Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1 ‐ CreERT2 ( left lane ) or mb1 ‐ CreERT2 ; Sykfl / fl ( right lane ) mice both treated with Tam as described ; blots were probed with anti ‐ Syk and anti ‐ GAPDH Ab , GAPDH being used as a loading control .

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Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl (right lane) mice both treated with Tam as described; blots were probed with anti‐Syk and anti‐GAPDH Ab, GAPDH being used as a loading control.


Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk ‐ deficient and control mice .

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Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk‐deficient and control mice.


Genomic DNA analysis of Tam ‐ treated mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / flmice ( as indicated ) . Upper row : amplification of floxed ( fl ) and wt ( + ) alleles . Middle row : amplification of the deleted ( d ) allele . Lower row : loading control ( TC21 , a gene unaffected by the deletion of Syk ) . Established control DNA was used to visualize the individual bands ( as indicated ) .

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Genomic DNA analysis of Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/flmice (as indicated). Upper row: amplification of floxed (fl) and wt (+) alleles. Middle row: amplification of the deleted (d) allele. Lower row: loading control (TC21, a gene unaffected by the deletion of Syk). Established control DNA was used to visualize the individual bands (as indicated).


A , B Flow cytometric analysis of B cells from ( A ) the BM and ( B ) the SP of mb1 ‐ CreERT2 control ( left ) and mb1 ‐ CreERT2 ; Sykfl / flmice ( right ) treated with Tam as described in the Materials and Methods section . The BM cells were stained with anti ‐ IgM and anti ‐ B220 , and the SP cells with anti ‐ CD19 and anti ‐ CD93 or anti ‐ CD23 and anti ‐ CD21 . The gated regions in the dot blots correspond to individual B ‐ cell populations : ( A ) Bone marrow : gate P ( B220 + IgM − ) pro ‐ / pre ‐ B cells , gate I ( B220loIgM + ) immature B cells , gate M ( B220hiIgM + ) mature B cells ; ( B ) spleen : gate T ( CD19 + CD93 + ) transitional B cells ( T ) , gate M ( CD19 + CD93 − ) mature B cells ; in CD93 − splenic B cells , gate M ( CD23hiCD21int CD93 − ) mature follicular B cells and gate MZ ( CD23lo CD21hi CD93 − ) marginal zone B cells . The numbers in the dot plots indicate the mean relative frequency of cells in the gate . C - E Statistical analysis of absolute cell numbers per Tam ‐ treated mouse : filled circles indicating cells obtained from mb1 ‐ CreERT2 control mice and open circles from mb1 ‐ CreERT2 ; Sykfl / flmice . ( C ) Statistical analysis of the absolute cell numbers of pro ‐ / pre ‐ , immature and mature recirculating B cells in the BM . ( D ) Absolute cell numbers of transitional , M and MZ B cells found in the SP . ( E ) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1 ‐ a and B1 ‐ b . An asterisk ( * ) marks statistically significant differences ( P 0 . 05 ) , two asterisks ( * * ) indicate P 0 . 01 , P ‐ values were obtained using two ‐ tailed Student ' s t ‐ test . Cell numbers of four to five mice per group are shown , with each dot representing an individual animal .

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A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/flmice (right) treated with Tam as described in the Materials and Methods section. The BM cells were stained with anti‐IgM and anti‐B220, and the SP cells with anti‐CD19 and anti‐CD93 or anti‐CD23 and anti‐CD21. The gated regions in the dot blots correspond to individual B‐cell populations: (A) Bone marrow: gate P (B220+IgM−) pro‐/pre‐B cells, gate I (B220loIgM+) immature B cells, gate M (B220hiIgM+) mature B cells; (B) spleen: gate T (CD19+ CD93+) transitional B cells (T), gate M (CD19+ CD93−) mature B cells; in CD93−splenic B cells, gate M (CD23hiCD21int CD93−) mature follicular B cells and gate MZ (CD23lo CD21hi CD93−) marginal zone B cells. The numbers in the dot plots indicate the mean relative frequency of cells in the gate.C-E Statistical analysis of absolute cell numbers per Tam‐treated mouse: filled circles indicating cells obtained from mb1‐CreERT2 control mice and open circles from mb1‐CreERT2;Sykfl/flmice. (C) Statistical analysis of the absolute cell numbers of pro‐/pre‐, immature and mature recirculating B cells in the BM. (D) Absolute cell numbers of transitional, M and MZ B cells found in the SP. (E) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1‐a and B1‐b. An asterisk (*) marks statistically significant differences (P 0.05), two asterisks (**) indicate P 0.01, P‐values were obtained using two‐tailed Student's t‐test. Cell numbers of four to five mice per group are shown, with each dot representing an individual animal.


A Flow cytometric analysis of IgM , CD19 and CD23 expression of splenic B cells from Tam ‐ treated mb1 ‐ CreERT2 control ( dashed line ) and mb1 ‐ CreERT2 ; Sykfl / flmice ( solid line ) .

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A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/flmice (solid line).


B Flow cytometric analysis of tyrosine phosphorylation ( pY ) in splenic B cells from Tam ‐ treated mb1 ‐ CreERT2 and mb1 ‐ CreERT2 ; Sykfl / flmice stimulated with medium ( dotted line ) or 50 μM pervanadate ( dashed and solid lines ) .

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B Flow cytometric analysis of tyrosine phosphorylation (pY) in splenic B cells from Tam‐treated mb1‐CreERT2 and mb1‐CreERT2;Sykfl/flmice stimulated with medium (dotted line) or 50 μM pervanadate (dashed and solid lines).


C , D Flow cytometric analysis of the intracellular Ca2 + influx in purified splenic B cells derived from Tam ‐ treated mice . mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / fl were treated with ( C ) 10 μg / ml anti ‐ Kappa F ( ab ′ ) 2 fragments or ( D ) 1 μM ionomycin .

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C, D Flow cytometric analysis of the intracellular Ca2+ influx in purified splenic B cells derived from Tam‐treated mice. mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl were treated with (C) 10 μg/ml anti‐Kappa F(ab′)2 fragments or (D) 1 μM ionomycin.


Ex vivo activation assay of splenic mature B cells from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . Purified splenic B cells were either left unstimulated ( medium ) or were stimulated with the indicated stimuli . The cells were analyzed by flow cytometry after 24 h . Shown are CD19 versus CD86 dot plots . 7 ‐ AAD was included to distinguish dead from viable cells . The numbers in the quadrants indicate the relative frequencies of cells in the gate .

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Ex vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). Purified splenic B cells were either left unstimulated (medium) or were stimulated with the indicated stimuli. The cells were analyzed by flow cytometry after 24 h. Shown are CD19 versus CD86 dot plots. 7‐AAD was included to distinguish dead from viable cells. The numbers in the quadrants indicate the relative frequencies of cells in the gate.


Ex vivo proliferation assay with mature B cells from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . The cells were incubated with the indicated stimuli for 90 h .

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

Ex vivo proliferation assay with mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). The cells were incubated with the indicated stimuli for 90 h.


A Flow cytometric analysis of splenic B cells from Rag2 − / − ; γc − / − mice . CD19 versus CD3ε dot plots are shown . The mice were injected i . v . with 5 × 106 splenic ( CD19 + CD93 − ) B cells and ( CD3ε + ) T cells from Tam ‐ untreated mb1 ‐ CreERT2 control or mb1 ‐ CreERT2 ; Sykfl / fl mice . The recipient mice were treated with Tam beginning 1 day after transfer as described in the Materials and Methods section . B Quantitative analysis of B and T cells from Rag2 − / − ; γc − / − mice repopulated with mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / fl splenocytes . Each symbol represents an individual mouse . Filled circles and squares represent Syk + / + B and T cells , respectively ; open circles and squares represent Sykfl / fl B and T cells .

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A Flow cytometric analysis of splenic B cells from Rag2−/−;γc−/− mice. CD19 versus CD3ε dot plots are shown. The mice were injected i.v. with 5 × 106 splenic (CD19+ CD93−) B cells and (CD3ε+) T cells from Tam‐untreated mb1‐CreERT2 control or mb1‐CreERT2;Sykfl/fl mice. The recipient mice were treated with Tam beginning 1 day after transfer as described in the Materials and Methods section.B Quantitative analysis of B and T cells from Rag2−/−;γc−/− mice repopulated with mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl splenocytes. Each symbol represents an individual mouse. Filled circles and squares represent Syk+/+ B and T cells, respectively; open circles and squares represent Sykfl/fl B and T cells.


A . AICD was induced in hPBT cells as described in Materials and methods . Apoptotic cells were detected at indicated times after AICD induction by flow cytometry as AnnexinV / PI double positive cells and the ratio between AICD and Ctrl values obtained are shown . Data represent mean ± SE of six independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. AICD was induced in hPBT cells as described in Materials and methods. Apoptotic cells were detected at indicated times after AICD induction by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown. Data represent mean ± SE of six independent experiments.


B . AICD was induced in Jurkat cells as described in Materials and methods . Apoptotic cells were detected as in ( A ) . Data represent mean ± SE of five independent experiments .

O O O O O O O O O O O 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. AICD was induced in Jurkat cells as described in Materials and methods. Apoptotic cells were detected as in (A). Data represent mean ± SE of five independent experiments.


C . hPBT cells were transfected with mtYFP and after 24h AICD was induced . Representative reconstructions of confocal z - stacks of the mtYFP fluorescence 30 ' after AICD induction is shown . Scale bar , 5μm .

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

C. hPBT cells were transfected with mtYFP and after 24h AICD was induced. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 30' after AICD induction is shown. Scale bar, 5μm.


D . Morphometric analysis of mitochondrial shape of cells treated as in ( B ) . Data represent mean ± SE of five independent experiments ( n = 30 cells per condition in each experiment ) .

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

D. Morphometric analysis of mitochondrial shape of cells treated as in (B). Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


E . Jurkat cells were transfected with mtYFP , 24h after AICD induction cells were fixed . Representative reconstructions of confocal z - stacks of the mtYFP fluorescence 24h after AICD induction are shown . Scale bar , 5μm . F . Morphometric analysis of mitochondrial shape in cells treated as in ( E ) is shown . Data represent mean ± SE of five independent experiments ( n = 30 cells per condition in each experiment ) .

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E. Jurkat cells were transfected with mtYFP, 24h after AICD induction cells were fixed. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.F. Morphometric analysis of mitochondrial shape in cells treated as in (E) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


G . Representative electron micrographs of hPBT cells treated as indicated . 30min after AICD induction cells were fixed and processed for electron microscopy . Scale bar , 2μm . H . 24h after AICD induction Jurkat cells were fixed and processed for electron microscopy . Representative electron micrographs of cells treated as indicated are shown . In EM images white arrowheads point to autophagic vesicles ( late autophagosomes or most likely autophagolysosomes , Aφ ) ; black arrows point to mitochondria ( mito ) with remodelled cristae , specifically touching a representative disorganized crista . Scale bar , 2μm .

G. Representative electron micrographs of hPBT cells treated as indicated. 30min after AICD induction cells were fixed and processed for electron microscopy. Scale bar, 2μm.H. 24h after AICD induction Jurkat cells were fixed and processed for electron microscopy. Representative electron micrographs of cells treated as indicated are shown. In EM images white arrowheads point to autophagic vesicles (late autophagosomes or most likely autophagolysosomes, Aφ); black arrows point to mitochondria (mito) with remodelled cristae, specifically touching a representative disorganized crista. Scale bar, 2μm.


I . Representative confocal microscopy images of hPBT cells transfected with mtYFP , fixed 30 ' after AICD induction and immunostained with an anti - cytochrome c antibody ( red ) . J . Cytochrome c localization index was calculated from 30 randomly selected cells treated as in ( I ) . Data represent mean ± SE of three independent experiments . K . Representative confocal images of Jurkat cells transfected with mtYFP , fixed at 32h after AICD induction and immunostained with an anti - cytochrome c antibody . L . Cytochrome c localization index was calculated from 30 randomly selected images per condition . Data represent mean ± SE of five independent experiments .

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I. Representative confocal microscopy images of hPBT cells transfected with mtYFP, fixed 30' after AICD induction and immunostained with an anti-cytochrome c antibody (red).J. Cytochrome c localization index was calculated from 30 randomly selected cells treated as in (I). Data represent mean ± SE of three independent experiments.K. Representative confocal images of Jurkat cells transfected with mtYFP, fixed at 32h after AICD induction and immunostained with an anti-cytochrome c antibody.L. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of five independent experiments.


A . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGEand immunoblotted with the indicated antibodies . WB are representative of at least three independent experiments .

O O O O O O O O O O O O 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. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGEand immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.


B . Jurkat cells were transfected with pE - GFP and the indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as the percentage of GFP - positive , annexin - V - PE - negative cells . Data represent mean ± SE of five independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Jurkat cells were transfected with pE-GFP and the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of five independent experiments.


C . Jurkat cells were transfected as indicated . Representative reconstructions of confocal z - stacks of the mtYFP fluorescence 24h after AICD induction are shown . Scale bar , 5μm . D . Morphometric analysis of mitochondrial shape of cells transfected as in ( C ) is shown . Data represent mean ± SE of five independent experiments ( n = 30 cells per condition in each experiment ) .

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C. Jurkat cells were transfected as indicated. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.D. Morphometric analysis of mitochondrial shape of cells transfected as in (C) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


E . AICD was induced in Jurkat cells over expressing pE - GFP and the indicated plasmids and sorted for GFP positive fluorescence . 24h after AICD induction cells were fixed and processed for electron microscopy . Representative electron micrographs of randomly selected mitochondria from cells treated as indicated are shown . White arrow points to tight cristae structure , black arrow points to remodelled cristae and to a swollen cristae junction . Scale bar , 0 . 2μm . F . Morphometric analysis of cristae width in cells transfected as in ( E ) .

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E. AICD was induced in Jurkat cells over expressing pE-GFP and the indicated plasmids and sorted for GFP positive fluorescence. 24h after AICD induction cells were fixed and processed for electron microscopy. Representative electron micrographs of randomly selected mitochondria from cells treated as indicated are shown. White arrow points to tight cristae structure, black arrow points to remodelled cristae and to a swollen cristae junction. Scale bar, 0.2μm.F. Morphometric analysis of cristae width in cells transfected as in (E).


G . Representative confocal microscopy images of cells transfected as indicated , fixed 32h after AICD induction and immunostained with an anti - cytochrome c antibody ( red ) . H . Cytochrome c localization index was calculated from 30 randomly selected cells ( per condition ) transfected as in ( G ) . Data represent mean ± SE of five independent experiments .

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G. Representative confocal microscopy images of cells transfected as indicated, fixed 32h after AICD induction and immunostained with an anti-cytochrome c antibody (red).H. Cytochrome c localization index was calculated from 30 randomly selected cells (per condition) transfected as in (G). Data represent mean ± SE of five independent experiments.


A . Representative confocal microscopy images of cells transfected GFP - LC3 and Parkin - Cherry , treated as indicated , fixed 24h after AICD induction and immunostained with anti - TOM20 antibody are shown . Scale bar , 5μm .

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

A. Representative confocal microscopy images of cells transfected GFP-LC3 and Parkin-Cherry, treated as indicated, fixed 24h after AICD induction and immunostained with anti-TOM20 antibody are shown. Scale bar, 5μm.


B . Statistical analysis of Parkin localization in cells subjected to AICD or to 10μM CCCP for the indicated times is shown . Data represent mean ± SE of three independent experiments ( n = 30 cells per condition in each experiment ) .

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

B. Statistical analysis of Parkin localization in cells subjected to AICD or to 10μM CCCP for the indicated times is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).


C . Cells were treated as in ( A ) . Where indicated , lysosomal proteases inhibitors E64D and PepA were added . LC3 - Parkin - mitochondria co - localization was calculated from 30 randomly selected cells per condition . Data represent mean ± SE of three independent experiments . Scale bar ,

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C. Cells were treated as in (A). Where indicated, lysosomal proteases inhibitors E64D and PepA were added. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected cells per condition. Data represent mean ± SE of three independent experiments. Scale bar, 5μm


D . hPBT cells were transfected with LC3 - cherry and treated as indicated . 30min after AICD induction cells were fixed and stained with TUNEL and DAPI . Representative reconstructions of confocal z - stacks of the LC3 - Cherry fluorescence and single stacks for DAPI are shown . Scale bar , 5μm . E . Statistical analysis of the autophagy in cells treated as in ( D ) is shown . Data represent mean ± SE of three independent experiments ( n = 20 cells per condition in each experiment ) .

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D. hPBT cells were transfected with LC3-cherry and treated as indicated. 30min after AICD induction cells were fixed and stained with TUNEL and DAPI. Representative reconstructions of confocal z-stacks of the LC3-Cherry fluorescence and single stacks for DAPI are shown. Scale bar, 5μm.E. Statistical analysis of the autophagy in cells treated as in (D) is shown. Data represent mean ± SE of three independent experiments (n = 20 cells per condition in each experiment).


F . Jurkat cells were transfected with GFP - LC3 and treated as indicated . Representative reconstructions of confocal z - stacks of the GFP - LC3 fluorescence 16h after AICD induction are shown . Scale bar , 5μm . G . Statistical analysis of the autophagic flux in cells treated as in ( F ) is shown . Data represent mean ± SE of three independent experiments ( n = 30 cells per condition in each experiment ) .

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F. Jurkat cells were transfected with GFP-LC3 and treated as indicated. Representative reconstructions of confocal z-stacks of the GFP-LC3 fluorescence 16h after AICD induction are shown. Scale bar, 5μm.G. Statistical analysis of the autophagic flux in cells treated as in (F) is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).


H . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Where indicated , lysosomal proteases inhibitors E64D and PepA were added . WB are representative of at least three independent experiments .

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

H. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated, lysosomal proteases inhibitors E64D and PepA were added. WB are representative of at least three independent experiments.


A - B . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Wb are representative of at least four independent experiments . Levels of proteins and phosphorylation status of AMPK were quantified and analyzed as shown . In these panels , Time states for " time , hours , after AICD treatment " and indicates at which time upon AICD induction samples have been collected .

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A-B. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Wb are representative of at least four independent experiments. Levels of proteins and phosphorylation status of AMPK were quantified and analyzed as shown. In these panels, Time states for "time, hours, after AICD treatment" and indicates at which time upon AICD induction samples have been collected.


C . Jurkat cells were transfected with the indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as the percentage of GFP - positive , annexin - V - PE - negative cells . Data represent mean ± SE of three independent experiments .

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

C. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.


D . Protein samples from Jurkat cells over expressing empty vector or AMPKS173A - S485A were immunoblotted for LC3 and LC3 - II / Actin ratio was quantified and analyzed as shown . Data represent mean ± SE of three independent experiments .

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

D. Protein samples from Jurkat cells over expressing empty vector or AMPKS173A-S485A were immunoblotted for LC3 and LC3-II/Actin ratio was quantified and analyzed as shown. Data represent mean ± SE of three independent experiments.


E - F . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . WB are representative of at least three independent experiments . Levels of proteins and phosphorylation status of LC3 were quantified and analyzed as shown .

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E-F. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments. Levels of proteins and phosphorylation status of LC3 were quantified and analyzed as shown.


G . Jurkat cells were transfected with the indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as described above .

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G. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as described above.


H - I . Cells where treated as in ( A ) and where indicated 10μM H89 was added . Quantitative analysis of P - AMPK Ser485 - 491 phosphorylation ( H ) and of LC3 - II / LC3 - I ratio ( I ) during AICD are shown .

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H-I. Cells where treated as in (A) and where indicated 10μM H89 was added. Quantitative analysis of P-AMPK Ser485-491 phosphorylation (H) and of LC3-II/LC3-I ratio (I) during AICD are shown.


J - K . AICD was induced in Jurkat cells ( J ) and in hPBT cells ( K ) in presence of 10μM H89 . At indicated time points ( hours ) after AICD induction , apoptotic cells were detected by flow cytometry as AnnexinV / PI double positive cells and the ratio between AICD and Ctrl values obtained are shown .

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J-K. AICD was induced in Jurkat cells (J) and in hPBT cells (K) in presence of 10μM H89. At indicated time points (hours) after AICD induction, apoptotic cells were detected by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown.


A . Apoptotic cells were detected at indicated time points after AICD induction in hPBT cells by flow cytometry as AnnexinV / PI double positive cells . Where indicated hPBT cells were pre - incubated with 100nM rapamycin for 24h before AICD induction . Data represent mean ± SE of six independent experiments .

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A. Apoptotic cells were detected at indicated time points after AICD induction in hPBT cells by flow cytometry as AnnexinV/PI double positive cells. Where indicated hPBT cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Data represent mean ± SE of six independent experiments.


B . AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction . Apoptotic cell death analysis was carried out as in ( A ) , except that cells were analysed 32h after AICD induction . Data represent mean ± SE of six independent experiments .

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B. AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of six independent experiments.


C . Morphometric analysis of mitochondrial shape 24h after AICD induction . Jurkat cells were transfected with mtYFP 24h before AICD induction and , where indicated , 100nM rapamycin was added . Data represent mean ± SE of four independent experiments ( n = 30 cells per condition in each experiment ) .

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C. Morphometric analysis of mitochondrial shape 24h after AICD induction. Jurkat cells were transfected with mtYFP 24h before AICD induction and, where indicated, 100nM rapamycin was added. Data represent mean ± SE of four independent experiments (n = 30 cells per condition in each experiment).


D . Representative electron micrographs of cells subjected to AICD untreated ( upper panel ) or treated with 100nM rapamycin ( lower panel ) . 24h after AICD induction cells were fixed and processed for electron microscopy , Scale bar , 2μm .

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D. Representative electron micrographs of cells subjected to AICD untreated (upper panel) or treated with 100nM rapamycin (lower panel). 24h after AICD induction cells were fixed and processed for electron microscopy, Scale bar, 2μm.


E . Jurkat cells were transfected with mtYFP and treated as indicated . 32h after AICD induction cells were fixed and immunostained with an anti - cytochrome c antibody . Cytochrome c localization index was calculated from 30 randomly selected images per condition . Data represent mean ± SE of four independent experiments .

E. Jurkat cells were transfected with mtYFP and treated as indicated. 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of four independent experiments.


F . Jurkat cells were transfected with DRP1S637A - GFP , 24h after transfection AICD was induced . 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP - positive , annexin - V - PE - negative cells . Data represent mean ± SE of three independent experiments .

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F. Jurkat cells were transfected with DRP1S637A-GFP, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.


G - H . Jurkat cells were co - transfected with mt - RFP and the indicated plasmids , 32h after AICD induction cells were fixed and immunostained with an anti - cytochrome c antibody . Cytochrome c localization index ( G ) and mitochondrial morphology ( H ) were calculated from 30 randomly selected images . Data represent mean ± SE of three independent experiments .

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G-H. Jurkat cells were co-transfected with mt-RFP and the indicated plasmids, 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index (G) and mitochondrial morphology (H) were calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments.


I . Jurkat cells were transfected with DRP1K38A , 24h after transfection AICD was induced . 32h after AICD induction viability was determined cytofluorimetrically as the percentage AnnexinV / PI double negative cells . Data represent mean ± SE of three independent experiments .

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I. Jurkat cells were transfected with DRP1K38A, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage AnnexinV/PI double negative cells. Data represent mean ± SE of three independent experiments.


A . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . WB are representative of at least three independent experiments . B - C . Quantitative analysis of the ratio between the protein levels of mitochondrial markers and Actin during AICD is shown . Data represent mean ± SE of three independent experiments .

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A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.B-C. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and Actin during AICD is shown. Data represent mean ± SE of three independent experiments.


D . Jurkat cells were co - transfected with GFP - LC3 and Parkin - Cherry , 32h after AICD induction cells were fixed and immunostained with an anti - TOM20 antibody . Representative images for each condition are presented . Scale bar , 5μm . E . LC3 - Parkin - mitochondria co - localization was calculated from 30 randomly selected images . Data represent mean ± SE of three independent experiments . Data referring to Ctrl and AICD - stimulated cells without rapamycin correspond to the ones presented in Fig . 3C .

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D. Jurkat cells were co-transfected with GFP-LC3 and Parkin-Cherry, 32h after AICD induction cells were fixed and immunostained with an anti-TOM20 antibody. Representative images for each condition are presented. Scale bar, 5μm.E. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments. Data referring to Ctrl and AICD-stimulated cells without rapamycin correspond to the ones presented in Fig.3C.


A . Splenocytes were isolated from Ambragt / + and wt C57 / BL6mice and activated as indicated in materials and methods . At day 7 after activation , AICD was induced with 0 . 1μg / ml of plate - bound anti - mCD3 ( Clone 17A2 ) . Apoptotic cells were detected 12h after AICD induction by flow cytometry as AnnexinV / PI double positive cells .

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A. Splenocytes were isolated from Ambragt/+ and wt C57/BL6mice and activated as indicated in materials and methods. At day 7 after activation, AICD was induced with 0.1μg/ml of plate-bound anti-mCD3 (Clone 17A2). Apoptotic cells were detected 12h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.


B . Splenocytes were isolated from ATG7 fl / fl and wt C57 / BL6mice . Activation of splenocytes and AICD were performed as previously described . At day 5 after activation , cells were infected with retroviral constructs expressing CRE recombinase . Where indicated , cells were pre - incubated with 100nM rapamycin for 24h before AICD induction . Apoptotic cells were detected 2h after AICD induction by flow cytometry as AnnexinV / PI double positive cells .

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B. Splenocytes were isolated from ATG7 fl/fl and wt C57/BL6mice. Activation of splenocytes and AICD were performed as previously described. At day 5 after activation, cells were infected with retroviral constructs expressing CRE recombinase. Where indicated, cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Apoptotic cells were detected 2h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.


C . Jurkat cells were transfected with scramble or ATG7 siRNA 24h before AICD induction . AICD was induced as described in materials and methods . Where indicated 100nM rapamycin was added at time 0h of AICD induction . Apoptotic cell death analysis was carried out as in ( A ) , except that cells were analysed 32h after AICD induction . Data represent mean ± SE of five independent experiments .

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C. Jurkat cells were transfected with scramble or ATG7 siRNA 24h before AICD induction. AICD was induced as described in materials and methods. Where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of five independent experiments.


D . Mitochondrial depolarization upon AICD induction was analysed in cells treated as indicated . Data represent mean ± SE of five independent experiments .

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D. Mitochondrial depolarization upon AICD induction was analysed in cells treated as indicated. Data represent mean ± SE of five independent experiments.


E . Protein samples ( 20μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . WB are representative of three independent experiments . F . Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown . Data represent mean ± SE of three independent experiments .

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E. Protein samples (20μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of three independent experiments.F. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown. Data represent mean ± SE of three independent experiments.


( B ) Cytoplasmic TDP - 43 immunostaining is elevated not only in poly - GA transduced neurons , but also in the non - transduced receiver cells . White and red arrows indicate cells with cytoplasmic TDP - 43 in GFP positive and negative cells , respectively .

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(B) Cytoplasmic TDP-43 immunostaining is elevated not only in poly-GA transduced neurons, but also in the non-transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively.


( C ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced ( donor ) , non - transduced ( receiver ) neurons . Cells with and without GFP signal were counted separately ( indicated by + / − ) . Two groups ( GFP negative donor , GFP positive receiver ) were excluded due to very high GFP transduction rate and very low GFP transmission rate . n = 4 biological replicates . In total 283 donor GFP , 273 donor GA175 - GFP , 284 receiver GFP and 266 receiver GA175 - GFP cells were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 .

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(C) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP negative donor, GFP positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n=4 biological replicates. In total 283 donor GFP, 273 donor GA175-GFP, 284 receiver GFP and 266 receiver GA175-GFP cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.


Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . Immunofluorescence staining of TDP - 43 ∆ NLS aggregate number per cell

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Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. Immunofluorescence staining of TDP-43∆NLS aggregate number per cell


Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . Immunofluorescence staining of TDP - 43 ∆ NLS aggregate number per

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Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . automatic quantification of TDP - 43 ∆ NLS aggregate number per cell In ( E ) n = 3 biological replicates with 368 donor iRFP , 251 donor GA175 - iRFP , 430 receiver iRFP and 328 GA175 - iRFP cells were analyzed . Cells with and without GFP signal were analyzed separately ( indicated by + / − ) . White and red arrows indicate cells with cytoplasmic TDP - 43 in GFP positive and negative cells , respectively . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. automatic quantification of TDP-43∆NLS aggregate number per cell In (E) n=3 biological replicates with 368 donor iRFP, 251 donor GA175-iRFP, 430 receiver iRFP and 328 GA175-iRFP cells were analyzed. Cells with and without GFP signal were analyzed separately (indicated by +/−). White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


( F ) in addition to filter trap assay of SDS - insoluble TDP - 43ΔNLS - GFP aggregates compared in iRFP or GA175 - iRFP transfected cells .

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(F) in addition to filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates compared in iRFP or GA175-iRFP transfected cells.


( G ) GFP mRNA expression levels were measured by qPCR . RNA levels were normalized to GAPDH , β - actin and β2 - microglobulin mRNA . Bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction . * denotes p < 0 . 05 , and * * denotes p < 0 . 01 .

(G) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. * denotes p<0.05, and ** denotes p<0.01.


( A ) Confocal imaging showed anti - GA antibody treatment reduces poly - GA aggregates and TDP - 43 mislocalization in hippocampal neurons . White and red arrows show cells with cytoplasmic TDP - 43 in GFP - positive and - negative cells , respectively . Scale bar denotes 30 µm .

(A) Confocal imaging showed anti-GA antibody treatment reduces poly-GA aggregates and TDP-43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP-43 in GFP-positive and -negative cells, respectively. Scale bar denotes 30 µm.


( B ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced ( donor ) , non - transduced ( receiver ) neurons . Four groups were excluded due to very high GFP transduction rate ( GFP - negative donor ) and very low GFP transmission rate ( GFP - positive receiver with IgG and anti - GA ) and complete prevention of GA - RFP transmission of anti - GA immunodepletion ( GA - GFP receiver with anti - GA ) . n = 3 biological replicates . In total 280 donor GFP , 284 receiver GFP with IgG , 317 receiver GFP with anti - GA , 277 donor GA175 - GFP , 294 receiver GA175 - GFP with IgG , 311 receiver GA175 - GFP with anti - GA cells were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 .

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(B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP-negative donor) and very low GFP transmission rate (GFP-positive receiver with IgG and anti-GA) and complete prevention of GA-RFP transmission of anti-GA immunodepletion (GA-GFP receiver with anti-GA). n=3 biological replicates. In total 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti-GA, 277 donor GA175-GFP, 294 receiver GA175-GFP with IgG, 311 receiver GA175-GFP with anti-GA cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.


( C ) Immunoblotting of poly - GA immunoprecipitated from conditioned media using antibody - coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media .

(C) Immunoblotting of poly-GA immunoprecipitated from conditioned media using antibody-coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media.


( D ) Poly - GA levels in conditioned media before and after immunodepletion with control IgG and anti - GA antibody were determined by immunoassay . n = 3 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * denotes p < 0 . 01 , and * * * denotes p < 0 . 001 .

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(D) Poly-GA levels in conditioned media before and after immunodepletion with control IgG and anti-GA antibody were determined by immunoassay. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ** denotes p<0.01, and *** denotes p<0.001.


Double immunofluorescence of the proteasome subunit PSMC4 and poly - GA inclusions in spinal cord of GA149 - CFP transgenic mouse Scale bar denotes 20 µm .

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Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in spinal cord of GA149-CFP transgenic mouse Scale bar denotes 20 µm.


Double immunofluorescence of the proteasome subunit PSMC4 and poly - GA inclusions in cortex of C9orf72 patients compared to controls . Scale bar denotes 20 µm .

Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in cortex of C9orf72 patients compared to controls. Scale bar denotes 20 µm.


Co - culture model of HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and an UbG76V - GFP proteostasis reporter in donor and receiver compartment ( 48 h ) . ( C ) Separate analysis of both compartments by immunoblot and ( D ) immunoblot quantification . For quantitative analysis of immunoblots UbG76V - GFP was normalized to calnexin . n = 3 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . Red dashed line indicates the control ' s expression level . * denotes p < 0 . 05 , * * * denotes p < 0 . 001 .

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Co-culture model of HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and an UbG76V-GFP proteostasis reporter in donor and receiver compartment (48 h). (C) Separate analysis of both compartments by immunoblot and (D) immunoblot quantification. For quantitative analysis of immunoblots UbG76V-GFP was normalized to calnexin. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Red dashed line indicates the control's expression level. * denotes p<0.05, *** denotes p<0.001.


( E ) GFP mRNA expression levels were measured by qPCR . RNA levels were normalized to GAPDH , β - actin and β2 - microglobulin mRNA . Bar - graphs of mean ± SD . n = 3 biological replicates . Unpaired two - tailed t - test with Welch ' s correction .

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(E) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. n=3 biological replicates. Unpaired two-tailed t-test with Welch's correction.


( F , G ) Flow cytometry analysis of a UbG76V - GFP reporter cell line incubated 48h with conditioned media from RFP or GA175 - RFP transfected cells upon immunodepletion of poly - GA or control depletion using unspecific IgG . n = 3 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * denotes p < 0 . 05 . ( G ) Comparisons of the corresponding histograms for compensated RFP and UbG76V - GFP fluorescence from one representative experiment that shows specific transmission of GA175 - RFP associated with accumulation of UbG76V - GFP in cells incubated with GA175 - RFP conditioned media .

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(F,G) Flow cytometry analysis of a UbG76V-GFP reporter cell line incubated 48h with conditioned media from RFP or GA175-RFP transfected cells upon immunodepletion of poly-GA or control depletion using unspecific IgG. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05. (G) Comparisons of the corresponding histograms for compensated RFP and UbG76V-GFP fluorescence from one representative experiment that shows specific transmission of GA175-RFP associated with accumulation of UbG76V-GFP in cells incubated with GA175-RFP conditioned media.


Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . ( A ) Immunofluorescence reveals enhanced cytoplasmic TDP - 43 levels in neurons with poly - GA aggregates or treated with MG132 . Arrows mark punctate TDP - 43 staining . Rolipram treatment reduced cytoplasmic TDP - 43 in GA175 - GFP neurons . Scale bar denotes 20 µm . ( B ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced neurons . n = 4 biological replicates . In total 462 GFP and 371 GA175 - GFP cells treated with vehicle , and 386 GFP and 529 GA175 - GFP cells treated with MG132 , 513 GFP and 434 GA175 - GFP cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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

Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP-43 levels in neurons with poly-GA aggregates or treated with MG132. Arrows mark punctate TDP-43 staining. Rolipram treatment reduced cytoplasmic TDP-43 in GA175-GFP neurons. Scale bar denotes 20 µm. (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced neurons. n=4 biological replicates. In total 462 GFP and 371 GA175-GFP cells treated with vehicle, and 386 GFP and 529 GA175-GFP cells treated with MG132, 513 GFP and 434 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . ( C ) Immunoblot to show effects of MG132 and rolipram on GA175 - GFP and GFP expression .

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

Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (C) Immunoblot to show effects of MG132 and rolipram on GA175-GFP and GFP expression.


Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . Filter trap assay with quantification of SDS - insoluble aggregated GA175 - GFP . n = 5 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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

Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. Filter trap assay with quantification of SDS-insoluble aggregated GA175-GFP. n=5 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


( F , G ) HeLa cells were co - transfected with RFP - TDP - CTF and GFP or GA175 - GFP for 2 days . For the final 16h , cells were treated with rolipram ( 30 µM ) or MG132 ( 10 µM ) . Filter trap assay of SDS - insoluble TDP - CTF aggregates quantified by densitometry . n = 4 biological replicates . Scatter dot plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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

(F,G) HeLa cells were co-transfected with RFP-TDP-CTF and GFP or GA175-GFP for 2 days. For the final 16h, cells were treated with rolipram (30 µM) or MG132 (10 µM). Filter trap assay of SDS-insoluble TDP-CTF aggregates quantified by densitometry. n=4 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


( H , I ) HeLa cells were co - transfected with TDP - 43ΔNLS - GFP and iRFP or GA175 - iRFP for 2 days . For the final 16h , cells were treated with either vehicle or rolipram ( 30 µM ) . Filter trap assay of SDS - insoluble TDP - 43ΔNLS - GFP aggregates quantified by densitometry . n = 3 biological replicates . Scatter dot plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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

(H,I) HeLa cells were co-transfected with TDP-43ΔNLS-GFP and iRFP or GA175-iRFP for 2 days. For the final 16h, cells were treated with either vehicle or rolipram (30 µM). Filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates quantified by densitometry. n=3 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Hela cells were co - transfected with an RFP - based TDP - NLS reporter and GFP or GA175 - GFP . 24 h after transfection , cells were treated with rolipram ( 30 µM ) for 16h . In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP - reporter . White arrows indicate cells with cytoplasmic TDP - 43 . ( B ) Automated quantification of cells with cytoplasmic TDP - NLS reporter in GFP and GA175 - GFP positive cells . n = 4 biological replicates . In total 345 GFP and 386 GA175 - GFP cells treated with vehicle , 371 GFP and 404 GA175 - GFP cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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

Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP-reporter. White arrows indicate cells with cytoplasmic TDP-43. (B) Automated quantification of cells with cytoplasmic TDP-NLS reporter in GFP and GA175-GFP positive cells. n=4 biological replicates. In total 345 GFP and 386 GA175-GFP cells treated with vehicle, 371 GFP and 404 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Hela cells were co - transfected with an RFP - based TDP - NLS reporter and GFP or GA175 - GFP . 24 h after transfection , cells were treated with rolipram ( 30 µM ) for 16h . ( C ) RFP - NLSTDPWT mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. (C) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Hela cells were co - transfected with the RFP - based TDP - NLS reporter , GFP or GA175 - GFP , as well as PSMD11 or empty vector . Image analysis as in ( A ) . n = 4 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . 354 GFP and 330 GA175 - GFP cells with vector , 367 GFP and 369 GA175 - GFP cells with PSMD11 in total were analyzed .

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Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. Image analysis as in (A). n=4 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA175-GFP cells with vector, 367 GFP and 369 GA175-GFP cells with PSMD11 in total were analyzed.


Hela cells were co - transfected with the RFP - based TDP - NLS reporter , GFP or GA175 - GFP , as well as PSMD11 or empty vector . F ) RFP - NLSTDPWT mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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

Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. F) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Immunofluorescence of Hela cells transfected with GFP or GA175 - GFP showing reduced poly - GA aggregation upon rolipram treatment ( 30 µM , 16 h ) . ( H ) Automated quantification of poly - GA aggregate number per cell . n = 3 biological replicates . In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction .

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 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

Immunofluorescence of Hela cells transfected with GFP or GA175-GFP showing reduced poly-GA aggregation upon rolipram treatment (30 µM, 16 h). (H) Automated quantification of poly-GA aggregate number per cell. n=3 biological replicates. In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.


( I ) GA - GFP mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction .

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(I) GA-GFP mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.


( B ) Immunoblot of HeLa cells transfected with RFP - based TDP - NLS wild - type ( WT ) or mutants ( K84A , K84R , K95A , K95R ) .

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(B) Immunoblot of HeLa cells transfected with RFP-based TDP-NLS wild-type (WT) or mutants (K84A, K84R, K95A, K95R).


( C HeLa cells were co - transfected with the indicated TDP - NLS reporters as well as GFP or GA175 - GFP , and treated with MG132 ( 10µM ) or vehicle for 16h .

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(C HeLa cells were co-transfected with the indicated TDP-NLS reporters as well as GFP or GA175-GFP, and treated with MG132 (10µM) or vehicle for 16h.


( D ) Automated quantification of RFP - NLS reporters in GFP positive cells . Note that K84A and K84R block overall import , while K95A and K95R allow import but are resistant to inhibition by poly - GA . n = 4 biological replicates . The total number of cells analyzed per group was ( from left to right ) : 667 , 581 , 789 , 783 , 809 , 708 , 628 , 721 , 938 , 557 , 857 , 861 , 886 , 699 , 789 , 539 , 636 , 577 , 638 and 870 . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . Scale bar denotes 20 µm .

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(D) Automated quantification of RFP-NLS reporters in GFP positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly-GA. n=4 biological replicates. The total number of cells analyzed per group was (from left to right): 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638 and 870. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. Scale bar denotes 20 µm.


( E , F ) HeLa cells were co - transfected with the indicated GFP - TDP NLS reporters and iRFP or GA175 - iRFP . Cell lysates were immunoprecipitated with anti - GFP and immunoblotted with indicated antibodies to detect co - immunoprecipitation of the TDP - 43 NLS with importin - α5 / KPNA1 nuclear import receptor . ( F ) Quantification of KPNA1 levels normalized to total GFP - NLSTDP reporter levels in anti - GFP immunoprecipitates . n = 3 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . See also Figure EV4 . Red dashed line indicates the control ' s expression level .

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(E,F) HeLa cells were co-transfected with the indicated GFP-TDP NLS reporters and iRFP or GA175-iRFP. Cell lysates were immunoprecipitated with anti-GFP and immunoblotted with indicated antibodies to detect co-immunoprecipitation of the TDP-43 NLS with importin-α5/KPNA1 nuclear import receptor. (F) Quantification of KPNA1 levels normalized to total GFP-NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. See also Figure EV4. Red dashed line indicates the control's expression level.


HeLa cells were co - transfected with wildtype or K95A GFP - TDP NLS , HA - ubiquitin and iRFP or GA175 - iRFP . 24h after transfection , cells were treated with rolipram ( 30 µM ) , MG132 ( 10 µM ) or DMSO ( vehicle ) for 16h . Lysates were immunoprecipitated with Protein G beads coupled with anti - GFP antibody . ( A ) Immunoblotting of input ( left panels ) and anti - GFP immunoprecipitates ( right panels ) to show GFP - reporter levels and poly - ubiquitination . ( B ) Quantification of HA - ubiquitin levels normalized to total GFP - NLSTDP reporter levels in anti - GFP immunoprecipitates . n = 3 biological replicates . Scatter plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * denotes p < 0 . 05 , and * * * denotes p < 0 . 001 . Red dashed line indicates the control ' s expression level .

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

HeLa cells were co-transfected with wildtype or K95A GFP-TDP NLS, HA-ubiquitin and iRFP or GA175-iRFP. 24h after transfection, cells were treated with rolipram (30 µM), MG132 (10 µM) or DMSO (vehicle) for 16h. Lysates were immunoprecipitated with Protein G beads coupled with anti-GFP antibody. (A) Immunoblotting of input (left panels) and anti-GFP immunoprecipitates (right panels) to show GFP-reporter levels and poly-ubiquitination. (B) Quantification of HA-ubiquitin levels normalized to total GFP- NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05, and *** denotes p<0.001. Red dashed line indicates the control's expression level.


HeLa cells were co - transfected with wildtype or K95A GFP - TDP NLS , HA - ubiquitin and iRFP or GA175 - iRFP . 24h after transfection , cells were treated with rolipram ( 30 µM ) , MG132 ( 10 µM ) or DMSO ( vehicle ) for 16h . Lysates were immunoprecipitated with Protein G beads coupled with anti - GFP antibody .

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

HeLa cells were co-transfected with wildtype or K95A GFP-TDP NLS, HA-ubiquitin and iRFP or GA175-iRFP. 24h after transfection, cells were treated with rolipram (30 µM), MG132 (10 µM) or DMSO (vehicle) for 16h. Lysates were immunoprecipitated with Protein G beads coupled with anti-GFP antibody.


( A - F ) Atg1 ‐ induced myosin II activation depends on the kinase activity of Atg1 . Third ‐ instar wing imaginal discs from ptc ‐ GAL4 UAS ‐ GFP controls or flies expressing indicated transgenes were stained with phospho ‐ MRLC ( blue ) and TRITC ‐ labelled phalloidin ( red ) . Low level of phospho ‐ MRLC staining was observed in controls ( A ) and cells overexpressing kinase ‐ deficient Atg1 ‐ KR ( C ) , whereas a robust increase in phospho ‐ MRLC was found in cells overexpressing Atg1 ( B ) . Co ‐ expression of Atg1 and spaghetti ‐ squash ( A20A21 ) exhibited low level of phospho ‐ MRLC staining ( D ) . Atg1 ‐ induced high level of phospho ‐ MRLC staining was not suppressed by expression of caspase inhibitor p35 ( E ) or by depletion of Atg12 ( Atg12RNAi ) ( F ) . Bar , 20 μm .

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(A-F) Atg1‐induced myosin II activation depends on the kinase activity of Atg1. Third‐instar wing imaginal discs from ptc‐GAL4 UAS‐GFP controls or flies expressing indicated transgenes were stained with phospho‐MRLC (blue) and TRITC‐labelled phalloidin (red). Low level of phospho‐MRLC staining was observed in controls (A) and cells overexpressing kinase‐deficient Atg1‐KR (C), whereas a robust increase in phospho‐MRLC was found in cells overexpressing Atg1 (B). Co‐expression of Atg1 and spaghetti‐squash (A20A21) exhibited low level of phospho‐MRLC staining (D). Atg1‐induced high level of phospho‐MRLC staining was not suppressed by expression of caspase inhibitor p35 (E) or by depletion of Atg12 (Atg12RNAi) (F). Bar, 20 μm.


( H ) Sqa , but not Atg1 , directly phosphorylated Sqh in vitro . Flag - Atg1 , Flag - Atg1 ‐ KR , HA - Sqa and HA - Sqa ‐ KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [ γ ‐ 32P ] ATP and bacterially expressed recombinant wild ‐ type Sqh or SqhA20A21 . As shown on the autoradiogram ( top panel ) , wild ‐ type but not the kinase ‐ deficient Atg1 ( Atg1 ‐ KR ) and Sqa ( Sqa ‐ KA ) was autophosphorylated . No phosphorylation was seen with SqhA20A21 . The equal input of His ‐ fusion proteins is shown on the Coomassie staining . Anti ‐ Flag and anti ‐ HA immunoblottings ( IBs ) were used as controls to quantify the amount of proteins precipitated .

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(H) Sqa, but not Atg1, directly phosphorylated Sqh in vitro. Flag-Atg1, Flag-Atg1‐KR, HA-Sqa and HA-Sqa‐KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [γ‐32P]ATP and bacterially expressed recombinant wild‐type Sqh or SqhA20A21. As shown on the autoradiogram (top panel), wild‐type but not the kinase‐deficient Atg1 (Atg1‐KR) and Sqa (Sqa‐KA) was autophosphorylated. No phosphorylation was seen with SqhA20A21. The equal input of His‐fusion proteins is shown on the Coomassie staining. Anti‐Flag and anti‐HA immunoblottings (IBs) were used as controls to quantify the amount of proteins precipitated.


( A - K ) Genetic interactions between Atg1 , Sqa , and spaghetti ‐ squash ( Sqh ) . Compared with the ptc ‐ GAL4 controls ( A ) , expression of Atg1 or Sqa by ptc ‐ GAL4 resulted in missing anterior cross ‐ vein phenotypes ( B , C ) . However , RNAi ‐ mediated downregulation of Atg1 ( Atg1RNAi ) or Sqa ( SqaRNAi ) did not cause wing vein defects ( D , E ) . Depletion of Atg1 and Sqa suppressed Atg1 and Sqa ‐ induced wing vein defects , respectively ( F , I ) . Atg1 ‐ induced wing defects were modulated by depletion of Sqa ( G ) or by co ‐ expression of SqhA20A21 ( H ) . Nevertheless , Sqa ‐ induced wing vein defects were suppressed by co ‐ expression of SqhA20A21 ( J ) but not Atg1RNAi ( K ) .

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(A-K) Genetic interactions between Atg1, Sqa, and spaghetti‐squash (Sqh). Compared with the ptc‐GAL4 controls (A), expression of Atg1 or Sqa by ptc‐GAL4 resulted in missing anterior cross‐vein phenotypes (B, C). However, RNAi‐mediated downregulation of Atg1 (Atg1RNAi) or Sqa (SqaRNAi) did not cause wing vein defects (D, E). Depletion of Atg1 and Sqa suppressed Atg1 and Sqa‐induced wing vein defects, respectively (F, I). Atg1‐induced wing defects were modulated by depletion of Sqa (G) or by co‐expression of SqhA20A21 (H). Nevertheless, Sqa‐induced wing vein defects were suppressed by co‐expression of SqhA20A21 (J) but not Atg1RNAi (K).


( M ) 293T cells were transfected with HA ‐ tagged Sqa WT ( wild ‐ type ) or KA , together with Flag ‐ tagged Atg1 WT or KR . The cells were lysed 48 h after transfection and immunoprecipitated ( IP ) with anti ‐ Flag antibodies . The immunoprecipitated proteins and the total cell lysates ( TCL ) were analysed by immunoblotting ( IB ) with antibodies as indicated .

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(M) 293T cells were transfected with HA‐tagged Sqa WT (wild‐type) or KA, together with Flag‐tagged Atg1 WT or KR. The cells were lysed 48 h after transfection and immunoprecipitated (IP) with anti‐Flag antibodies. The immunoprecipitated proteins and the total cell lysates (TCL) were analysed by immunoblotting (IB) with antibodies as indicated.


( N ) 293T cells transfected with Flag - Atg1 ‐ KR and various HA ‐ tagged Sqa contracts were subjected to immunoprecipitations with anti ‐ HA antibody . The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated .

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(N) 293T cells transfected with Flag-Atg1‐KR and various HA‐tagged Sqa contracts were subjected to immunoprecipitations with anti‐HA antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated.


( O ) Atg1 directly phosphorylated Sqa in vitro . Flag ‐ tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa ‐ K1 , Sqa ‐ K2 , and Sqa ‐ C in an in vitro kinase assay . The lower panels represent equal input of His ‐ fusion proteins and Atg1 immunoprecipitates .

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(O) Atg1 directly phosphorylated Sqa in vitro. Flag‐tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa‐K1, Sqa‐K2, and Sqa‐C in an in vitro kinase assay. The lower panels represent equal input of His‐fusion proteins and Atg1 immunoprecipitates.


( A ) Characterization of Atg1 ‐ dependent phosphorylation sites on Sqa . 293T cells transfected with Flag ‐ tagged Atg1 or Atg1 ‐ KR were subjected to immunoprecipitation with anti ‐ Flag antibodies , followed by in vitro kinase assays with bacterially expressed Sqa ‐ K2 , Sqa ‐ K2 ‐ T194A , Sqa ‐ K2 ‐ T239A , and Sqa ‐ K2 ‐ T279A as substrates . Atg1 but not Atg1 ‐ KR was autophosphorylated ( top panel ) . Relative phosphorylation levels of substrates were quantified . Data are represented as mean ± s . e . of triplicates . ( B - D ) Phosphorylation of Sqa at Thr ‐ 279 is critical for the kinase activity of Sqa .

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(A) Characterization of Atg1‐dependent phosphorylation sites on Sqa. 293T cells transfected with Flag‐tagged Atg1 or Atg1‐KR were subjected to immunoprecipitation with anti‐Flag antibodies, followed by in vitro kinase assays with bacterially expressed Sqa‐K2, Sqa‐K2‐T194A, Sqa‐K2‐T239A, and Sqa‐K2‐T279A as substrates. Atg1 but not Atg1‐KR was autophosphorylated (top panel). Relative phosphorylation levels of substrates were quantified. Data are represented as mean±s.e. of triplicates. (B-D) Phosphorylation of Sqa at Thr‐279 is critical for the kinase activity of Sqa.


( B ) HA ‐ tagged Sqa or Sqa ‐ T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti ‐ squash ( Sqh ) WT and A20A21 , in an in vitro kinase assay .

(B) HA‐tagged Sqa or Sqa‐T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti‐squash (Sqh) WT and A20A21, in an in vitro kinase assay.


( C ) Clonal expression of Sqa but not Sqa ‐ T279A ( GFP ‐ positive cells ) in the larval wing imaginal discs resulted in a marked increase in phospho ‐ MRLC staining ( blue ) and actin reorganization ( red ) . Bar , 20 μm .

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(C) Clonal expression of Sqa but not Sqa‐T279A (GFP‐positive cells) in the larval wing imaginal discs resulted in a marked increase in phospho‐MRLC staining (blue) and actin reorganization (red). Bar, 20 μm.


( D ) The larval fat body of denoted genotypes were dissected , lysed , and subjected to western blot analysis using anti ‐ phospho ‐ MRLC and anti ‐ Sqh antibodies . For quantification , the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh

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(D) The larval fat body of denoted genotypes were dissected, lysed, and subjected to western blot analysis using anti‐phospho‐MRLC and anti‐Sqh antibodies. For quantification, the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh levels


( A , B ) Activation of myosin II on nutrient deprivation . The larval fat body of denoted genotypes under fed or starved conditions were dissected , lysed , and subjected to western blot analysis using antibodies specific for phospho ‐ myosin regulatory light chain ( MRLC ) and total MRLC . The Rheb , SqaRNAi , Sqa ‐ T279A , spaghetti ‐ squash ( SqhA20A21 ) , and Atg7RNAi transgenes were expressed under the control of hs - GAL4 driver ( B ) . For quantification , the relative phosphorylation levels of MRLC were quantified as in Figure 3D . Data are represented as mean ± s . e . of triplicates .

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(A, B) Activation of myosin II on nutrient deprivation. The larval fat body of denoted genotypes under fed or starved conditions were dissected, lysed, and subjected to western blot analysis using antibodies specific for phospho‐myosin regulatory light chain (MRLC) and total MRLC. The Rheb, SqaRNAi, Sqa‐T279A, spaghetti‐squash (SqhA20A21), and Atg7RNAi transgenes were expressed under the control of hs-GAL4 driver (B). For quantification, the relative phosphorylation levels of MRLC were quantified as in Figure 3D. Data are represented as mean±s.e. of triplicates.


( C - J ) Starvation ‐ induced autophagosome formation was compromised by inhibition of myosin II activation . Compared with the fed condition ( C ) , starvation induced a robust formation of GFP - Atg8a puncta in larval fat ‐ body cells ( D ) . Clonal expression of Atg12RNAi ( E ) , Sqa ‐ T279A ( F ) , SqaRNAi ( G ) , or SqhA20A21 ( H ) markedly suppressed GFP - Atg8a puncta formation during starvation . The autophagic defects caused by Sqa ‐ T279A and SqaRNAi were rescued by co ‐ expression of the constitutively active SqhE20E21 ( I ) and SqhD20D21 ( J ) , respectively . Bar , 20 μm .

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(C-J) Starvation‐induced autophagosome formation was compromised by inhibition of myosin II activation. Compared with the fed condition (C), starvation induced a robust formation of GFP-Atg8a puncta in larval fat‐body cells (D). Clonal expression of Atg12RNAi (E), Sqa‐T279A (F), SqaRNAi (G), or SqhA20A21 (H) markedly suppressed GFP-Atg8a puncta formation during starvation. The autophagic defects caused by Sqa‐T279A and SqaRNAi were rescued by co‐expression of the constitutively active SqhE20E21 (I) and SqhD20D21 (J), respectively. Bar, 20 μm.


( K ) The average number of GFP - Atg8a marked autophagosomes per cell is shown ( data are represented as mean ± s . e . of 20 fat ‐ body samples imaged per genotype ; * * * P0 . 001 ) .

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(K) The average number of GFP-Atg8a marked autophagosomes per cell is shown (data are represented as mean±s.e. of 20 fat‐body samples imaged per genotype; ***P0.001).


( L ) Inhibition of myosin II activity increased sensitivity to starvation . Flies carrying transgenes under the control of fat body ‐ specific Cg - GAL4 driver were used for further analysis . Histogram illustrating the survival curve of adult female flies of denoted genotypes when placed under starved conditions . Data are mean ± s . e . from triplicate experiments ( n = 100 flies / genotype / treatment ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . See Supplementary data for genotypes .

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(L) Inhibition of myosin II activity increased sensitivity to starvation. Flies carrying transgenes under the control of fat body‐specific Cg-GAL4 driver were used for further analysis. Histogram illustrating the survival curve of adult female flies of denoted genotypes when placed under starved conditions. Data are mean±s.e. from triplicate experiments (n=100 flies/genotype/treatment). *P0.05, **P0.01, ***P0.001. See Supplementary data for genotypes.


( A ) Ulk1 interacted with ZIPK . 293T cells transfected with HA ‐ tagged ZIPK together with Flag ‐ tagged Ulk1 or Ulk1 ‐ KI were subjected to immunoprecipitations with anti ‐ Flag antibody . The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated .

(A) Ulk1 interacted with ZIPK. 293T cells transfected with HA‐tagged ZIPK together with Flag‐tagged Ulk1 or Ulk1‐KI were subjected to immunoprecipitations with anti‐Flag antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated.


( B ) HA ‐ tagged kinase ‐ inactive ZIPK ‐ KA ( K42A ) was expressed in 293T cells with Flag ‐ tagged Ulk1 or Ulk1 ‐ KI . Cell lysates were incubated with or without alkaline phosphatase ( CIP ) and analysed by immunoblotting with antibodies as indicated .

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(B) HA‐tagged kinase‐inactive ZIPK‐KA (K42A) was expressed in 293T cells with Flag‐tagged Ulk1 or Ulk1‐KI. Cell lysates were incubated with or without alkaline phosphatase (CIP) and analysed by immunoblotting with antibodies as indicated.


( C ) Myosin II was activated upon nutrient deprivation in MCF7 cells . MCF7 cells were cultured in serum containing DMEM medium ( nutrient ‐ rich condition ) or in Earle ' s balanced salt solution ( EBSS ; starved condition ) for 2 h . The myosin II activity was significantly downregulated after EBSS ‐ starved cells were replenished with fresh DMEM medium containing 10 % FBS for 2 h . The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls . Each value represents mean ± s . e . of triplicates .

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(C) Myosin II was activated upon nutrient deprivation in MCF7 cells. MCF7 cells were cultured in serum containing DMEM medium (nutrient‐rich condition) or in Earle's balanced salt solution (EBSS; starved condition) for 2 h. The myosin II activity was significantly downregulated after EBSS‐starved cells were replenished with fresh DMEM medium containing 10% FBS for 2 h. The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls. Each value represents mean±s.e. of triplicates.


( D ) MCF7 cells stably infected with lentivirus expressing control ( shLuc ) , Ulk1 or ZIPK shRNA were cultured in nutrient ‐ rich DMEM medium ( F ) or EBSS ( S ) for 2 h . Effects of Ulk1 and ZIPK knockdown on starvation ‐ induced myosin II activation were analysed by immunoblotting with antibodies as indicated . Data are mean ± s . e . of triplicates .

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(D) MCF7 cells stably infected with lentivirus expressing control (shLuc), Ulk1 or ZIPK shRNA were cultured in nutrient‐rich DMEM medium (F) or EBSS (S) for 2 h. Effects of Ulk1 and ZIPK knockdown on starvation‐induced myosin II activation were analysed by immunoblotting with antibodies as indicated. Data are mean±s.e. of triplicates.


( A ) MCF7 / GFP - LC3 cells stably infected with lentivirus expressing control ( shLuc ) , ZIPK , or non ‐ muscle myosin heavy chain ‐ IIA ( NMHC ‐ IIA ) shRNA were cultured in nutrient ‐ rich medium ( DMEM ) or starvation medium ( Earle ' s balanced salt solution ; EBSS ) in the presence or absence of lysosomal inhibitor bafilomycin A1 ( BafA1 ) for 2 h . Depletion of ZIPK and NMHC ‐ IIA markedly inhibited starvation ‐ induced GFP - LC3 puncta formation . Quantification of the number of GFP - LC3 dots per cell ( lower panel ) was shown ( data are represented as mean ± s . e . of 100 cells , * * * P0 . 001 ) .

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(A) MCF7/GFP-LC3 cells stably infected with lentivirus expressing control (shLuc), ZIPK, or non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) shRNA were cultured in nutrient‐rich medium (DMEM) or starvation medium (Earle's balanced salt solution; EBSS) in the presence or absence of lysosomal inhibitor bafilomycin A1 (BafA1) for 2 h. Depletion of ZIPK and NMHC‐IIA markedly inhibited starvation‐induced GFP-LC3 puncta formation. Quantification of the number of GFP-LC3 dots per cell (lower panel) was shown (data are represented as mean±s.e. of 100 cells, ***P0.001).


( B ) Cells as in ( A ) were cultured in nutrient ‐ rich medium ( F ) or EBSS ( S ) with or without BafA1 for 2 h . Effects of ZIPK and NMHC ‐ IIA knockdown on starvation ‐ induced GFP - LC3 conversion were assessed by immunoblotting with anti ‐ LC3 , anti ‐ ZIPK , anti ‐ NMHC ‐ IIA , and anti ‐ tubulin antibodies . The relative ratio of LC3II / LC3I is shown at the right panel . Data are mean ± s . e . of triplicates .

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(B) Cells as in (A) were cultured in nutrient‐rich medium (F) or EBSS (S) with or without BafA1 for 2 h. Effects of ZIPK and NMHC‐IIA knockdown on starvation‐induced GFP-LC3 conversion were assessed by immunoblotting with anti‐LC3, anti‐ZIPK, anti‐NMHC‐IIA, and anti‐tubulin antibodies. The relative ratio of LC3II/LC3I is shown at the right panel. Data are mean±s.e. of triplicates.


( A ) The fat body of spaghetti ‐ squash ( sqhAX3 ) ; sqh - GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis . Sqh - GFP and phospho ‐ myosin regulatory light chain ( MRLC ) were enriched in cell - cell junction under nutrient ‐ rich ( fed ) condition . Under starvation conditions , Sqh - GFP and phospho ‐ MRLC were localized to both cell - cell junction and perinuclear region . Actin was stained with TRITC ‐ labelled phalloidin ( red ) and nucleus was stained with DAPI ( blue ) . Bar , 20 μm .

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(A) The fat body of spaghetti‐squash (sqhAX3); sqh-GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis. Sqh-GFP and phospho‐myosin regulatory light chain (MRLC) were enriched in cell-cell junction under nutrient‐rich (fed) condition. Under starvation conditions, Sqh-GFP and phospho‐MRLC were localized to both cell-cell junction and perinuclear region. Actin was stained with TRITC‐labelled phalloidin (red) and nucleus was stained with DAPI (blue). Bar, 20 μm.


( B ) Starvation induced redistribution of myosin II in MCF7 / GFP - LC3 cells . Both phospho ‐ MRLC and MRLC were localized in peripheral region of cells in nutrient ‐ rich medium ( DMEM ) , whereas they redistributed to the perinuclear region and co ‐ localized with GFP - LC3 under starvation conditions ( Earle ' s balanced salt solution ; EBSS ) . Bar , 10 μm .

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(B) Starvation induced redistribution of myosin II in MCF7/GFP-LC3 cells. Both phospho‐MRLC and MRLC were localized in peripheral region of cells in nutrient‐rich medium (DMEM), whereas they redistributed to the perinuclear region and co‐localized with GFP-LC3 under starvation conditions (Earle's balanced salt solution; EBSS). Bar, 10 μm.


( C ) MCF7 / GFP - LC3 cells cultured in nutrient ‐ rich ( DMEM ) or starved ( EBSS ) conditions were homogenized and subjected to centrifugation , and the resulting post ‐ nuclear supernatant ( PNS ) was fractionated by high ‐ speed centrifugation into membrane pellet and cytosol . Proteins were resolved by SDS - PAGE and immunoblotted with anti ‐ TGN46 antibody as a control for membrane ‐ association proteins , anti ‐ caspase ‐ 3 as a control for cytosolic proteins . The levels of phospho ‐ MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS ( n = 3 ) . Each value represents mean ± s . e . of three experiments . * P0 . 05 , * * P0 . 01 .

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(C) MCF7/GFP-LC3 cells cultured in nutrient‐rich (DMEM) or starved (EBSS) conditions were homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by high‐speed centrifugation into membrane pellet and cytosol. Proteins were resolved by SDS-PAGE and immunoblotted with anti‐TGN46 antibody as a control for membrane‐association proteins, anti‐caspase‐3 as a control for cytosolic proteins. The levels of phospho‐MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS (n=3). Each value represents mean±s.e. of three experiments. *P0.05, **P0.01.