( 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 ) .

(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 .

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(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 .

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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 ) RNA ISH for circLMP2A . U6 and 18S are mainly localized in the nucleus and cytoplasm , respectively , and 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 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 .

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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 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 .

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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 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 .

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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 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.


( 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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

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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 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 .

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(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 .

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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 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 .

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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 .

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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.


( D ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . TDP - 43 levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 .

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(D) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. TDP-43 levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1.


( E ) iPSC - derived human neurons ( DIV7 + 3 ) were transduced with shTDP or shCtrl and GFP - RAB11 and at least three dendrite segments per neuron analyzed as in ( a ) .

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(E) iPSC-derived human neurons (DIV7+3) were transduced with shTDP or shCtrl and GFP-RAB11 and at least three dendrite segments per neuron analyzed as in (a).


Quantitative analysis of vesicle motility ( F ) and number ( G ) from kymographs . Mean + / - s . e . m . , n = 3 , unpaired two - tailed t - test : * denotes p < 0 . 05 , * * denotes p < 0 . 01 , * * * denotes p < 0 . 001 .

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Quantitative analysis of vesicle motility (F) and number (G) from kymographs. Mean +/- s.e.m., n = 3, unpaired two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 or a control together with tagRFP ( not depicted ) . Representative images of neurons subjected to Alexa - transferrin ( Alexa - Tf ) for 20 min ( pulse ) and chased for 0 , 20 or 60 min in complete media . Scale bar represents 100 µm . ( B ) Quantification of labeled cellular Alexa - Tf normalized to cell area ( using tagRFP ) after chase period . At least ten images per condition were analyzed per experiment in three independent experiments .

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(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 or a control together with tagRFP (not depicted). Representative images of neurons subjected to Alexa-transferrin (Alexa-Tf) for 20 min (pulse) and chased for 0, 20 or 60 min in complete media. Scale bar represents 100 µm. (B) Quantification of labeled cellular Alexa-Tf normalized to cell area (using tagRFP) after chase period. At least ten images per condition were analyzed per experiment in three independent experiments.


( C ) ELISA measurement of β2 - transferrin in CSF of control subjects ( n = 65 ) and sporadic ALS patients ( n = 55 ) , FTD - ALS patients ( n = 12 ) and behavioral variant FTD patients ( bvFTD , n = 16 ) .

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(C) ELISA measurement of β2-transferrin in CSF of control subjects (n=65) and sporadic ALS patients (n=55), FTD-ALS patients (n=12) and behavioral variant FTD patients (bvFTD, n=16).


( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected shTDP or shCtrl and GFP as transfection marker . After 3 days neurons were subjected to Alexa - Tf for 20 min and intracellular Alexa - Tf movement was imaged as in Figure 1 afterwards . Quantitative analysis of Alexa - Tf movement ( E ) and vesicle number ( F ) from kymographs .

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(D) Primary hippocampal neurons (DIV6+3) were transfected shTDP or shCtrl and GFP as transfection marker. After 3 days neurons were subjected to Alexa-Tf for 20 min and intracellular Alexa-Tf movement was imaged as in Figure 1 afterwards. Quantitative analysis of Alexa-Tf movement (E) and vesicle number (F) from kymographs.


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transfected with myc - tagged TDP - 43 wild - type or a mutant lacking the nuclear localization signal ( ΔNLS ) or an empty vector control . Immunostaining with the indicated antibodies and DAPI to label nuclei . Scale bar represents 50 µm .

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(A) Primary hippocampal neurons (DIV6+4) were transfected with myc-tagged TDP-43 wild-type or a mutant lacking the nuclear localization signal (ΔNLS) or an empty vector control. Immunostaining with the indicated antibodies and DAPI to label nuclei. Scale bar represents 50 µm.


( B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either TDP - 43 wild - type , TDP - 43ΔNLS or an empty vector control together with GFP - RAB11 to visualize recycling endosomes and analyzed as in Figure 1 . Quantitative analysis of recycling endosome movement ( C ) and vesicle number ( D ) .

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(B) Primary hippocampal neurons (DIV6+3) were transfected with either TDP-43 wild-type, TDP-43ΔNLS or an empty vector control together with GFP-RAB11 to visualize recycling endosomes and analyzed as in Figure 1. Quantitative analysis of recycling endosome movement (C) and vesicle number (D).


( A ) Primary cortical neurons were transduced with shTDP or control ( DIV2 + 7 ) and subjected to quantitative proteomic analysis . Volcano plot shows up and down regulated proteins from three biological replicates . Significant changes are colored . Compare Table EV1 .

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(A) Primary cortical neurons were transduced with shTDP or control (DIV2+7) and subjected to quantitative proteomic analysis. Volcano plot shows up and down regulated proteins from three biological replicates. Significant changes are colored. Compare Table EV1.


( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . Immunoblots with the indicated antibodies ( C ) . Quantification of VPS4B and TDP - 43 protein level using densitometry ( n = 4 ) . Expression was normalized to the housekeeping gene β - actin ( D ) .

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(C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. Immunoblots with the indicated antibodies (C). Quantification of VPS4B and TDP-43 protein level using densitometry (n = 4). Expression was normalized to the housekeeping gene β-actin (D).


( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . VPS4B mRNA level were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping genes YWHAZ and PGK1 ( E ) .

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(C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. VPS4B mRNA level were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping genes YWHAZ and PGK1 (E).


( F ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either shTDP or shCtrl and iRFP670 . Immunostaining with the indicated antibodies . Scale bar represents 50 µm .

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(F) Primary hippocampal neurons (DIV6+3) were transfected with either shTDP or shCtrl and iRFP670. Immunostaining with the indicated antibodies. Scale bar represents 50 µm.


( G ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . VPS4B levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 ( n = 3 ) .

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(G) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. VPS4B levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1 (n = 3).


( A - B ) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP - 43 . ( B ) HEK293 cells were transfected with shRNAs targeting human TDP - 43 , a control shRNA , TDP - 43 wild - type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter . VPS4B promoter - driven renilla luciferase activity was normalized to TK promoter - driven firefly luciferase . Quantification from six independent experiments .

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

(A-B) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP-43. (B) HEK293 cells were transfected with shRNAs targeting human TDP-43, a control shRNA, TDP-43 wild-type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter. VPS4B promoter-driven renilla luciferase activity was normalized to TK promoter-driven firefly luciferase. Quantification from six independent experiments.


( C - D ) ChIP assay to analyze binding of TDP - 43 to VPS4B promoter region in rat cortical neurons , HEK293 cells and human brain tissue . PCR reaction from input , negative control and TDP - 43 immunoprecipitates . Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input .

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(C-D) ChIP assay to analyze binding of TDP-43 to VPS4B promoter region in rat cortical neurons, HEK293 cells and human brain tissue. PCR reaction from input, negative control and TDP-43 immunoprecipitates. Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input.


( E - H ) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD / ALS - TDP and five healthy controls . ( E , G ) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord . Scale bar represents 50 µm . ( F , H ) Quantification of cellular VPS4B levels . At least 50 cells per case were analyzed .

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(E-H) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD/ALS-TDP and five healthy controls. (E, G) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord. Scale bar represents 50 µm. (F, H) Quantification of cellular VPS4B levels. At least 50 cells per case were analyzed.


( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either VPS4B or an empty vector control together with GFP - RAB11 to visualize recycling endosomes . Neurons were imaged as in Figure 1 to analyze recycling endosome transport . Quantitative analysis of vesicle motility ( B ) and number ( C ) from kymographs ( n = 4 ) .

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(A) Primary hippocampal neurons (DIV6+3) were transfected with either VPS4B or an empty vector control together with GFP-RAB11 to visualize recycling endosomes. Neurons were imaged as in Figure 1 to analyze recycling endosome transport. Quantitative analysis of vesicle motility (B) and number (C) from kymographs (n = 4).


( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with the indicated combinations of shRNA targeting TDP - 43 , VPS4B and control together with GFP - RAB11 and imaged as in Figure 1A . Quantitative analysis of vesicle motility ( E ) and number ( F ) from kymographs ( n = 3 ) .

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(D) Primary hippocampal neurons (DIV6+3) were transfected with the indicated combinations of shRNA targeting TDP-43, VPS4B and control together with GFP-RAB11 and imaged as in Figure 1A. Quantitative analysis of vesicle motility (E) and number (F) from kymographs (n=3).


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification . Volcano plot depicts proteins that are significantly ( p < 0 . 01 , t - test , Benjamini - Hochberg , FDR : 0 . 05 ) decreased or increased on the cell surface in three independent experiments with two technical replicates . For full dataset see Table EV2 .

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(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 (shTDP) or control (shCtrl) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification. Volcano plot depicts proteins that are significantly (p < 0.01, t-test, Benjamini-Hochberg, FDR: 0.05) decreased or increased on the cell surface in three independent experiments with two technical replicates. For full dataset see Table EV2.


( B - C ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the ErbB4 ligand NRG1 ( 10 nM ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( B ) . Quantification of total and phosho - ERBB4 ( Y1056 ) using densitometry 3 - 7 biological replicates ( C ) .

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(B-C) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the ErbB4 ligand NRG1 (10 nM) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (B). Quantification of total and phosho-ERBB4 (Y1056) using densitometry 3-7 biological replicates (C).


( D - E ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the FGFR1 ligand FGF1 ( 10 ng / ml ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( D ) . Quantification of total and phosho - FGFR1 ( Y654 ) using densitometry 3 - 7 biological replicates ( E ) .

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(D-E) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the FGFR1 ligand FGF1 (10 ng/ml) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (D). Quantification of total and phosho-FGFR1 (Y654) using densitometry 3-7 biological replicates (E).


( F ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shTDP or shCtrl . Gene expression was analyzed by quantitative RT - PCR normalized to the housekeeping genes YWHAZ and GAPDH ( n = 3 )

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

(F) Primary hippocampal neurons (DIV6+4) were transduced with shTDP or shCtrl. Gene expression was analyzed by quantitative RT-PCR normalized to the housekeeping genes YWHAZ and GAPDH (n=3)


( G ) Hippocampal neurons were transfected with ErbB4 containing a luminal HA - tag ( HA / T - ErbB4 ) and shCtrl or shTDP ( DIV9 + 3 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with HA - antibodies . ( H ) Quantification of HA / T - ErbB4 surface levels . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 50 µm .

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(G) Hippocampal neurons were transfected with ErbB4 containing a luminal HA-tag (HA/T-ErbB4) and shCtrl or shTDP (DIV9+3). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with HA-antibodies. (H) Quantification of HA/T-ErbB4 surface levels. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.


( A , B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shCtrl or shTDP and ErbB4 containing an N - terminal HA epitope tag and followed by thrombin cleavage site ( HA / T - ErbB4 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with two different HA - antibodies . After removing the HA - signal of surface ErbB4 ( 0 min ) using thrombin cleavage ( 5min , 1U / ml ) membrane insertion of HA / T - ErbB4 was measured at the indicated time points . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 50 µm .

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

(A,B) Primary hippocampal neurons (DIV6+3) were transfected with shCtrl or shTDP and ErbB4 containing an N-terminal HA epitope tag and followed by thrombin cleavage site (HA/T-ErbB4). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with two different HA-antibodies. After removing the HA-signal of surface ErbB4 (0 min) using thrombin cleavage (5min, 1U/ml) membrane insertion of HA/T-ErbB4 was measured at the indicated time points. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( A , B ) TDP - 43 knockdown ( DIV6 + 5 ) significantly reduces dendrite branching compared to control : at 25 , 37 . 5 and 87 . 5 µm radius p < 0 . 05 , at 50 µm p < 0 . 01 and from 62 . 5 to 75 µm p < 0 . 001 .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (A, B) TDP-43 knockdown (DIV6+5) significantly reduces dendrite branching compared to control: at 25, 37.5 and 87.5 µm radius p < 0.05, at 50 µm p < 0.01 and from 62.5 to 75 µm p < 0.001.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( C , D ) VPS4B transfection ( DIV6 + 4 ) significantly reduces dendrite complexity : at 25 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 and from 87 . 5 to 100 µm p < 0 . 01 .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (C, D) VPS4B transfection (DIV6+4) significantly reduces dendrite complexity: at 25 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001 and from 87.5 to 100 µm p < 0.01.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( E , F ) Two days after shRNA transfection neurons were treated with 1 nM NRG1 - beta 1 ( NRG1 ) or vehicle and analyzed at DIV6 + 5 . shCtrl vs . shTDP : at 25 and 87 . 5 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 . shCtrl vs . shCtrl + NRG1 : from 37 . 5 to 62 . 5 µm p < 0 . 05 . shTDP vs . shTDP + NRG1 : no significant difference .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (E, F) Two days after shRNA transfection neurons were treated with 1 nM NRG1-beta 1 (NRG1) or vehicle and analyzed at DIV6+5. shCtrl vs. shTDP: at 25 and 87.5 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001. shCtrl vs. shCtrl + NRG1: from 37.5 to 62.5 µm p < 0.05. shTDP vs. shTDP+NRG1: no significant difference.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( G , H ) Transfection of the indicated combinations of shCtrl , shTDP , ErbB4 or vector control ( DIV6 + 5 ) . shCtrl + Ctrl vs . shTDP + Ctrl : from 25 to 75 µm radius p < 0 . 001 , at 87 and 112 . 5 µm p < 0 . 01 and at 100 µm p < 0 . 05 . shTDP + Ctrl vs . shTDP + ErbB4 : from 25 to 100 µm radius p < 0 . 001 , at 112 . 5 µm p < 0 . 01 . shCtrl + Ctrl vs . shTDP + ErbB4 : no significant differences .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (G, H) Transfection of the indicated combinations of shCtrl, shTDP, ErbB4 or vector control (DIV6+5). shCtrl + Ctrl vs. shTDP + Ctrl: from 25 to 75 µm radius p < 0.001, at 87 and 112.5 µm p < 0.01 and at 100 µm p < 0.05. shTDP + Ctrl vs. shTDP + ErbB4: from 25 to 100 µm radius p < 0.001, at 112.5 µm p < 0.01. shCtrl + Ctrl vs. shTDP + ErbB4: no significant differences.


Confocal z - projections of the indicated genotypes and age , labeled with membrane bound GFP ( mCD8 - GFP ; CD8 ) driven by the γ - specific Gal4 driver GMR71G10 - Gal4 ( γ - Gal4 ) . While γ - axons of control flies project through the entire lobe ( C is the RNAi control ; n = 12 / 12 , E ; is the tsCRISPR control ; n = 14 / 14 ) , knockdown of dpr12 by RNAi ( D ; n = 12 / 12 ) or knockout by tsCRISPR ( F ; n = 14 / 14 ) resulted in short axons . At L3 , γ - axons in dpr12 ∆ 50 - 81 homozygotus mutant animals ( K ; n = 20 / 20 ) resemble WT γ - axons ( G ; n = 20 / 20 ) . At 48hr APF , γ - axons normally re - extend to form the adult lobe ( H ; n = 12 / 12 ) . dpr12 ∆ 50 - 81 γ - axons ( L ; n = 14 / 14 ) fail to extend to the end of the lobe . This defect persists to adult ( I ; n = 11 / 11 vs . M ; n = 18 / 18 ) . Expressing a UAS - Dpr12 transgene within γ - KCs in dpr12 ∆ 50 - 81 homozygote mutant animals rescued the axon regrowth defect ( N ; n = 23 / 24 , J ; n = 14 / 14 ) . The adult γ - lobe and α / β lobes are outlined in ( C , D ) in yellow and orange , respectively , for clarity . Asterisks demarcate the distal part of the lobe . Green and white indicate mCD8 - GFP . Magenta represents FasII staining . Scale bar is 20 µm .

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Confocal z-projections of the indicated genotypes and age, labeled with membrane bound GFP (mCD8-GFP; CD8) driven by the γ-specific Gal4 driver GMR71G10-Gal4 (γ-Gal4). While γ-axons of control flies project through the entire lobe (C is the RNAi control; n=12/12, E; is the tsCRISPR control; n=14/14), knockdown of dpr12 by RNAi (D; n=12/12) or knockout by tsCRISPR (F; n=14/14) resulted in short axons. At L3, γ-axons in dpr12∆50-81 homozygotus mutant animals (K; n=20/20) resemble WT γ-axons (G; n=20/20). At 48hr APF, γ-axons normally re-extend to form the adult lobe (H; n=12/12). dpr12∆50-81 γ-axons (L; n=14/14) fail to extend to the end of the lobe. This defect persists to adult (I; n=11/11 vs. M; n=18/18). Expressing a UAS-Dpr12 transgene within γ-KCs in dpr12∆50-81 homozygote mutant animals rescued the axon regrowth defect (N; n=23/24, J; n=14/14). The adult γ-lobe and α/β lobes are outlined in (C, D) in yellow and orange, respectively, for clarity. Asterisks demarcate the distal part of the lobe. Green and white indicate mCD8-GFP. Magenta represents FasII staining. Scale bar is 20 µm.