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 .

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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 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 .

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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 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

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(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 ) 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 .

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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 ) 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . Heatmap summary of Aft1 induction experiment showing all 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 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 .

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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 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 .

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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 the localization of pri - miR - 31 . The 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 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 .

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

(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 the fluorescence of FAM - miPEP31 in anti - CD3 / 28 activated CD4 + T cells treated with 10 μM FAM - miPEP31 . Scale bar 5 μm .

(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 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 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 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 . .

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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 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 .

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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 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 .

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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 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 .

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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 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 . * * * *

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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 the 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 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 .

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 ) and T2 ( H ) ( n = 8 individuals ) upon indicated stimuli .

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


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 .

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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 . 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 .

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 cells . 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 the same samples . Negative log10 padj - values are plotted against the 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 ) 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 ) .

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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 ( 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 .

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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 ) 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 .

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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 and 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 . 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 .

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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 analysis of phospho - NF - κB and NLRP3 in cell 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 ) 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 ) .

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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 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 ) .

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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 primary 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 . Quantification of cell death 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 staining at T0 and 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 staining at T0 and 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 analysis of SOD2 in macrophages at T0 and 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 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 )

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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 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 ) .

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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 ) 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 .

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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 ] and T2 [ n = 11 individuals ] ) ( K ) and 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 weeks after 2nd vaccination ( first booster ) ( T4 = 7 individuals ) and 2 weeks after the 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 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 .

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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 proteins were mixed , as indicated , and incubated as above . The reactions were stopped , subjected to SDS - PAGE and 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 proteins were mixed , as indicated , incubated as above and analyzed with α - His antibody .

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


( 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 .

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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 ) 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 .

(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 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 .

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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 ) 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

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 ) 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 ) .

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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 morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein 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 staining of promastigotes with both MTR ( 0 . 1 µM ) and MTG ( 0 . 2 µM ) . Scale bar , 10 µm .

(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 . All data are means ± SD from three independent experiments . * , Alamar blue reduction was significantly different ( p < 0 . 05 ) .

(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 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 ) .

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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 ) and Δatg5 ( B ) promastigotes extracted for lipids and analysed by ES - MS , as described in Materials and 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 the PNA assay . Values shown are the means ± SD from three independent experiments . * , differed significantly ( p < 0 . 05 ) .

(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 analysis 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 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 ) .

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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 the means ± SD from 5 mice . * , infection level of Δatg5 differed significantly from WT ( p < 0 . 01 ) and 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 cells isolated from mouse lesions and analysed by SEM . Scale bar : 2 µm .

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


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 .

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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 and body lengths of stationary phase promastigotes . Data represent measurements from ∼ 200 cells 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 .

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


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

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


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 .

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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 assay of SNU719 and SNU - 4th cells to determine cell proliferation under the 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 ) and drug resistance genes ( ABCG2 , Mrp1 ) evaluated by real - time PCR ( G ) in SNU719 and SNU - 4th cells .

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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 ) and drug resistance genes ( ABCG2 , Mrp1 ) evaluated by WB ( H ) in SNU719 and SNU - 4th cells .

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

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

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

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


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

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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 images of the flow cytometric analysis of SP cells .

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


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

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


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 .

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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 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 ) .

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(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 .

(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 analysis of the expression of circLMP2A in SNU - 4th , SNU719 and YCCEL1 cells .

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


( 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 .

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

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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 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 .

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

(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 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 .

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(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 analysis of the interfering efficacy and linear LMP2A mRNA levels 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 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 .

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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 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 .

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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 and drug resistance genes were evaluated by WB ( D ) in SNU - 4th cells 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 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 .

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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 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 .

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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 the 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 weight ( J ) of the xenografts were remarkably inhibited by down - regulation of circLMP2A . Six mice per group .

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

(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 the over - expression of circLMP2A in SNU719 and YCCEL1 cells 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 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 .

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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 and drug resistance genes were assessed by real - time PCR ( C ) in SNU719 and YCCEL1 cells 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 and drug resistance genes were assessed by WB ( D ) in SNU719 and YCCEL1 cells 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 analysis of the rate of SP cells ( E ) and apoptosis ( F ) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector .

(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 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 .

(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 the 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 weight ( J ) of the 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.


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

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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 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 .

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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 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 .

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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 ) 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 .

(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 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 .

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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 analysis of the tumour sphere formation ability of SNU - 4th cells transfected with the 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 ) analysis of EMT - related markers , stemness markers and drug resistance genes expression in SNU - 4th cells transfected with the above different mimics .

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 ) analysis of EMT - related markers , stemness markers and drug resistance genes expression in SNU - 4th cells transfected with the 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 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 .

(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 analysis of the rate of apoptosis ( F ) and SP cells ( G ) in SNU - 4th cells transfected with the 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 analysis of the correlation between the expression of circLMP2A and miR - 3908 . n = 69 , P = 0 . 039 , Pearson ' s correlation coefficient analysis .

(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 analysis of the correlation between miR - 3908 expression and OS . n = 69 , P = 0 . 049 , log - rank test .

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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 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 .

(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 analysis of the mRNA level of TRIM59 in SNU719 and SNU - 4th cells .

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


( 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 .

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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 analysis of the expression of TRIM59 and p53 in SNU - 4th ( G ) , SNU719 ( H ) and YCCEL1 cells ( I ) transfected with the 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 staining showing that over - expression of circLMP2A could lead to increased expression of TRIM59 and 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 analysis of the expression of TRIM59 and p53 in tumour xenografts .

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


( 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 .

(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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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


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 .

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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 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 .

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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.


Human WAS patient CD4 + T cells - APC conjugates display synapse symmetry defects similar to those observed in mouse WASP - / - CD4 + T cells . CD4 + T cells purified from healthy controls or WAS patients were incubated with superantigen loaded HUVEC cells for 5 ' ( APC ; see ' Methods ' ) and imaged using confocal microscopy . Each image represents a maximum intensity projection of the synaptic area . Graph in the middle shows values normalized to the mean value of the ' Healthy ' case . In the case of ' actin ' and ' actin foci ' , n = 87 for healthy and n = 63 for WAS ; in the case of pCasL , n = 56 for healthy and n = 21 for WAS ; for ' AR ' graph , n = 88 for healthy and n = 63 for WAS case . P values ; * * * P < 0 . 001 ; n . s . P = 0 . 09 , as determined by using Mann - Whitney two - tailed test between populations of cells within the same experiment .

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Human WAS patient CD4+T cells- APC conjugates display synapse symmetry defects similar to those observed in mouse WASP-/- CD4+ T cells. CD4+T cells purified from healthy controls or WAS patients were incubated with superantigen loaded HUVEC cells for 5' (APC; see 'Methods') and imaged using confocal microscopy. Each image represents a maximum intensity projection of the synaptic area. Graph in the middle shows values normalized to the mean value of the 'Healthy' case. In the case of 'actin' and 'actin foci', n= 87 for healthy and n= 63 for WAS; in the case of pCasL, n=56 for healthy and n= 21 for WAS; for 'AR' graph, n= 88 for healthy and n= 63 for WAS case. P values; ***P <0.001; n.s. P=0.09, as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Simulation of T cell IS F - actin behavior and resultant mechanical tension incorporating the differential dynamics and positioning of foci and lamella across the synaptic interface , using Brownian dynamics equation with no inertia ( see ' Methods ' ) . The images show simulation snapshots at the beginning ( 0 min ; soon after the attachment and spreading of T cells on the substrates ) and the end of the simulations for the three IS cases - with persistent foci ( WT , left most panels ) , with no foci ( WASP - / - , middle right panels ) , and with WASP and consequently foci downregulated after a period of synapse maturation ( WT transitional ) . The colors in the images indicate myosin II motor ( pseudocolored teal ) , F - actin crosslinking proteins ( pseudocolored yellow ) , and the F - actin filaments ( pseudocolored blue for the low tension and the red for thigh tension actin filaments ) ; middle left image panels show the distribution of the aforementioned proteins at individual foci site at a higher magnification .

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Simulation of T cell IS F-actin behavior and resultant mechanical tension incorporating the differential dynamics and positioning of foci and lamella across the synaptic interface, using Brownian dynamics equation with no inertia (see 'Methods'). The images show simulation snapshots at the beginning (0 min; soon after the attachment and spreading of T cells on the substrates) and the end of the simulations for the three IS cases - with persistent foci (WT, left most panels), with no foci (WASP-/-, middle right panels), and with WASP and consequently foci downregulated after a period of synapse maturation (WT transitional). The colors in the images indicate myosin II motor (pseudocolored teal), F-actin crosslinking proteins (pseudocolored yellow), and the F-actin filaments (pseudocolored blue for the low tension and the red for thigh tension actin filaments); middle left image panels show the distribution of the aforementioned proteins at individual foci site at a higher magnification.


Predicted stress ( tension normalized to the area ) profiles within the cytoskeletal architecture as the synaptic contact progresses in time , corresponding to the three cases shown in ( D ) .

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Predicted stress (tension normalized to the area) profiles within the cytoskeletal architecture as the synaptic contact progresses in time, corresponding to the three cases shown in (D).


Live imaging of a T cell using SIM shows the inter - foci connections ( arrows in the inset ) as predicted in the simulations ; and their loss concomitant with loss of actin foci , as the T cell polarizes to initiate motility ( compare insets between 0 and 120 sec ) . ' 0 sec ' refers to the beginning of the observation of the cell , after it has attached to the substrate , spread and maintained synapse for 12 min . Scale bar , 5µm in main images and 1 . 5μm in the magnified insets .

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Live imaging of a T cell using SIM shows the inter-foci connections (arrows in the inset) as predicted in the simulations; and their loss concomitant with loss of actin foci, as the T cell polarizes to initiate motility (compare insets between 0 and 120 sec). '0 sec' refers to the beginning of the observation of the cell, after it has attached to the substrate, spread and maintained synapse for 12 min. Scale bar, 5µm in main images and 1.5μm in the magnified insets.


Ultrastructural visualization of detergent extracted WT and WASP - / - T cell synaptic cytoskeleton using platinum replica electron microscopy ( top images ) . Each image shows individual synapse , dark zone in the middle of the synapse represents cell nucleus . Scale bar , 2 . 5μm . The bottom left micrograph is the magnified inset from WT synapse and shows actin foci ( identified as globular cluster of short filaments in the inset , marked with arrows ) - dependent interconnected cytoskeletal architecture that is associated with synapse symmetry as predicted in the model , and is lacking in the WASP - / - cell synapse ( bottom right micrograph - magnified inset from WASP - / - synapse ) . Scale bars , 1μm . This experiment was repeated at least twice with similar results .

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Ultrastructural visualization of detergent extracted WT and WASP-/- T cell synaptic cytoskeleton using platinum replica electron microscopy (top images). Each image shows individual synapse, dark zone in the middle of the synapse represents cell nucleus. Scale bar, 2.5μm. The bottom left micrograph is the magnified inset from WT synapse and shows actin foci (identified as globular cluster of short filaments in the inset, marked with arrows)-dependent interconnected cytoskeletal architecture that is associated with synapse symmetry as predicted in the model, and is lacking in the WASP-/- cell synapse (bottom right micrograph- magnified inset from WASP-/- synapse). Scale bars, 1μm.This experiment was repeated at least twice with similar results.


Schematic of the cytoskeletal inhibitors and their targets used in the experiments . SIM imaging of mouse T cells activated for 5 ' on APS , then treated with the indicated inhibitors or vehicle control . Shown are the representative SIM images ( B ) , and quantification of actin foci ( from the phalloidin images ) , talin , or pCasL in the contact interface ( normalized to the mean value in the control case , C ) , and measured cell morphology aspect ratios ( D , E ) . Since CK666 treatment reduces F - actin intensity Kumari et al . , 2015 ( ) , the ' actin ' image of the CK666 treated cell in ( B ) was contrasted differently than the ' control ' and ' Blebb . ' cases for a clearer visualization of F - actin . p values , * P = 0 . 02 , * * P = 0 . 001 , n . s . > 0 . 05 . For the rest of the comparisons between the control and treatment cases , p < 0 . 0001 . For the bar graph in ( B ) , n for foci groups are 214 , 83 , 92 , 98 ; for pCasL groups are 100 , 68 , 91 , 92 ; and for Talin groups are 102 , 70 , 89 , 93 respectively . p value for the n . s . cases is > 0 . 05 , * P = 0 . 02 . In the foci , pCasL and talin groups , when not indicated , the p values are < 0 . 0001 using Mann - Whitney two - tailed test . Central values in the graphs represent Mean and error bars represent ± SEM . These experiments were repeated at least thrice with similar results . Scale bar , 5µm .

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Schematic of the cytoskeletal inhibitors and their targets used in the experiments. SIM imaging of mouse T cells activated for 5' on APS, then treated with the indicated inhibitors or vehicle control. Shown are the representative SIM images (B), and quantification of actin foci (from the phalloidin images), talin, or pCasL in the contact interface (normalized to the mean value in the control case, C), and measured cell morphology aspect ratios (D, E). Since CK666 treatment reduces F-actin intensity Kumari et al., 2015(), the 'actin' image of the CK666 treated cell in (B) was contrasted differently than the 'control' and 'Blebb.' cases for a clearer visualization of F-actin. p values, *P =0.02, **P =0.001, n.s. >0.05. For the rest of the comparisons between the control and treatment cases, p <0.0001. For the bar graph in (B), n for foci groups are 214, 83, 92, 98; for pCasL groups are 100, 68, 91, 92; and for Talin groups are 102, 70, 89, 93 respectively. p value for the n.s. cases is > 0.05, *P =0.02. In the foci, pCasL and talin groups, when not indicated, the p values are <0.0001 using Mann-Whitney two-tailed test. Central values in the graphs represent Mean and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5µm.


Simulation of synaptic F - actin network following localized myosin inactivation and inactivated myosin - mediated F - actin cross - linking within the area marked with dotted line in the left image . The graph shows the predicted alteration in mechanical stress profile , as myosin is inactivated crosslinked in the defined region ( marked by an arrow ) , ( G ) .

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Simulation of synaptic F-actin network following localized myosin inactivation and inactivated myosin-mediated F-actin cross-linking within the area marked with dotted line in the left image. The graph shows the predicted alteration in mechanical stress profile, as myosin is inactivated crosslinked in the defined region (marked by an arrow), (G).


Experimental confirmation of localized actomyosin crosslinking and symmetry breaking . LifeAct - GFP expressing T cells were incubated with activating substrates , treated with vehicle control or 1µM Azido - blebb and their contact interface imaged using low excitation laser power ( 0 . 5 mW ) , using a spinning disc confocal microscope ( H ) . The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido - Blebb and actomyosin network within the illuminated area ( shown with a box in the image ) . The deflection in center of mass ( COM ) within a minute was calculated and plotted for at least 10 cells in each case ( graphs in I ) . The left graphs in ( I ) show individual COM trajectory , and the graph on the right shows average COM movement of cells within a minute post photo - inactivation of myosinII . This experiment was repeated twice with similar results . Scale bar , 5µm .

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Experimental confirmation of localized actomyosin crosslinking and symmetry breaking. LifeAct-GFP expressing T cells were incubated with activating substrates, treated with vehicle control or 1µM Azido-blebb and their contact interface imaged using low excitation laser power (0.5 mW), using a spinning disc confocal microscope (H). The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido-Blebb and actomyosin network within the illuminated area (shown with a box in the image). The deflection in center of mass (COM) within a minute was calculated and plotted for at least 10 cells in each case (graphs in I). The left graphs in (I) show individual COM trajectory, and the graph on the right shows average COM movement of cells within a minute post photo-inactivation of myosinII. This experiment was repeated twice with similar results. Scale bar, 5µm.


T cells were activated using Alexa568 - 2C11 and ICAM1 reconstituted lipid bilayers for 5 min , fixed , stained with phalloidin and imaged using SIM . In the graph , n = 50 for WT and 61 for WASP - / - , P value , P * * * < 0 . 0001 , obtained using Mann - Whitney two - tailed test . The graphs in ( B ) show synaptic levels of indicated proteins , normalized to the mean levels of ' + WASP ' in each case . For the left graph , n = 48 , 46 , 52 , 46 respectively , and p value , P * = 0 . 03 . For the right graph , n = 50 for WT and n = 61 for WASP - / - in ' Actin ' , ' Foci ' , as well as ' pCasL ' cases . P values P * * * < 0 . 0001 , obtained using Mann - Whitney two - tailed test .

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T cells were activated using Alexa568-2C11 and ICAM1 reconstituted lipid bilayers for 5 min, fixed, stained with phalloidin and imaged using SIM. In the graph, n= 50 for WT and 61 for WASP-/-, P value, P***<0.0001, obtained using Mann-Whitney two-tailed test. The graphs in (B) show synaptic levels of indicated proteins, normalized to the mean levels of '+WASP' in each case. For the left graph, n= 48, 46, 52, 46 respectively, and p value, P *= 0.03. For the right graph, n= 50 for WT and n= 61 for WASP-/- in 'Actin', 'Foci', as well as 'pCasL' cases. P values P*** <0.0001, obtained using Mann-Whitney two-tailed test.


Synapse breaking behavior of T cells on hydrogels of indicated stiffness , covalently conjugated with 2C11 and ICAM1 . These experiments were repeated at least thrice with similar results . In the left graph , n for WT = 14 , for WASP - / - , n = 19 ; P * * = 0 . 0015 ; in the graph on the right , n for WT = 40 , n for WASP - / - = 36 , P * * * = 0 . 0006 , as assessed using Mann - Whitney two - tailed test .

Synapse breaking behavior of T cells on hydrogels of indicated stiffness, covalently conjugated with 2C11 and ICAM1. These experiments were repeated at least thrice with similar results. In the left graph, n for WT= 14, for WASP-/-, n= 19; P**= 0.0015; in the graph on the right, n for WT= 40, n for WASP-/-= 36, P***= 0.0006, as assessed using Mann-Whitney two-tailed test.


( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) together with GFP - RAB11 to visualize trafficking of recycling endosomes ( RE ) . At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport . Representative dendrite segments and kymographs of GFP - RAB11 vesicle movement . Quantitative analysis of vesicle motility ( B ) and total number ( C ) from kymographs .

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(A) Primary hippocampal neurons (DIV6+3) were transfected with shRNA targeting TDP-43 (shTDP) or control (shCtrl) together with GFP-RAB11 to visualize trafficking of recycling endosomes (RE). At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport. Representative dendrite segments and kymographs of GFP-RAB11 vesicle movement. Quantitative analysis of vesicle motility (B) and total number (C) from kymographs.