , 10 μM FAM - labeled miPEP31 was added for 20 min at 4 ℃ or 37 ℃ . Confocal Microscopy of . Scale bar 10 μm . localization of miPEP31 with DAPI was shown in ( E ) . Data are representative co - localization of miPEP31 in four representative from four biological experiments . ( mean ± ) . * * * * p < 0 . 0001 , unpaired two - Student ' s t - .

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(D, E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, 10 μM FAM-labeled miPEP31 was added for 20 min at 4℃ or 37℃. Confocal Microscopy analysis of the fluorescence. Scale bar 10 μm. The localization of miPEP31 with DAPI was shown in (E). Data are representative nuclear co-localization of miPEP31 in four representative images from four biological experiments. (mean ± SEM). ****p < 0.0001, unpaired two-tailed Student's t-test.


scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization . ( D , E ) H & E Fast blue of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization . Histopathology scores of inflammation demyelination were shown in ( E ) . Scale bars , 70 μm . Data in ( C , E ) are presented as mean ± of three independent experiments with nine to ten mice per group in each . * * * *

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scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization. (D, E) H&E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization. Histopathology scores of inflammation and demyelination were shown in (E). Scale bars, 70 μm. Data in (C, E) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each. ****


IL - 1β concentrations in supernatants of macrophages , unstimulated ( control ) ( B ) or stimulated with SP / N ( C ) or LPS / N ( D ) .

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IL-1β concentrations in supernatants of macrophages, unstimulated (control) (B) or stimulated with SP/N (C) or LPS/N (D).


E . SARS - CoV - 2 spike ELISA of corresponding plasma : ( EC50 titer ) of samples collected at T0 ( n = 35 individuals ) , T1 ( n = 28 individuals ) or T2 ( n = 44 individuals ) .

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E. SARS-CoV-2 spike ELISA of the corresponding plasma: (EC50 titer) of samples collected at T0 (n = 35 individuals), T1 (n = 28 individuals) or T2 (n = 44 individuals).


F . Immunofluorescence microscopy of ASC specks ( green ) in T2 macrophages . Nuclei were stained with DAPI ( blue ) . Scale bar indicates 20 µm . Arrow indicates ASC speck formation with magnified illustration in right corner of image .

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F. Immunofluorescence microscopy images of ASC specks (green) in T2 macrophages. Nuclei were stained with DAPI (blue). Scale bar indicates 20 µm. Arrow indicates ASC speck formation with magnified illustration in the right corner of the image.


G , H . ASC specks were quantified microscopically at T0 ( G ) ( n = 7 individuals ) T2 ( H ) ( n = 8 individuals ) upon indicated stimuli .

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G, H. ASC specks were quantified microscopically at T0 (G) (n = 7 individuals) and T2 (H) (n = 8 individuals) upon indicated stimuli.


I . gasdermin D ( lysate ) IL - 1β ( supernatant ) detected by Western Blot in macrophages stimulated with LPS / N or SP / N at T0 T2 .

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I. Cleaved gasdermin D (lysate) and cleaved IL-1β (supernatant) detected by Western Blot in macrophages stimulated with LPS/N or SP/N at T0 and T2.


J , K . of macrophages by flow cytometry in unstimulated or SP / N stimulated at T0 ( n = 5 individuals ) ( J ) T2 ( n = 5 individuals ) ( K ) using viability dye Zombie UV . LPS / N was used as positive control .

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J, K. Cell death quantification of macrophages by flow cytometry in unstimulated or SP/N stimulated cells at T0 (n = 5 individuals) (J) and T2 (n = 5 individuals) (K) using the viability dye Zombie UV. LPS/N was used as positive control.


L . LDH release of macrophages ( n = 6 individuals ) stimulated with SP / N at T2 compared to unstimulated . M . IL - 1β concentrations of MCC950 treated T2 macrophage ( n = 44 individuals ) stimulation with SP / N .

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L. LDH release of macrophages (n = 6 individuals) stimulated with SP/N at T2 compared to unstimulated cells. M. IL-1β concentrations of MCC950 treated T2 macrophage (n = 44 individuals) stimulation with SP/N.


B . Volcano plot showing DEGs in same samples . Negative log10 padj - values are plotted against log2 fold - change . Selected genes are labeled .

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B. Volcano plot showing DEGs in the same samples. Negative log10 padj-values are plotted against the log2 fold-change. Selected genes are labeled.


C , D . CLEC4D ( C ) CLEC4E ( D ) surface expression on - derived macrophages prior ( n = 5 individuals ) or post ( n = 5 individuals ) vaccination were quantified by flow cytometry . Graphs indicate mean ( MFI ) .

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C, D. CLEC4D (C) and CLEC4E (D) surface expression on patient-derived macrophages prior (n = 5 individuals) or post (n = 5 individuals) vaccination were quantified by flow cytometry. Graphs indicate the mean fluorescence intensity (MFI).


E , F . SP or LPS as a control PAMP were incubated with affinity purified CLRs ( : 10 vs . 1 for SP ) for 1h then added to T2 macrophages ( n = 5 individuals ) . Subsequently , IL - 1β was quantified in supernatants . ( E ) shows absolute values for SP ; ( F ) shows fold change calculated with corresponding unstimulated control for SP LPS .

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E, F. SP or LPS as a control PAMP were incubated with affinity purified CLRs (ratio: 10 vs. 1 for SP) for 1h and then added to primary T2 macrophages (n = 5 individuals). Subsequently, IL-1β was quantified in the supernatants. (E) shows absolute values for SP; (F) shows the fold change calculated with the corresponding unstimulated control for SP and LPS.


G . Monocytes were isolated prior ( n = 5 individuals ) or after vaccination ( n = 5 individuals ) differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages were measured after stimulation with SP S1 subunit SP nigericin . H . Monocytes were isolated prior ( T0 ; n = 35 individuals ) or after vaccination [ 2 after 1st ( T1 ; n = 28 individuals ) , 2 ( T2 ; n = 38 individuals ) 10 ( T3 ; n = 33 individuals ) after 2nd vaccination ] differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages , stimulated with SP S2 subunit SP are shown .

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G. Monocytes were isolated prior (n = 5 individuals) or after vaccination (n = 5 individuals) and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin. H. Monocytes were isolated prior (T0; n = 35 individuals) or after vaccination [2 weeks after 1st (T1; n = 28 individuals), 2 weeks (T2; n = 38 individuals) and 10 weeks (T3; n =33 individuals) after 2nd vaccination] and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages, stimulated with the SP S2 subunit SP are shown.


B . Phospho - SYK Immunofluorescence - microscopy in macrophages before after vaccination . Nuclei stained with DAPI . Scale bars indicate 20 µm .

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B. Phospho-SYK Immunofluorescence-microscopy in macrophages before and after vaccination. Nuclei stained with DAPI. Scale bars indicate 20 µm.


C . of pSYK positive macrophages by flow cytometry before ( n = 3 individuals ) 14 d after vaccination ( n = 3 individuals ) . Total SYK was measured as control .

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C. Quantification of pSYK positive macrophages by flow cytometry before (n = 3 individuals) and 14 d after vaccination (n = 3 individuals). Total SYK was measured as control.


D . Western blot of phospho - NF - κB NLRP3 in lysates of macrophages ( T0 ; T2 ) stimulated with LPS / N or SP / N .

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D. Western blot analysis of phospho-NF-κB and NLRP3 in cell lysates of macrophages (T0; T2) stimulated with LPS/N or SP/N.


E . Macrophages were incubated with IKK - β - inhibitor KINK - 1 ( n = 10 individuals ) SYK - inhibitors entospletinib ( n = 20 individuals ) , R406 ( n = 13 individuals ) prior to SP / nigericin stimulation . IL - 1β concentrations measured with ELISA . DMSO - treated , SP / N - stimulated untreated unstimulated macrophages were used as control ( both n = 44 individuals ) .

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E. Macrophages were incubated with IKK-β-inhibitor KINK-1 (n = 10 individuals) and SYK-inhibitors entospletinib (n = 20 individuals), R406 (n = 13 individuals) prior to SP/nigericin stimulation. IL-1β concentrations measured with ELISA. DMSO-treated, SP/N-stimulated and untreated unstimulated macrophages were used as control (both n = 44 individuals).


F . IL - 1β in THP - 1 wild - type incubated with SYK inhibitors entospletinib ( n = 4 independent experiments ) R406 ( n = 2 independent experiments ) or in THP - 1 SYKKO stimulated with SP / N ( n = 5 independent experiments ) .

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F. IL-1β quantification in THP-1 wild-type cells incubated with SYK inhibitors entospletinib (n = 4 independent experiments) and R406 (n = 2 independent experiments) or in THP-1 SYKKO cells stimulated with SP/N (n = 5 independent experiments).


G . ASC formation quantified by immunofluorescence - microscopy in SP / N stimulated macrophages after vaccination ( n = 5 individuals ) .

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G. ASC formation quantified by immunofluorescence-microscopy in SP/N stimulated primary macrophages after vaccination (n = 5 individuals).


H . of in T2 macrophages ( n = 4 individuals ) treated with R406 prior to SP / N - stimulation .

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H. Quantification of cell death in T2 macrophages (n = 4 individuals) treated with R406 prior to SP/N-stimulation.


I - K . mROS quantified by flow cytometry using MitoSOX red at T0 T2 in SP / N - stimulated macrophages without inhibitor ( I [ exemplary results ] , J [ T0 : n = 5 individuals ; T2 : n = 5 individuals ] )

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I-K. mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages without inhibitor (I [exemplary results], J [T0: n = 5 individuals; T2: n = 5 individuals])


mROS quantified MitoSOX red at T0 T2 in SP / N - stimulated macrophages with R406 treatment ( n = 4 individuals ) ( K ) .

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mROS quantified MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages with R406 treatment (n = 4 individuals) (K).


L . Western blot of SOD2 in macrophages at T0 T2 treated with SP / N or LPS / N .

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L. Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP/N or LPS/N.


M . Mitochondrial potential determined with flow cytometry of tetramethylrhodamine methyl ester ( TMRM ) stained macrophages at T0 ( n = 5 individuals ) T2 ( n = 5 individuals ) stimulated with SP / N . N . T2 - macrophages ( n = 4 individuals ) were treated with SYK - inhibitor R406 . Data information : For statistical , one - or two - way ANOVA with tukey post hoc was used . Box plots indicate median upper lower quartile . Outliers are plotted as dots ( outside 10 - 90 percentile ) . Scatter dot plots show mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 . ( S - : SP , lipopolysaccharide : LPS , nigericin : N )

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M. Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester (TMRM) stained macrophages at T0 (n = 5 individuals) and T2 (n = 5 individuals) stimulated with SP/N. N. T2-macrophages (n = 4 individuals) were treated with the SYK-inhibitor R406. Data information: For statistical analysis, one- or two-way ANOVA with tukey post hoc test was used. Box plots indicate the median and the upper and lower quartile. Outliers are plotted as individual dots (outside the 10-90 percentile). Scatter dot plots show mean ± SD. *p<0.05; **p<0.01, ***p<0.001. (S-protein: SP, lipopolysaccharide: LPS, nigericin: N)


A - H . ( A ) T at T0 ( n = 6 individuals ) T2 ( n = 10 individuals ) were incubated with SP / N - stimulated macrophages . Co - cultures of unstimulated macrophages were used as control ( con ) . CD4 + subpopulation analyses performed with flow cytometry ( Experimental setup : Fig EV3 ) . ( B ) of fold change of CD4 + T subpopulation upon SP / N stimulation ( T0 ; T2 ) . Dotted indicates baseline ( fold change = 1 . 0 ) . ( C ) Fold change of CD4 + naïve T CD4 + effector memory T populations ( D ) upon co - culture of macrophages stimulated with SP / N in presence of MCC950 or R406 . Identical analyses performed with CD8 + T ( E , F , G , H ) .

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A-H. (A) T cells at T0 (n = 6 individuals) and T2 (n = 10 individuals) were incubated with SP/N-stimulated macrophages. Co-cultures of unstimulated macrophages were used as control (con). CD4+ subpopulation analyses performed with flow cytometry (Experimental setup: Fig EV3). (B) Quantification of fold change of the CD4+ T cell subpopulation upon SP/N stimulation (T0; T2). Dotted line indicates the baseline (fold change = 1.0). (C) Fold change of CD4+ naïve T cell and CD4+ effector memory T cell populations (D) upon co-culture of macrophages stimulated with SP/N in presence of MCC950 or R406. Identical analyses performed with CD8+ T cells (E, F, G, H).


I , J . PD - 1 expression was analyzed in CD4 + ( I ) CD8 + T ( J ) by flow cytometry upon co - culture with SP / N - stimulated T0 ( n = 6 individuals ) or T2 ( n = 11 individuals ) macrophages . Co - cultures of unstimulated macrophages were used as control .

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I, J. PD-1 expression was analyzed in CD4+ (I) and CD8+ T cells (J) by flow cytometry upon co-culture with SP/N-stimulated T0 (n = 6 individuals) or T2 (n = 11 individuals) macrophages. Co-cultures of unstimulated macrophages were used as control.


K , L . Interferon - γ quantified in supernatants of corresponding co - cultures , untreated ( T0 [ n = 6 individuals ] T2 [ n = 11 individuals ] ) ( K ) treated with MCC950 or R406 ( L ) at T2 ( n = 11 individuals ) .

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K, L. Interferon-γ quantified in supernatants of corresponding co-cultures, untreated (T0 [n = 6 individuals] and T2 [n = 11 individuals]) (K) and treated with MCC950 or R406 (L) at T2 (n = 11 individuals).


M . IL - 1β concentrations determined in SP / N - stimulated monocyte - derived macrophages at 34 after 2nd vaccination ( first booster ) ( T4 = 7 individuals ) 2 after 3rd vaccination ( second booster ) ( T5 = 11 individuals ) .

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M. IL-1β concentrations determined in SP/N-stimulated monocyte-derived macrophages at 34 weeks after 2nd vaccination (first booster) (T4 = 7 individuals) and 2 weeks after the 3rd vaccination (second booster) (T5 = 11 individuals).


( A ) Recombinant were mixed , as indicated , in presence ( lane 5 ) or absence ( lane 4 ) of ATP incubated for 1 h at 30 ° C before being separated by SDS - PAGE analyzed by western blotting using α - His antibody . Components present ( + ) or absent ( − ) from are indicated .

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(A) Recombinant proteins were mixed, as indicated, in the presence (lane 5) or absence (lane 4) of ATP and incubated for 1 h at 30°C before being separated by SDS-PAGE and analyzed by western blotting using the α-His antibody. Components present (+) or absent (−) from the assay are indicated.


( B ) Recombinant were mixed , as indicated , incubated as above . reactions were stopped , subjected to SDS - PAGE stained with Coomassie Blue .

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(B) Recombinant proteins were mixed, as indicated, and incubated as above. The reactions were stopped, subjected to SDS-PAGE and stained with Coomassie Blue.


( C ) Recombinant were mixed , as indicated , incubated as above analyzed with α - His antibody .

(C) Recombinant proteins were mixed, as indicated, incubated as above and analyzed with α-His antibody.


( A ) occurrence of mC - ATG5 in WT L . major promastigotes expressing mC - ATG5 incubated in nutrient - rich medium at log phase at 26 ° C .

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(A) The occurrence of mC-ATG5 puncta in WT L. major promastigotes expressing mC-ATG5 incubated in nutrient-rich medium at log phase at 26°C.


( B ) multiplicity of in promastigotes expressing mC - ATG5 after starvation for up to 1 h in PBS at 26 ° C . time ranges indicated reflect starvation period together with 30 min period during which observations on microscope were undertaken .

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(B) The multiplicity of puncta in promastigotes expressing mC-ATG5 after starvation for up to 1 h in PBS at 26°C. The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken.


( C ) Co - labelling of with mC - ATG5 GFP - ATG12 co - expressed in promastigotes incubated in PBS for 2 h at 26 ° C . ( D ) In nutrient - rich medium , GFP - ATG8 punctum without mC - ATG5 is arrowed . ( E ) Incubated in PBS for 30 min at 26 ° C . ( F ) In nutrient - rich medium at 26 ° C . ( G ) Incubated in PBS for 30 min at 26 ° C , small panels are enlargements of merged panels . ( H - I ) Promastigotes in nutrient - rich medium . ( J - K ) Promastigotes at late log phase in nutrient - rich conditions . ( L ) Promastigotes at stationary phase in nutrient - rich conditions . Scale bar throughout , 10 µm .

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(C) Co-labelling of puncta with mC-ATG5 and GFP-ATG12 co-expressed in promastigotes incubated in PBS for 2 h at 26°C. (D) In nutrient-rich medium, GFP-ATG8 punctum without mC-ATG5 staining is arrowed. (E) Incubated in PBS for 30 min at 26°C. (F) In nutrient-rich medium at 26°C. (G) Incubated in PBS for 30 min at 26°C, the small panels are enlargements of the merged panels. (H-I) Promastigotes in nutrient-rich medium. (J-K) Promastigotes at late log phase in nutrient-rich conditions. (L) Promastigotes at stationary phase in nutrient-rich conditions. Scale bar throughout, 10 µm.


( A ) Growth curve of Leishmania promastigotes in HOMEM medium at 26 ° C . * , Δatg5 differed significantly from WT ( p < 0 . 05 ) .

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(A) Growth curve of Leishmania promastigotes in HOMEM medium at 26°C. *, Δatg5 differed significantly from WT (p<0.05).


( B ) occurrence of GFP - ATG8 in promastigotes after incubation in nutrient - deprived ( PBS , ND ) nutrient - rich ( HOMEM medium , NR ) conditions for 2 h at 26 ° C . Scale bar , 10

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(B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm


( C ) Occurrence of GFP - ATG8 in promastigotes when incubated in conditions detailed in ( B ) . Means ± SD from four independent experiments . * * * , occurrence of GFP - ATG8 in Δatg5 were significantly different from in WT in nutrient - deprived nutrient - rich conditions ( p < 0 . 05 ) .

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(C) Occurrence of GFP-ATG8 puncta in promastigotes when incubated in the conditions detailed in (B). Means ± SD from four independent experiments. * and **, occurrence of GFP-ATG8 puncta in Δatg5 were significantly different from in WT in nutrient-deprived and nutrient-rich conditions (p<0.05).


( D ) Western blot of extracts of promastigotes expressing GFP - ATG8 at logarithmic growth under standard conditions probed with α - GFP antibody . faster migrating , lipidated band is labelled GFP - ATG8 - II while un - lipidated band migrating more slowly is labelled GFP - ATG8 - I .

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(D) Western blot analysis of extracts of promastigotes expressing GFP-ATG8 at logarithmic growth under standard conditions and probed with α-GFP antibody. The faster migrating, lipidated band is labelled GFP-ATG8-II while the un-lipidated band migrating more slowly is labelled GFP-ATG8-I.


( A ) Enlarged ( EM ) swollen ( WM ) mitochondria seen by transmission electron microscopy ( ) in Δatg5 promastigotes under standard growth conditions . WT is shown at bottom right . Scale bar , 500 nm .

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(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm.


( B ) from MitoTracker Red ( MTR , 0 . 1 µM ) MitoTracker Green ( MTG , 0 . 2 µM ) in 2 × 106promastigotes after 30 min incubation at 26 ° C . Values shown are means ± SD from three independent experiments . * * * , was significantly different between WT Δatg5 ( p < 0 . 05 ) .

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(B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05).


( C ) Types of mitochondrial observed by microscopy of Δatg5 promastigotes expressing mitochondrial marker MUP - GFP . Scale bar , 10 µm .

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(C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm.


( D ) Differential of promastigotes with both MTR ( 0 . 1 µM ) MTG ( 0 . 2 µM ) . Scale bar , 10 µm .

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(D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm.


( E ) Viability , as measured by Alamar Blue reduction , of promastigotes . data are means ± SD from three independent experiments . * , Alamar blue reduction was significantly different ( p < 0 . 05 ) .

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(E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05).


( F ) Spectrometric analyses of DCF resulting from incubating promastigotesat 2 × 106 / ml with H2DCFDA at 0 . 1 mM for 2 h at 26 ° C . Values shown are means ± SD from three independent experiments . * , DCF was significantly different ( p < 0 . 05 ) .

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(F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).


Phospholipid accumulation in Δatg5 promastigotes . Negative ion survey scans ( 650 - 900 m / z ) of WT ( A ) Δatg5 ( B ) promastigotes extracted for lipids analysed by ES - MS , as described in Materials Methods . a = ( alkylacyl ) .

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Phospholipid accumulation in Δatg5 promastigotes.Negative ion survey scans (650-900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).


( A ) Proportion of metacyclic promastigotes in stationary phase cultures , assessed using PNA . Values shown are means ± SD from three independent experiments . * , differed significantly ( p < 0 . 05 ) .

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(A) Proportion of metacyclic promastigotes in stationary phase cultures, assessed using the PNA assay. Values shown are the means ± SD from three independent experiments. *, differed significantly (p<0.05).


( B ) Western blot of extracts of 107promastigotes at stationary phase of growth probed with α - HASPB . α - Cysteine Synthase was used as a loading control [ 56 ] .

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(B) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α-HASPB. α-Cysteine Synthase was used as a loading control [56].


( C ) Infectivity survival of promastigotes in peritoneal macrophages in vitro , infected at a 5 ∶ 1 , with infection rates being assessed after 1 5 days . * , differed significantly ( p < 0 . 01 ) .

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(C) Infectivity and survival of promastigotes in peritoneal macrophages in vitro, infected at a ratio 5∶1, with the infection rates being assessed after 1 and 5 days. *, differed significantly (p<0.01).


( D ) Lesion progression in BALB / c mice inoculated with 5 × 105 stationary phase promastigotes . Values shown are means ± SD from 5 mice . * , infection level of Δatg5 differed significantly from WT ( p < 0 . 01 ) Δatg5 : : ATG5 ( p < 0 . 05 ) .

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(D) Lesion progression in BALB/c mice inoculated with 5×105 stationary phase promastigotes. Values shown are the means ± SD from 5 mice. *, infection level of Δatg5 differed significantly from WT (p<0.01) and Δatg5::ATG5 (p<0.05).


( E ) Morphologies of isolated from mouse lesions analysed by . Scale bar : 2 µm .

(E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.


A ) of promastigote culture initiated with Δatg5 isolated from a mouse lesion cultured in nutrient - rich medium . Shown are ovoid amastigote - like form ( left ) ; spindled - shaped form without an external flagellum ( centre left panel ) with an external flagellum of varying lengths ( centre right right panels ) . Scale bar , 10 µm .

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A) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient-rich medium. Shown are ovoid and amastigote-like form (left); spindled-shaped form without an external flagellum (centre left panel) and with an external flagellum of varying lengths (centre right and right panels). Scale bar, 10 µm.


( B ) Distribution of flagella lengths lengths of stationary phase promastigotes . Data represent measurements from ∼ 200 from each promastigote population .

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(B) Distribution of flagella lengths and body lengths of stationary phase promastigotes. Data represent measurements from ∼200 cells from each promastigote population.


B ) Tumour growth curves for successive four - generation xenografts treated with 5 - Fu or PBS . Twelve mice per group .

B) Tumour growth curves for successive four-generation xenografts treated with 5-Fu or PBS. Twelve mice per group.


C ) Representative cases show first fourth generation xenograft H & E ( left ) EBER - 1 ISH ( right ) .

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C) Representative cases show the first and fourth generation xenograft H&E staining (left) and EBER-1 ISH staining (right).


D) The morphology of parental SNU719 and SNU-4th cells.


E ) Tumour sphere culture was performed to evaluate sphere formation capacity in SNU719 SNU - 4th , data are shown as mean numbers sizes of tumour spheres . central horizontal lines represent median , top bottom positions of box represent upper lower quartiles , error bars represent mean ± SD , average number maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates .

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E) Tumour sphere culture was performed to evaluate the sphere formation capacity in SNU719 and SNU-4th cells, and the data are shown as the mean numbers and sizes of the tumour spheres. The central horizontal lines represent the median, the top and the bottom positions of the box represent upper and lower quartiles, error bars represent the mean ± SD, and the average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates.


F ) CCK8 of SNU719 SNU - 4th to determine proliferation under indicated culture conditions ( 10 % FBS or 0 . 1 % FBS ) .

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F) CCK8 assay of SNU719 and SNU-4th cells to determine cell proliferation under the indicated culture conditions (10% FBS or 0.1% FBS).


Expression of stemness markers ( Sox2 , Klf4 , Bmi1 , Oct4 ) drug resistance genes ( ABCG2 , Mrp1 ) evaluated by real - time PCR ( G ) in SNU719 SNU - 4th .

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Expression of stemness markers (Sox2, Klf4, Bmi1, Oct4) and drug resistance genes (ABCG2, Mrp1) evaluated by real-time PCR (G) in SNU719 and SNU-4th cells.


Expression of stemness markers ( Sox2 , Klf4 , Bmi1 , Oct4 ) drug resistance genes ( ABCG2 , Mrp1 ) evaluated by WB ( H ) in SNU719 SNU - 4th .

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Expression of stemness markers (Sox2, Klf4, Bmi1, Oct4) and drug resistance genes (ABCG2, Mrp1) evaluated by WB (H) in SNU719 and SNU-4th cells.


I ) Real - time PCR of expression of EMT - associated markers ( E - cad , vimentin ) transcription factors ( Twist1 , Snail , Slug , Zeb1 ) in SNU719 SNU - 4th .

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I) Real-time PCR analysis of the expression of EMT-associated markers (E-cad, vimentin) and transcription factors (Twist1, Snail, Slug, Zeb1) in SNU719 and SNU-4th cells.


J ) WB of expression of E - cad , vimentin Mmp7 in SNU719 SNU - 4th .

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J) WB analysis of the expression of E-cad, vimentin and Mmp7 in SNU719 and SNU-4th cells.


K ) Colony formation of colony - forming ability in SNU719 SNU - 4th .

K) Colony formation assay analysis of the colony-forming ability in SNU719 and SNU-4th cells.


L ) Transwell migration Matrigel invasion were performed to assess migratory invasive capability of SNU719 SNU - 4th .

L) Transwell migration and the Matrigel invasion assay were performed to assess the migratory and invasive capability of SNU719 and SNU-4th cells.


M ) Representative of flow cytometric of SP .

M) Representative images of the flow cytometric analysis of SP cells.


N ) Representative of flow cytometric of surface antigen phenotype .

N) Representative images of the flow cytometric analysis of the surface antigen phenotype.


A ) Heat map of expression of circRNAs ( both human circRNAs ebv - circRNAs ) in fourth passage xenografts treated with 5 - Fu or PBS . Red colour represents upregulated circRNAs , green colour represents downregulated circRNAs .

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A) Heat map analysis of the expression of circRNAs (both human circRNAs and ebv-circRNAs) in the fourth passage xenografts treated with 5-Fu or PBS. Red colour represents upregulated circRNAs, and green colour represents downregulated circRNAs.


( B ) Volcano plot representing expression fold changes in circRNAs . red dots represent upregulated circRNAs , green dots represent downregulated circRNAs . ( C ) back - spliced read distribution of ebv - circRNAs ( n = 261 ) .

(B) Volcano plot representing the expression fold changes in circRNAs. The red dots represent upregulated circRNAs, and green dots represent downregulated circRNAs. (C) The back-spliced read distribution of ebv-circRNAs (n=261).


(D) The length distribution of ebv-circRNAs.


( E ) Genomic origin of ebv - circRNAs .

(E) Genomic origin of ebv-circRNAs.


( F ) Number of ebv - circRNAs produced from one gene .

(F) Number of ebv-circRNAs produced from one gene.


( B ) Real - time PCR of expression of circLMP2A in SNU - 4th , SNU719 YCCEL1 .

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(B) Real-time PCR analysis of the expression of circLMP2A in SNU-4th, SNU719 and YCCEL1 cells.


( C ) existence of circLMP2A was validated by RT - PCR ( up ) real - time PCR ( down ) in SNU - 4th , SNU719 YCCEL1 treated with or without RNase R digestion . GAPDH was used as a negative control .

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(C) The existence of circLMP2A was validated by RT-PCR (up) and real-time PCR (down) in SNU-4th, SNU719 and YCCEL1 cells treated with or without RNase R digestion. GAPDH was used as a negative control.


( D ) Real - time PCR of circLMP2A LMP2A expression after treatment with actinomycin D at indicated time points in SNU - 4th .

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(D) Real-time PCR analysis of circLMP2A and LMP2A expression after treatment with actinomycin D at the indicated time points in SNU-4th cells.


( E ) Real - time PCR of circLMP2A LMP2A expression in either cytoplasm or nucleus in SNU - 4th . GAPDH U6 were used as controls in cytoplasm nucleus , respectively .

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(E) Real-time PCR analysis of circLMP2A and LMP2A expression in either the cytoplasm or nucleus in SNU-4th cells. GAPDH and U6 were used as controls in cytoplasm and nucleus, respectively.


( F ) ISH for circLMP2A . U6 18S are mainly localized in nucleus cytoplasm , respectively , were used as controls .

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(F) RNA ISH for circLMP2A. U6 and 18S are mainly localized in the nucleus and cytoplasm, respectively, and were used as controls.


( G ) Kaplan - Meier survival curve showing correlation between circLMP2A expression OS . n = 69 , P < 0 . 001 , log - rank . ( H ) Kaplan - Meier survival curve showing correlation between circLMP2A expression progression - free survival ( PFS ) . n = 69 , P < 0 . 001 , log - rank .

(G) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and OS. n= 69, P < 0.001, log-rank test. (H) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and progression-free survival (PFS). n = 69, P < 0.001, log-rank test.


( A ) Real - time PCR of interfering efficacy linear LMP2A mRNA after transfection of three circLMP2A shRNAs .

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(A) Real-time PCR analysis of the interfering efficacy and linear LMP2A mRNA levels after transfection of three circLMP2A shRNAs.


( B ) Tumour sphere culture of tumour sphere formation in SNU - 4th transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . central horizontal lines represent median , top bottom positions of box represent upper lower quartiles , error bars represent mean ± SD . average number maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . Scale bars = 100µm .

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(B) Tumour sphere culture analysis of the tumour sphere formation in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. The central horizontal lines represent the median, top and bottom positions of the box represent upper and lower quartiles, and error bars represent the mean ± SD. The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates. Scale bars =100µm.


EMT - related markers , stemness markers drug resistance genes were evaluated by real - time PCR ( C ) in SNU - 4th transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control .

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EMT-related markers, stemness markers and drug resistance genes were evaluated by real-time PCR (C) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.


EMT - related markers , stemness markers drug resistance genes were evaluated by WB ( D ) in SNU - 4th transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control .

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EMT-related markers, stemness markers and drug resistance genes were evaluated by WB (D) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.


( E - F ) Transwell ( E ) colony formation ( F ) of migratory invasive capability colony - forming ability in SNU - 4th transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . Scale bars = 100µm .

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(E-F) Transwell assay (E) and colony formation assay (F) analysis of the migratory and invasive capability and colony-forming ability in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. Scale bars =100µm.


( G - H ) Flow cytometric of rate of SP ( G ) apoptosis ( H ) in SNU - 4th transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control .

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(G-H) Flow cytometric analysis of the rate of SP cells (G) and apoptosis (H) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.


Subcutaneously established tumour xenografts in NOD / SCID mice . Tumour volume ( I ) of xenografts were remarkably inhibited by down - regulation of circLMP2A . Six mice per group .

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Subcutaneously established tumour xenografts in NOD/SCID mice. Tumour volume (I) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group.


Subcutaneously established tumour xenografts in NOD / SCID mice . tumour ( J ) of xenografts were remarkably inhibited by down - regulation of circLMP2A . Six mice per group .

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Subcutaneously established tumour xenografts in NOD/SCID mice. tumour weight (J) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group.


( K - L ) Representative of bioluminescence ( K ) lung metastases ( L ) are presented to measure metastatic colonies . Six mice per group , scale bars = 50µm .

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(K-L) Representative images of bioluminescence (K) and lung metastases (L) are presented to measure the metastatic colonies. Six mice per group, scale bars =50µm.


( A ) Real - time PCR confirmed over - expression of circLMP2A in SNU719 YCCEL1 after transfection .

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(A) Real-time PCR confirmed the over-expression of circLMP2A in SNU719 and YCCEL1 cells after transfection.


( B ) Over - expression of circLMP2A induced formation of tumour spheres in SNU719 YCCEL1 . central horizontal lines represent median , top bottom positions of box represent upper lower quartiles , error bars represent mean ± SD . average number maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . Scale bars = 100µm .

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(B) Over-expression of circLMP2A induced the formation of tumour spheres in SNU719 and YCCEL1 cells. The central horizontal lines represent the median, top and bottom positions of the box represent upper and lower quartiles, and error bars represent the mean ± SD. The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates. Scale bars =100µm.


EMT - related markers , stemness markers drug resistance genes were assessed by real - time PCR ( C ) in SNU719 YCCEL1 transfected with circLMP2A or vector .

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EMT-related markers, stemness markers and drug resistance genes were assessed by real-time PCR (C) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.


EMT - related markers , stemness markers drug resistance genes were assessed by WB ( D ) in SNU719 YCCEL1 transfected with circLMP2A or vector .

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EMT-related markers, stemness markers and drug resistance genes were assessed by WB (D) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.


( E - F ) Flow cytometric of rate of SP ( E ) apoptosis ( F ) in SNU719 YCCEL1 transfected with circLMP2A or vector .

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(E-F) Flow cytometric analysis of the rate of SP cells (E) and apoptosis (F) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.


( G - H ) Transwell ( G ) colony formation ( H ) of migratory invasive capability colony - forming ability in SNU719 YCCEL1 transfected with circLMP2A or vector . Scale bars = 100µm .

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(G-H) Transwell assay (G) and colony formation assay (H) analysis of the migratory and invasive capability and colony-forming ability in SNU719 and YCCEL1 cells transfected with circLMP2A or vector. Scale bars =100µm.


Subcutaneously established tumour xenografts in NOD / SCID mice . Tumour volume ( I ) of xenografts were significantly increased by over - expression of circLMP2A . Six mice per group .

Subcutaneously established tumour xenografts in NOD/SCID mice. Tumour volume (I) of the xenografts were significantly increased by over-expression of circLMP2A. Six mice per group.


Subcutaneously established tumour xenografts in NOD / SCID mice . tumour ( J ) of xenografts were significantly increased by over - expression of circLMP2A . Six mice per group .

Subcutaneously established tumour xenografts in NOD/SCID mice. tumour weight (J) of the xenografts were significantly increased by over-expression of circLMP2A. Six mice per group.


( B ) relative of 7 miRNA candidates were detected by real - time PCR in SNU719 SNU - 4th .

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(B) The relative levels of 7 miRNA candidates were detected by real-time PCR in SNU719 and SNU-4th cells.


( C - E ) Real - time PCR of 7 miRNA expression in SNU - 4th transfected with sh - circLMP2A - 2 or sh - control ( C ) , SNU719 ( D ) YCCEL1 ( E ) transfected with circLMP2A or vector .

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(C-E) Real-time PCR analysis of 7 miRNA expression in SNU-4th cells transfected with sh-circLMP2A-2 or sh-control (C), and SNU719 (D) and YCCEL1 cells (E) transfected with circLMP2A or vector.


( F - H ) circ LMP2Ain specific probe detected by real - time PCR ( F ) RT - PCR ( H ) . relative of 5 miRNA candidates were detected by real - time PCR ( G ) RT - PCR ( H ) . GAPDH was used as a negative control .

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(F-H) circ LMP2Ain specific probe and detected by real-time PCR (F) and RT-PCR (H). The relative levels of 5 miRNA candidates were detected by real-time PCR (G) and RT-PCR (H). GAPDH was used as a negative control.


( I ) ISH showing co - localization between circLMP2A miR - 3908 in SNU - 4th . circLMP2A probes were labelled with Cy3 , miR - 3908 probes were labelled with FITC . Nuclei were stained with DAPI .

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(I) RNA ISH showing the co-localization between circLMP2A and miR-3908 in SNU-4th cells. circLMP2A probes were labelled with Cy3, and miR-3908 probes were labelled with FITC. Nuclei were stained with DAPI.


( K ) Dual luciferase reporter for luciferase activity of indicated plasmids ( circLMP2A Wt , circLMP2A mut1 , circLMP2A mut2 , circLMP2Amut3 luciferase reporter ) in HEK - 293T transfected with mimic - NC or miR - 3908 .

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(K) Dual luciferase reporter assay for luciferase activity of the indicated plasmids (circLMP2A Wt, circLMP2A mut1, circLMP2A mut2, circLMP2Amut3 luciferase reporter) in HEK-293T cells transfected with mimic-NC or miR-3908.


( A ) Tumour sphere culture of tumour sphere formation ability of SNU - 4th transfected with above different mimics .

(A) Tumour sphere culture analysis of the tumour sphere formation ability of SNU-4th cells transfected with the above different mimics.


Real - time PCR ( B ) of EMT - related markers , stemness markers drug resistance genes expression in SNU - 4th transfected with above different mimics .

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Real-time PCR (B) analysis of EMT-related markers, stemness markers and drug resistance genes expression in SNU-4th cells transfected with the above different mimics.


WB ( C ) of EMT - related markers , stemness markers drug resistance genes expression in SNU - 4th transfected with above different mimics .

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WB (C) analysis of EMT-related markers, stemness markers and drug resistance genes expression in SNU-4th cells transfected with the above different mimics.


( D - E ) Colony formation ( D ) transwell ( E ) of colony - forming ability migratory invasive capability of SNU - 4th transfected with above different mimics .

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(D-E) Colony formation assay (D) and transwell assay (E) analysis of the colony-forming ability and migratory and invasive capability of SNU-4th cells transfected with the above different mimics.


( F - G ) Flow cytometric of rate of apoptosis ( F ) SP ( G ) in SNU - 4th transfected with above different mimics .

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(F-G) Flow cytometric analysis of the rate of apoptosis (F) and SP cells (G) in SNU-4th cells transfected with the above different mimics.


( H ) Pearson correlation of correlation between expression of circLMP2A miR - 3908 . n = 69 , P = 0 . 039 , Pearson ' s correlation coefficient .

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(H) Pearson correlation analysis of the correlation between the expression of circLMP2A and miR-3908. n = 69, P = 0.039, Pearson's correlation coefficient analysis.


( I ) Kaplan - Meier survival curves of correlation between miR - 3908 expression OS . n = 69 , P = 0 . 049 , log - rank .

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(I) Kaplan-Meier survival curves analysis of the correlation between miR-3908 expression and OS. n = 69, P = 0.049, log-rank test.


( B ) Dual luciferase reporter for luciferase activity of indicated plasmids ( TRIM59 - Wt TRIM59 - Mut luciferase reporter ) in HEK - 293T transfected with mimic - NC or miR - 3908 .

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(B) Dual luciferase reporter assay for the luciferase activity of the indicated plasmids (TRIM59-Wt and TRIM59-Mut luciferase reporter) in HEK-293T cells transfected with mimic-NC or miR-3908.


( C ) Real - time PCR of mRNA level of TRIM59 in SNU719 SNU - 4th .

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(C) Real-time PCR analysis of the mRNA level of TRIM59 in SNU719 and SNU-4th cells.


( D - F ) Real - time PCR of expression of TRIM59 in SNU - 4th ( D ) , SNU719 ( E ) YCCEL1 ( F ) transfected with above different mimics .

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(D-F) Real-time PCR analysis of the expression of TRIM59 in SNU-4th (D), SNU719 (E) and YCCEL1 cells (F) transfected with the above different mimics.


( G - I ) WB of expression of TRIM59 p53 in SNU - 4th ( G ) , SNU719 ( H ) YCCEL1 ( I ) transfected with above different mimics .

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(G-I) WB analysis of the expression of TRIM59 and p53 in SNU-4th (G), SNU719 (H) and YCCEL1 cells (I) transfected with the above different mimics.


( J ) Immunohistochemical showing that over - expression of circLMP2A could lead to increased expression of TRIM59 ERG but decreased expression of p53 .

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(J) Immunohistochemical staining showing that over-expression of circLMP2A could lead to increased expression of TRIM59 and ERG but decreased expression of p53.


( K ) WB of expression of TRIM59 p53 in tumour xenografts .

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(K) WB analysis of the expression of TRIM59 and p53 in tumour xenografts.


( L ) Immunohistochemical showing that high expression of circLMP2A in EBVaGC tissues resulted in significant increases in CD44 + CD24 - significant decreases in p53 + .

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(L)Immunohistochemical staining showing that high expression of circLMP2A in EBVaGC tissues resulted in significant increases in CD44+ CD24- cells and significant decreases in p53+ cells.


T interface actin ( phalloidin ) re - organization during synapse symmetry breaking , imaged using SIM . of shape elongation ( AR ; B , n = 44 for 5 ' , 39 for 20 ' ) , or relative signal of indicated in SIM ( C , D ) normalized to mean of values at 5 ' ; points represent values for . Arrowheads in ( C ) indicate foci . p values are : * * * P < 0 . 0001 for AR , * * P = 0 . 003 for foci , * * P = 0 . 008 for talin , p = 0 . 36 for total F - actin using Mann - Whitney two - between populations of within same experiment . Relationship between AR foci at level , each point in scatter plot represents value obtained from a single .

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T cell interface actin (phalloidin) re-organization during synapse symmetry breaking, imaged using SIM. Quantification of the shape elongation (AR; B, n= 44 for 5', 39 for 20'), or relative fluorescence signal of the indicated proteins in the SIM images (C, D) normalized to the mean of values at 5'; points represent values for individual cells. Arrowheads in (C) indicate foci. p values are: ***P< 0.0001 for AR, **P =0.003 for foci, **P= 0.008 for talin, and p =0.36 for total F-actin using Mann-Whitney two-tailed test between populations of cells within the same experiment. Relationship between AR and foci at individual cell level, each point in the scatter plot represents value obtained from a single cell.


Integrin augmentation does not rescue synapse symmetry breaking . were allowed to adhere to APS for 5 ' or 10 ' , were then treated with vehicle control , 0 . 5mM MnCl2 or 100nM A286982 for subsequent 10 ' . were fixed , stained with phalloidin anti - Talin antibody , imaged using SIM ( F ) analyzed for AR ( G ) as well as talin , total F - actin actin foci at synapse ( H ) . In ( E - H ) , n for 5 ' = 40 , for 20 ' = 62 , for MnCl2 = 59 , for A286982 = 53 . values in plots represent values normalized to mean of 5 ' in each set . p values in graph , n . s . > 0 . 05 ; * * * P < 0 . 001 ; * P = 0 . 01 ; * * P < 0 . 009 using Mann - Whitney two - between populations of within same experiment .

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Integrin augmentation does not rescue synapse symmetry breaking. Cells were allowed to adhere to the APS for 5' or 10', and were then treated with vehicle control, 0.5mM MnCl2 or 100nM A286982 for subsequent 10'. Cells were fixed, stained with phalloidin and anti-Talin antibody, and imaged using SIM (F) and analyzed for AR (G) as well as talin, total F-actin and actin foci at the synapse (H). In (E-H), n for 5'= 40, for 20'= 62, for MnCl2= 59, for A286982= 53. The values in the plots represent the intensity values normalized to the mean of 5' in each set. p values in the graph, n.s. > 0.05; ***P <0.001; *P= 0.01; **P<0.009 using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Time - lapse imaging of LifeAct - GFP expressing T using Lattice lightsheet microscopy ( LLSM ) during synapse polarization . Shown are snapshots at selected timepoints ( left ) a kymograph of actin foci ( denoted by arrows in first image panel ) over time ( right ) for a representative T . of changes in actin foci aspect in LLSM during symmetry breaking . values in plot are raw values normalized to their mean in each case .

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Time-lapse imaging of LifeAct-GFP expressing T cell using Lattice lightsheet microscopy (LLSM) during synapse polarization. Shown are snapshots at selected timepoints (left) and a kymograph of actin foci (denoted by arrows in the first image panel) over time (right) for a representative T cell. Quantification of changes in actin foci and cellular aspect ratio in LLSM images during symmetry breaking. The values in the plot are the raw values normalized to their mean in each case.


T incubated with APS for indicated time periods were processed for phalloidin - or immune - ( C , D ) Graph in ( D ) shows relative of total active WASP ( WASP - phosphorylated at Y293 , pWASP ) , normalized to mean at 5 ' ; for pWASP n in 5 ' = 49 , in 20 ' = 44 ; for WASP , n in 5 ' = 56 , 20 ' = 56 . P values , P * * = 0 . 0027 for pWASP P * * = 0 . 0035 for WASP using Mann - Whitney two - . scatterplot in ( D ) shows relationship between active WASP foci on a per basis .

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T cells incubated with APS for the indicated time periods were processed for phalloidin- or immune-staining (C, D) Graph in (D) shows relative levels of total and active WASP (WASP-phosphorylated at Y293, pWASP), normalized to the mean levels at 5'; for pWASP n in 5'= 49, in 20'= 44; for WASP, n in 5'= 56, 20'= 56. P values, P**= 0.0027 for pWASP and P**= 0.0035 for WASP using Mann-Whitney two-tailed test. The scatterplot in (D) shows the relationship between active WASP and foci on a per cell basis.


T incubated with APS for indicated time periods were processed for for western blotting ( E ) . Numbers in graph in ( E ) indicate WASP band divided by actin band , resulting ratios normalized to values at 0 ' time point ( non - activated ) , from three independent experiments ( n = 3 ) . P value * = 0 . 025 , using paired two t - .

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T cells incubated with APS for the indicated time periods were processed for for western blotting (E). Numbers in the graph in (E) indicate WASP band intensity divided by the actin band intensity, and resulting ratios normalized to the values at the 0' time point (non-activated cells), from three independent experiments (n=3). P value *= 0.025, using paired two tailed t-test.


Actin dynamics in mature synapse of LifeAct - GFP expressing T using TIRFM . show foci ( pseudocolored red ) extracted from total synaptic F - actin ( F - G ) , kymograph shows time course of foci lamellar activity over a period of 210 sec . Histograms of foci lamellar protrusion retraction ( fluctuations ) dynamics in WT T ( H ) ; Lifetime of foci lamellar events ( graph on bottom right , each dot represents a single foci / lamellar protrusion - retraction event n = 126 for foci n = 77 for lamellar protrusions ) . * * * P < 0 . 0001 using Mann - Whitney two - between populations of within same experiment .

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Actin dynamics in mature synapse of LifeAct-GFP expressing T cell using TIRFM. The images show the foci (pseudocolored red) extracted from the total synaptic F-actin (F-G), and kymograph shows the time course of foci and lamellar activity over a period of 210 sec. Histograms of foci and lamellar protrusion and retraction (fluctuations) dynamics in WT T cell (H); Lifetime of individual foci and lamellar events (graph on the bottom right, each dot represents a single foci/lamellar protrusion-retraction event n= 126 for foci and n= 77 for lamellar protrusions). ***P<0.0001 using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Kymograph of lamellar activity of WASP - deficient that lack actin foci .

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Kymograph of the lamellar activity of WASP-deficient cells that lack actin foci.


Foci - deficient display poor traction forces in their synapse . WT or WASP - / - T were incubated on polyacrylamide substrates covalently functionalized with anti - CD3 ICAM1 , traction force measurements were carried out as described in ' Methods ' . in right show traction force maps without ( left panels ) or with ( right panels ) force vectors . p value , * * P < 0 . 005 as determined by using Mann - Whitney two - between populations of within same experiment , n = 9 11 .

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Foci -deficient cells display poor traction forces in their synapse. WT or WASP-/- T cells were incubated on polyacrylamide substrates covalently functionalized with anti-CD3 and ICAM1, and traction force measurements were carried out as described in 'Methods'. The images in the right show traction force maps without (left panels) or with (right panels) force vectors. p value, **P<0.005 as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment, n = 9 and 11.


SIM imaging of WT or WASP - / - T activated by APS for 5 ' . graphs show of actin foci ( derived from phalloidin ) , pCasL talin in synapse normalized to ' WT ' mean value in each case ( Middle graph ; in case of foci , n = 63 for WT 86 for WASP - / - ; in case of pCasL n = 63 for WT 78 for WASP - / - ; in case of Talin , n = 59 for WT 73 for WASP - / - ; in case of AR , n = 46 for WT 42 for WASP - / - ) ; right graph shows AR measurement . * * * P < 0 . 0001 ; p value for talin * P = 0 . 07 , as determined by using Mann - Whitney two - between populations of within same experiment .

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SIM imaging of WT or WASP-/- T cells activated by APS for 5'. The graphs show quantification of actin foci (derived from phalloidin staining), pCasL and talin levels in the synapse normalized to the 'WT' mean value in each case (Middle graph; in the case of foci, n= 63 for WT and 86 for WASP-/-; in the case of pCasL n= 63 for WT and 78 for WASP-/-; in the case of Talin, n= 59 for WT and 73 for WASP-/-; in the case of AR, n= 46 for WT and 42 for WASP-/-); right graph shows AR measurement. ***P <0.0001; p value for talin *P=0.07, as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment.