A . ( Left ) Hematoxylin and eosin ( H & E ) - stained cross section of an Ewing sarcoma ( ES ) of the shoulder tumor . Annotated regions are annotated as T - tumor and M - muscle . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . The image displays a heatmap gradient of intensity from white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A - E . B . ( Left ) H & E stained cross section of an ES of the chest wall tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . C . ( Left ) H & E stained cross section of an ES of the rib tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . D . ( Left ) H & E stained cross section of an ES of the abdomen tumor . Annotated regions are T - tumor , S - stroma , and A - adipose tissue . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . E . ( Left ) H & E stained cross section of an ES of the bladder tumor . Annotated regions are T - tumor and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . F . Magnified H & E cross section of ( top , left ) tumor , and ( top , right ) muscle tissue from ES rib with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , and ( bottom , right ) muscle tissues . G . Magnified H & E cross section of ( top , left ) tumor , ( top , middle ) stroma , and ( top , right ) adipose tissues from ES abdomen with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , ( bottom , middle ) stroma and ( bottom , right ) adipose tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (Left) Hematoxylin and eosin (H&E)-stained cross section of an Ewing sarcoma (ES) of the shoulder tumor. Annotated regions are annotated as T-tumor and M-muscle. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A-E. B. (Left) H&E stained cross section of an ES of the chest wall tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. C. (Left) H&E stained cross section of an ES of the rib tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. D. (Left) H&E stained cross section of an ES of the abdomen tumor. Annotated regions are T-tumor, S-stroma, and A-adipose tissue. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. E. (Left) H&E stained cross section of an ES of the bladder tumor. Annotated regions are T-tumor and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. F. Magnified H&E cross section of (top, left) tumor, and (top, right) muscle tissue from ES rib with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, and (bottom, right) muscle tissues. G. Magnified H&E cross section of (top, left) tumor, (top, middle) stroma, and (top, right) adipose tissues from ES abdomen with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, (bottom, middle) stroma and (bottom, right) adipose tissues.


A . ( Left ) Hematoxylin and eosin ( H & E ) stained cross section of a normal human tibia ( Top ) resected at the same time as the Ewing sarcoma ( ES ) of the tibia tumor ( Bottom ) . Annotated regions are CT - connective tissue , B - decalcified bone , BM - bone marrow , T - tumor , and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . The image displays a heatmap gradient of intensity with white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A . B . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from normal tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . # 0 . 01 < P < 0 . 05 ; * * * * P < 0 . 0001 , analyzed by two - way ANOVA with Tukey ' s multiple comparison for each glycogen chain length . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from the ES of the tibia tumor in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * 0 . 001 < P < 0 . 01 ; * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (Left) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A. B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


F . In vivo tumor growth of A673 EV or GYS1 - KO injected in the flanks of athymic nude mice . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test .

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

F. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


G . Half maximal effective concentration ( EC50 ) of the small molecular inhibitor guaiacol in A673 cells in vitro .

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

G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.


J . Spatial distribution of phosphorylated CL5 of human tibia ( top ) and tibial ES tumor ( bottom ) . The image displays a heatmap gradient of intensity with black ( least abundant ) to yellow ( most abundant ) . K . Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length .

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

J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant). K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


N . Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor ( T ) and stroma ( S ) tissues . Tissues were scanned digitally using the Axio Scan . Z1 side scanner . Scale bar is shown below the image . O . Quantification of immunohistochemical laforin staining between normal tissue ( n = 3 technical replicates per region ) , tibial ES tumor ( n = 7 technical replicates per region ) , and stroma of five ES tissues ( n = 7 ) . Values are presented as mean + / - standard error . * * 0 . 001 < P < 0 . 0 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison .

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

N. Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor (T) and stroma (S) tissues. Tissues were scanned digitally using the Axio Scan.Z1 side scanner. Scale bar is shown below the image. O. Quantification of immunohistochemical laforin staining between normal tissue (n=3 technical replicates per region), tibial ES tumor (n=7 technical replicates per region), and stroma of five ES tissues (n=7). Values are presented as mean +/- standard error. **0.001 < P < 0.0; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.


P . ( Top ) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors . ( Bottom ) Tumor growth of xenografts of A673 empty vector ( EV ) and laforin overexpression ( LAOE ) . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test .

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-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

P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


( B ) HEK293T cells transiently expressing FLAG and FLAG - tagged TEX264 were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies .

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

(B) HEK293T cells transiently expressing FLAG and FLAG-tagged TEX264 were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies.


( C ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies .

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

(C) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( D and E ) MEFs stably expressing TEX264 - GFP or its mutant were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 1 µm ( insets ) ( D ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 48 cells for each cell type ( E ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E) MEFs stably expressing TEX264-GFP or its mutant were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 1 µm (insets) (D). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 48 cells for each cell type (E).


( F and G ) WT and TEX264 - KO HeLa cells ( with or without the indicated TEX264 mutants with a C - terminal FLAG tag ) stably expressing the ER - phagy reporter ( ss - RFP - GFP - KDEL ) were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured in starvation medium lacking amino acids and serum for 9 h ( F ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± standard error of the mean ( SEM ) of three independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( G ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G) WT and TEX264-KO HeLa cells (with or without the indicated TEX264 mutants with a C-terminal FLAG tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured in starvation medium lacking amino acids and serum for 9 h (F). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).


( A and B ) MEFs stably expressing TEX264 - GFP were cultured in starvation media with or without bafilomycin A1 ( Baf A1 ) and immunostained with anti - pTEX264 and anti - LC3 antibodies . Bars : 10 µm and 1 µm ( insets ) ( A ) . Quantification of the number of pTEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 35 cells for each cell type ( B ) .

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

(A and B) MEFs stably expressing TEX264-GFP were cultured in starvation media with or without bafilomycin A1 (Baf A1) and immunostained with anti-pTEX264 and anti-LC3 antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).


( C ) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h , and cell homogenates were subjected to an OptiPrep membrane flotation analysis .

O O O O O O O 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) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.


( D , E and F ) WT , ATG3 - KO , and FIP200 - KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies ( D ) . Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown ( E and F ) . Data represent the mean ± SEM of three independent experiments .

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

(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown (E and F). Data represent the mean ± SEM of three independent experiments.


( A ) HeLa cells were cultured with 20 nM CK1 inhibitor ( D4476 ) or CK2 inhibitor ( CX4945 ) for 3 h , followed by starvation ( with the inhibitors ) for 3 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies .

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

(A) HeLa cells were cultured with 20 nM CK1 inhibitor (D4476) or CK2 inhibitor (CX4945) for 3 h, followed by starvation (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.


( B ) Recombinant TEX264 - FLAG was incubated with or without CK2 and the CK2 inhibitor ( CX4945 ) in the presence of 200 µM ATP for 1 h .

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

(B) Recombinant TEX264-FLAG was incubated with or without CK2 and the CK2 inhibitor (CX4945) in the presence of 200 µM ATP for 1 h.


( C ) HEK293T cells transiently expressing TEX264 - FLAG and one of the three Halo - CK2A isoforms or Halo - CK2B were subjected to immunoprecipitation with an anti - FLAG antibody and detected with anti - Halo and anti - FLAG antibodies .

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 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

(C) HEK293T cells transiently expressing TEX264-FLAG and one of the three Halo-CK2A isoforms or Halo-CK2B were subjected to immunoprecipitation with an anti-FLAG antibody and detected with anti-Halo and anti-FLAG antibodies.


( D and E ) HEK293T cells transiently expressing WT or TEX264 - FLAG mutants were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies ( D ) . The band intensities in the IP fractions were quantified , and the phospho - serine : FLAG ratio was calculated . Data represent the mean ± SEM of five independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( E ) .

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

(D and E) HEK293T cells transiently expressing WT or TEX264-FLAG mutants were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies (D). The band intensities in the IP fractions were quantified, and the phospho-serine:FLAG ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).


( F and G ) MEFs stably expressing TEX264 - GFP were cultured with the CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) and immunostained with an anti - LC3 antibody . Scale bars represent 10 μm and 1 μm ( insets ) ( F ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test . Data were collected from 85 cells for each cell type ( G ) .

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

(F and G) MEFs stably expressing TEX264-GFP were cultured with the CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) and immunostained with an anti-LC3 antibody. Scale bars represent 10 μm and 1 μm (insets) (F). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by an unpaired two-tailed Student's t-test. Data were collected from 85 cells for each cell type (G).


( H and I ) WT HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) for 6 h ( H ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of six independent experiments . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test ( I ) .

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

(H and I) WT HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) for 6 h (H). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).


( B ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies .

(B) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( C and D ) MEFs stably expressing TEX264 - GFP or its mutants were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 5 µm ( insets ) ( C ) . The intensity of the GFP signal under starvation conditions was quantified at TEX264 - GFP puncta and the ER . The puncta : ER signal ratio was calculated . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 70 puncta for each cell type ( D ) .

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

(C and D) MEFs stably expressing TEX264-GFP or its mutants were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264-GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 70 puncta for each cell type (D).


( E and F ) WT and TEX264 - KO ( expressing WT or TEX264 - FLAG mutants ) HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , the cells were cultured in starvation medium lacking amino acids and serum for 9 h ( E ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of four independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( F ) .

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(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG mutants) HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).


A Transfected GFP − Atg8 homologs were immunoprecipitated with μMACS ™ from total U2OS cell extracts followed by immunoblot analysis with anti − GFP and anti − ALFY antibodies . Data are representative of two independent experiments .

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A Transfected GFP−Atg8 homologs were immunoprecipitated with μMACS™ from total U2OS cell extracts followed by immunoblot analysis with anti−GFP and anti−ALFY antibodies. Data are representative of two independent experiments.


C The indicated 35S − labelled in vitro − translated GFP − tagged constructs were incubated with GST , GST − LC3B or GST − GABARAP conjugated to glutathione Sepharose , and their binding was evaluated by autoradiography ( ARG ) . 5 % of the in vitro − translated protein ( arrow head ) used was loaded . Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue ( CBB ) staining . Data are representative of three independent experiments .

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C The indicated 35S−labelled in vitro−translated GFP−tagged constructs were incubated with GST, GST−LC3B or GST−GABARAP conjugated to glutathione Sepharose, and their binding was evaluated by autoradiography (ARG). 5% of the in vitro−translated protein (arrow head) used was loaded. Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments.


D Recombinant ALFY ( aa 2981 - 3526 ) was incubated with GST ‐ Atg8 proteins conjugated to glutathione Sepharose . The pulled ‐ down complexes were subjected to SDS - PAGE and anti ‐ ALFY and anti ‐ GST immunoblotting . 5 % of the recombinant ALFY protein used was loaded as input . Data are representative of three independent experiments .

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D Recombinant ALFY (aa 2981-3526) was incubated with GST‐Atg8 proteins conjugated to glutathione Sepharose. The pulled‐down complexes were subjected to SDS-PAGE and anti‐ALFY and anti‐GST immunoblotting. 5% of the recombinant ALFY protein used was loaded as input. Data are representative of three independent experiments.


B 35S − labelled in vitro − translated GFP − ALFY ( aa 2981 - 3526 ) wild − type and different LIR mutants were incubated with GST − GABARAP or − LC3C and binding evaluated by ARG . 10 and 2 % of the in vitro − translated proteins used were loaded to illustrate binding affinity . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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B 35S−labelled in vitro−translated GFP−ALFY (aa 2981-3526) wild−type and different LIR mutants were incubated with GST−GABARAP or −LC3C and binding evaluated by ARG. 10 and 2% of the in vitro−translated proteins used were loaded to illustrate binding affinity. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


C MBP , MBP − ALFY ( aa 3255 - 3526 ) or the ALFY − LIR mutant ( F3346A ) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP . The pulled − down complexes were subjected to SDS - PAGE and visualized by CBB staining . Data are representative of three independent experiments .

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C MBP, MBP−ALFY (aa 3255-3526) or the ALFY−LIR mutant (F3346A) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP. The pulled−down complexes were subjected to SDS-PAGE and visualized by CBB staining. Data are representative of three independent experiments.


D Thermodynamic parameters of ALFY − LIR peptide ( aa 3341 - 3354 ) to Atg8 homologs . All titrations were performed at 25 ° C as described in Supplementary Methods . ITC data were fitted to a one − site binding model . ND , not detected .

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D Thermodynamic parameters of ALFY−LIR peptide (aa 3341-3354) to Atg8 homologs. All titrations were performed at 25°C as described in Supplementary Methods. ITC data were fitted to a one−site binding model. ND, not detected.


E 35S − labelled in vitro − translated GFP or GFP − Bchs ( aa 3326 - 3341 ) were incubated with GST or GST − dAtg8a and binding evaluated by ARG . 5 % of the in vitro − translated protein used was loaded . Ponceau staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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E 35S−labelled in vitro−translated GFP or GFP−Bchs (aa 3326-3341) were incubated with GST or GST−dAtg8a and binding evaluated by ARG. 5% of the in vitro−translated protein used was loaded. Ponceau staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


D GFP − LC3B and GFP − LC3B ( Q26K / H27Y / H57D ) were immunoprecipitated with GFP − TRAP ® from total cell lysate of stably transfected HeLa FlpIn cells , followed by SDS - PAGE and immunoblotting with the indicated antibodies . Data are representative of three independent experiments .

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D GFP−LC3B and GFP−LC3B (Q26K/H27Y/H57D) were immunoprecipitated with GFP−TRAP® from total cell lysate of stably transfected HeLa FlpIn cells, followed by SDS-PAGE and immunoblotting with the indicated antibodies. Data are representative of three independent experiments.


E MBP ‐ ALFY3255 - 3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants . The pulled ‐ down complexes were subjected to SDS - PAGE and visualized by immunoblotting with anti ‐ MBP and anti ‐ GABARAP antibodies . Data are representative of three independent experiments .

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E MBP‐ALFY3255-3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants. The pulled‐down complexes were subjected to SDS-PAGE and visualized by immunoblotting with anti‐MBP and anti‐GABARAP antibodies. Data are representative of three independent experiments.


F MBP − tagged ALFY3255 - 3526 and p62168 - 391 or their corresponding LIR mutants ( F3346A and W340A , respectively ) were conjugated to amylose resin and incubated with purified GST , GABARAP , GABARAP mutant ( K24Q / Y25H / D54H ) , LC3B or LC3B mutant ( Q26K / H27Y / H57D ) . The bound proteins were visualized by immunoblotting with anti − GABARAP , anti − LC3 and anti − MBP antibodies . Data are representative of three independent experiments .

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F MBP−tagged ALFY3255-3526 and p62168-391 or their corresponding LIR mutants (F3346A and W340A, respectively) were conjugated to amylose resin and incubated with purified GST, GABARAP, GABARAP mutant (K24Q/Y25H/D54H), LC3B or LC3B mutant (Q26K/H27Y/H57D). The bound proteins were visualized by immunoblotting with anti−GABARAP, anti−LC3 and anti−MBP antibodies. Data are representative of three independent experiments.


G 35S ‐ labelled in vitro ‐ translated GFP ‐ ALFY ( aa 2981 - 3526 ) wild ‐ type and K3343A / D3344A / Y3351A mutant were incubated with GST ‐ LC3B and GABARAP and binding evaluated by ARG . 10 % of the in vitro ‐ translated proteins used was loaded . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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G 35S‐labelled in vitro‐translated GFP‐ALFY (aa 2981-3526) wild‐type and K3343A/D3344A/Y3351A mutant were incubated with GST‐LC3B and GABARAP and binding evaluated by ARG. 10% of the in vitro‐translated proteins used was loaded. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


A HeLa FlpIn GFP − GABARAP cells were treated with proteasomal inhibitor ( MG132 , 2 h ) or subjected to amino acid starvation ( EBSS , 2 h ) , before staining with anti − ALFY antibodies . Scale bar , 10 μm .

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A HeLa FlpIn GFP−GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti−ALFY antibodies. Scale bar, 10 μm.


B GFP − tagged full − length ALFY wild − type or LC3 − interacting region ( LIR ) − mutant was expressed into Alfy − deficient MEFs using an adenovirus system . At 48 h after infection , the MEFs were cultured in normal media or EBSS for 1 . 5 h before staining with anti − LC3B or anti − GABARAP antibodies . Scale bars 10 μm .

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B GFP−tagged full−length ALFY wild−type or LC3−interacting region (LIR)−mutant was expressed into Alfy−deficient MEFs using an adenovirus system. At 48 h after infection, the MEFs were cultured in normal media or EBSS for 1.5 h before staining with anti−LC3B or anti−GABARAP antibodies. Scale bars 10 μm.


C HeLa cells were treated with control or siRNA targeting GABARAP , GABARAPL1 and GABARAPL2 . 72 h after transfection cells were immunostained with anti − LC3B , anti − ALFY and anti − p62 antibodies . Scale bars , 10 μm .

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C HeLa cells were treated with control or siRNA targeting GABARAP, GABARAPL1 and GABARAPL2. 72 h after transfection cells were immunostained with anti−LC3B, anti−ALFY and anti−p62 antibodies. Scale bars, 10 μm.


D HeLa cells were treated with control or siRNA targeting ALFY . 72 h after transfection , cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX − 100 − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments .

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D HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX−100−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.


E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X − 100 ( TX − 100 ) − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments .

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E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X−100 (TX−100)−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.


B . ERK71 mutants have elevated triacylglycerol ( TAG ) levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . C . Fat body specific depletion of ERK7 by RNAi ( BDSC 56939 ) leads to increased TAG levels ( N = 4 replicates of 10 larvae / replicate for each genotype ) .

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B. ERK71 mutants have elevated triacylglycerol (TAG) levels (N=4 replicates of ≥10 larvae/ replicate for each genotype). C. Fat body specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels (N=4 replicates of 10 larvae/replicate for each genotype).


D . Representative immunofluorescent images of lipid droplet ( LipidTOX ) and nuclear ( DAPI ) staining in control and ERK71 mutant fat bodies of third instar larvae . Scale bar : 50 µm . E . ERK71 mutant fat body cells contain more lipid droplets than control cells ( N = 30 cells for each genotype ) .

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D. Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK71 mutant fat bodies of third instar larvae. Scale bar: 50 µm. E. ERK71 mutant fat body cells contain more lipid droplets than control cells (N=30 cells for each genotype).


F . After fed [ 13C ] glucose , the ERK71 mutant fat bodies display elevated [ 13C ] TAG / PE and unlabeled TAG / PE molar ratios ( measured by mass spectrometry - based lipidomics , N = 3 replicates of 15 fat bodies / replicate , TAG = triacylglycerol , PE = phosphatidylethanolamine )

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F. After fed [13C]glucose, the ERK71 mutant fat bodies display elevated [13C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry-based lipidomics, N=3 replicates of 15 fat bodies/replicate, TAG=triacylglycerol, PE=phosphatidylethanolamine)


G . ERK7 overexpression in the fat body results in reduced TAG levels , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the TAG storage ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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G. ERK7 overexpression in the fat body results in reduced TAG levels, while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage (N=4 replicates of ≥10 larvae/replicate for each genotype).


H - ERK7 expressing , GFP - marked , fat body clone ( H , marked by yellow dotted line ; scale bar : 50 µm ) visualized by LipidTOX staining ( N = 11 for control and 4 for ERK7 overexpression ) .

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H- ERK7 expressing, GFP-marked, fat body clone (H, marked by yellow dotted line; scale bar: 50 µm) visualized by LipidTOX staining (N=11 for control and 4 for ERK7 overexpression).


ERK7 expressing , fat body clone contains less ( I ) and smaller ( J ) lipid droplets than control cells ( N = 11 for control and 4 for ERK7 overexpression ) .

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ERK7 expressing, fat body clone contains less (I) and smaller (J) lipid droplets than control cells (N=11 for control and 4 for ERK7 overexpression).


K . qRT - PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation . Expression of RP49 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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K. qRT-PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation. Expression of RP49 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


A . Fatty acid composition of total lipids in CG > control and CG > ERK7 larvae , measured by gas chromatography ( N = 3 replicates of 15 larvae / replicate for each genotype ) .

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A. Fatty acid composition of total lipids in CG>control and CG>ERK7 larvae, measured by gas chromatography (N=3 replicates of 15 larvae/replicate for each genotype).


C . Amount of glucose - derived [ 13C ] - labeled carbons incorporated into the TAG pool of control and CG > ERK7 fat bodies ( N = 3 replicates of 15 fat bodies / replicate for each genotype and diet ) .

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C. Amount of glucose-derived [13C]-labeled carbons incorporated into the TAG pool of control and CG>ERK7 fat bodies (N=3 replicates of 15 fat bodies/replicate for each genotype and diet).


D . Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels ( N = 3 replicates of 15 larvae / replicate for each genotype ) . E . Circulating glucose levels are decreased in ERK71 mutants ( N = 4 replicates of 15 larvae / replicate for each genotype ) .

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D. Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels (N=3 replicates of 15 larvae/replicate for each genotype). E. Circulating glucose levels are decreased in ERK71 mutants (N=4 replicates of 15 larvae/replicate for each genotype).


A . Four - way plot ( t - statistics ) presenting the ERK7 - dependent fasting - responsive genes ( adj . p . val < 0 . 05 ) . Dark blue indicates significant in both comparisons ; dark turquoise , significant in one comparison ; light turquoise , not significant .

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A. Four-way plot (t-statistics) presenting the ERK7-dependent fasting-responsive genes (adj.p.val < 0.05). Dark blue indicates significant in both comparisons; dark turquoise, significant in one comparison; light turquoise, not significant.


D . Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism . Color key displays scaled log2 gene expression .

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D. Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism. Color key displays scaled log2 gene expression.


E , F . Expression analysis of Seipin , Agpat3 , ATPCL , fabp , Lip4 and CG34448 by qRT - PCR . RP49 was used for normalization ( N = 3 replicates of ≥ 10 larvae / replicate for each genotype ) .

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E, F. Expression analysis of Seipin, Agpat3, ATPCL, fabp, Lip4 and CG34448 by qRT-PCR. RP49 was used for normalization (N=3 replicates of ≥10 larvae/replicate for each genotype).


A . Weight of control and ERK71 mutant larvae at 72h after egg deposition ( AED ) ( N = 4 replicates of 12 larvae / replicate for each genotype ) .

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A. Weight of control and ERK71 mutant larvae at 72h after egg deposition (AED) (N=4 replicates of 12 larvae/replicate for each genotype).


B . Pupariation kinetics of control and ERK71 mutants ( N = 4 replicates of 30 larvae / replicate for each genotype ) . p < 0 . 0001 , analyzed by Log - rank test .

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B. Pupariation kinetics of control and ERK71 mutants (N=4 replicates of 30 larvae/replicate for each genotype). p<0.0001, analyzed by Log-rank test.


C . ERK71 mutants fail to suppress growth upon 24 h of starvation ( N ≥ 4 replicates of 15 larvae / replicate for each genotype ) .

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C. ERK71 mutants fail to suppress growth upon 24 h of starvation (N≥4 replicates of 15 larvae/replicate for each genotype).


D . ERK71 mutant larvae ( N = 4 replicates of 24 larvae / replicate for each genotype , p < 0 . 01 analyzed by Log - rank test ) are sensitive to nutrient deprivation .

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D. ERK71 mutant larvae (N=4 replicates of 24 larvae/replicate for each genotype, p<0.01 analyzed by Log-rank test) are sensitive to nutrient deprivation.


Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to a moderately accelerated pupariation rate ( E , p < 0 . 001 analyzed by Log - rank test , N = 8 replicates of 30 larvae / replicate for each genotype )

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Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to a moderately accelerated pupariation rate (E, p<0.001 analyzed by Log-rank test, N=8 replicates of 30 larvae/replicate for each genotype)


F . Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to an increase in larval weight at 72h AED ( F , N = 5 replicates of 15 larvae / replicate for each genotype ) .

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F. Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to an increase in larval weight at 72h AED (F, N=5 replicates of 15 larvae/replicate for each genotype).


G . A representative image showing that fat body - specific overexpression of ERK7 by the CG - GAL4 driver ( CG > ERK7 ) impairs larval growth . Scale bar : 0 . 5 mm .

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G. A representative image showing that fat body-specific overexpression of ERK7 by the CG-GAL4 driver (CG>ERK7) impairs larval growth. Scale bar: 0.5 mm.


H . Ectopic expression of ERK7 leads to delayed pupariation ( p < 0 . 0001 , analyzed by Log - rank test ) , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence pupariation rate ( N = 4 replicates of 30 larvae / replicate for each genotype ) .

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H. Ectopic expression of ERK7 leads to delayed pupariation (p<0.0001, analyzed by Log-rank test), while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence pupariation rate (N=4 replicates of 30 larvae/replicate for each genotype).


I . In contrast to ERK7 wt , kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the pupal volume ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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I. In contrast to ERK7 wt, kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the pupal volume (N=4 replicates of 10 pupae/replicate for each genotype).


A . Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus . Scale bar : 30 µm . B , C . ERK71 mutant fat bodies display increased nuclear area ( B , N > 160 cells , obtained from 5 independent fat bodies ) and increased nucleolar / nuclear area ratio ( C , N > 80 cells for nucleolus / nucleus ratio , obtained from 5 independent fat bodies ) , when compared to control fat bodies .

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A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies.


D - F . ERK7 expressing fat body clones ( D , marked by GFP , scale bar : 20 µm ) display reduced nuclear area ( E ) and reduced nucleolar / nuclear area ratio ( F ) , when compared to control cells . Nuclei were visualized by DAPI staining , nucleoli were visualized by anti - Fibrillarin antibodies ( N = 10 ) .

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D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10).


G , H . Expression analysis of Pol I ( G ) and Pol III ( H ) target RNAs by qRT - PCR . Expression of CDK7 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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G, H. Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT-PCR. Expression of CDK7 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


A . ERK7 overexpression leads to phosphorylation of PWP1 , as shown by appearance of an extra band on Phos - tag Western blot ( marked with red arrow ) . Insulin - inducible bands ( dark blue ) remain unresponsive to ERK7 .

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A. ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos-tag Western blot (marked with red arrow). Insulin-inducible bands (dark blue) remain unresponsive to ERK7.


B . Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae . Scale bar : 20 µm . C . PWP1 localization displays increased nucleolar / nuclear ratio in ERK71 mutant fat bodies compared to control ( N = 20 cells from 5 independent fat bodies per genotype ) .

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B. Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae. Scale bar: 20 µm. C. PWP1 localization displays increased nucleolar/nuclear ratio in ERK71 mutant fat bodies compared to control (N=20 cells from 5 independent fat bodies per genotype).


D . Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae . ERK7 overexpression ( GFP marked clones ) alters the subcellular localization of PWP1 . Scale bar : 30 µm . E . PWP1 localization displays reduced nuclear / cytoplasmic ratio in ERK7 overexpressing clones , compared to control cells ( N = 10 cells from at least 8 different fat bodies per genotype ) .

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D. Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae. ERK7 overexpression (GFP marked clones) alters the subcellular localization of PWP1. Scale bar: 30 µm. E. PWP1 localization displays reduced nuclear/cytoplasmic ratio in ERK7 overexpressing clones, compared to control cells (N=10 cells from at least 8 different fat bodies per genotype).


F . Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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F. Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels (N=4 replicates of ≥10 larvae/replicate for each genotype).


G . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body ( CG - GAL4 ) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . H , I . Fat body specific knockdown of PWP1 suppresses increased lipid droplet number ( H ; N > 30 cells per genotype ) and increased nuclear area ( I ; N > 45 cells per genotype ) caused by ERK7 knockdown ( BDSC 56939 ) .

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G. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG-GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. H, I. Fat body specific knockdown of PWP1 suppresses increased lipid droplet number (H; N>30 cells per genotype) and increased nuclear area (I; N>45 cells per genotype) caused by ERK7 knockdown (BDSC 56939).


A , B . Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 ( A ) and ectopic expression of ERK7 ( B ) ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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A, B. Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 (A) and ectopic expression of ERK7 (B) (N=3 replicates of 10 fat bodies/replicate for each genotype).


C . ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting ( N = 4 replicates of 10 larvae / replicate for each genotype ) .

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C. ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting (N=4 replicates of 10 larvae/replicate for each genotype).


D . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants ( sug17Δ / Df ( 2R ) Exel7123 ) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . E . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased lipid droplet number observed in ERK71 mutants ( N > 20 ) .

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D. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants (sug17Δ/Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. E. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK71 mutants (N>20).


F . Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG > ERK7 larvae ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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F. Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae (N=4 replicates of ≥10 larvae/replicate for each genotype).


G . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells ( N ≥ 25 cells per genotype ) .

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G. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells (N ≥25 cells per genotype).


Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate ( H ) ( p < 0 . 001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype ) of CG > ERK7

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Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate (H) (p<0.001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype) of CG>ERK7 larvae


I . Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG > ERK7 larvae ( I ) ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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I. Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG>ERK7 larvae (I) (N=4 replicates of 10 pupae/replicate for each genotype).


J . Addition of 20 % sucrose improves the growth rate and survival of CG > ERK7 larvae . Pupariation kinetics of CG > ERK7 larvae on 20 % yeast or 20 % yeast + 20 % sucrose diet ( p < 0 . 0001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype and diet ) .

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J. Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet (p<0.0001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype and diet).


( F ) Representative images of GFP - positive HEK293 cells at day 7 post - transduction using 1x106 TU / cell of RGD4C . TPA . GFP or RGD4C . AAVP . GFP particles . Non - targeted ( NT ) particles were included as control . Data shown are representative of five independent experiments , performed each in triplicates . Scale bar , 100 μm .

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(F) Representative images of GFP-positive HEK293 cells at day 7 post-transduction using 1x106 TU/cell of RGD4C.TPA.GFP or RGD4C.AAVP.GFP particles. Non-targeted (NT) particles were included as control. Data shown are representative of five independent experiments, performed each in triplicates. Scale bar, 100 μm.


( A ) Quantification of gene expression in HEK293 cells after transduction with 0 . 5x106 , 1x106 , and 2x106 TU / cell of RGD4C . TPA , RGD4C . AAVP or non - targeted ( NT ) particles carrying Lucia . Luciferase activity was measured at days 1 to 6 post - transduction . Data shown are representative of three independent experiments ( n = 3 ) , and are expressed as mean + SEM . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(A) Quantification of gene expression in HEK293 cells after transduction with 0.5x106, 1x106, and 2x106 TU/cell of RGD4C.TPA, RGD4C.AAVP or non-targeted (NT) particles carrying Lucia. Luciferase activity was measured at days 1 to 6 post-transduction. Data shown are representative of three independent experiments (n = 3), and are expressed as mean + SEM. One-way ANOVA with Tukey's HSD test was used for data analysis.


( B ) Representative images of FITC - labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post - inoculation ( n = 3 ) . Scale bar , 100 μm . ( C ) Particle diffusion using a vertical trans - well system partitioned by Matrigel . Samples were collected from the wells below the Matrigel at days 1 to 7 , n = 3 , technical replicates . Data are expressed as mean + SEM , independent t - test was used for data analysis .

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(B) Representative images of FITC-labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post-inoculation (n = 3). Scale bar, 100 μm. (C) Particle diffusion using a vertical trans-well system partitioned by Matrigel. Samples were collected from the wells below the Matrigel at days 1 to 7, n = 3, technical replicates. Data are expressed as mean + SEM, independent t-test was used for data analysis.


( D ) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins , then quantified by FACS at 2 and 4 hours post - transduction , 0 . 1X106TU / cell , and normalised to particle length . Data are expressed as mean + SEM of n = 3 independent experiments , independent t - test was used for data analysis .

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(D) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins, then quantified by FACS at 2 and 4 hours post-transduction, 0.1X106TU/cell, and normalised to particle length. Data are expressed as mean + SEM of n = 3 independent experiments, independent t-test was used for data analysis.


( E ) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post - transduction with 0 . 5x106 TU / cell of RGD4C . TPA and RGD4C . AAVP , using qPCR with primers specific to AAV - 2 ITRs . Data are representative of n = 3 independent experiments and expressed as mean + SEM , independent t - test was used for data analysis .

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(E) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post-transduction with 0.5x106 TU/cell of RGD4C.TPA and RGD4C.AAVP, using qPCR with primers specific to AAV-2 ITRs. Data are representative of n = 3 independent experiments and expressed as mean + SEM, independent t-test was used for data analysis.


( F ) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma - derived neural stem cells with 1x106 TU / cell . Data are expressed as mean + SEM of n = 3 independent experiments . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(F) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma-derived neural stem cells with 1x106 TU/cell. Data are expressed as mean + SEM of n = 3 independent experiments. One-way ANOVA with Tukey's HSD test was used for data analysis.


( G ) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia . Experiments were repeated three times and performed in triplicates . Data are shown as mean + SEM and are representative of one experiment . Independent t - test was used for data analysis .

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(G) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia. Experiments were repeated three times and performed in triplicates. Data are shown as mean + SEM and are representative of one experiment. Independent t-test was used for data analysis.


( B ) ELISA quantification of TNFα production in media collected on days 3 to 5 post - transduction of human primary GBM001 tumor spheres at 1x106 TU / cell of targeted ( RGD4C ) or non - targeted ( NT ) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(B) ELISA quantification of TNFα production in media collected on days 3 to 5 post-transduction of human primary GBM001 tumor spheres at 1x106 TU/cell of targeted (RGD4C) or non-targeted (NT) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis.


( C ) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C . TPA . TNFαIL2 to NOD / SCID mice bearing subcutaneous human GBM001 . Tumors and healthy organs were harvested at day 18 post - transduction and analysed using RT - qPCR to quantify the TNFαIL2 transcripts . Data shown are expressed as mean + SEM , from n = 3 mice . Two - way ANOVA was used for data analysis .

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(C) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C.TPA.TNFαIL2 to NOD/SCID mice bearing subcutaneous human GBM001. Tumors and healthy organs were harvested at day 18 post-transduction and analysed using RT-qPCR to quantify the TNFαIL2 transcripts. Data shown are expressed as mean + SEM, from n = 3 mice. Two-way ANOVA was used for data analysis.


( D ) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene , at day 18 post targeted TNFαIL2 cytokine treatment . ( E ) Analysis of tumor growth in representative mice , from D , using the tumor bioluminescence data ( total flux ) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0 .

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(D) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene, at day 18 post targeted TNFαIL2 cytokine treatment. (E) Analysis of tumor growth in representative mice, from D, using the tumor bioluminescence data (total flux) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0.


( B ) Investigation of the TPA . IL15IgK in comparison to the native TPA . IL15 in B16 . F1 and CT26 . CL25 tumor cells . The TPA DNA constructs were transfected into tumor cells , then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL - 2 . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice and performed in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data

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(B) Investigation of the TPA.IL15IgK in comparison to the native TPA.IL15 in B16.F1 and CT26.CL25 tumor cells. The TPA DNA constructs were transfected into tumor cells, then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL-2. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice and performed in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis


( C ) Targeted ( RGD4C ) and non - targeted ( NT ) TPA particles carrying IL15IgK were produced and used to transduce B16 . F1 or CT26 . CL25 cells at 0 . 5x106 or 1x106 TU / cell . Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post - transduction . RGD4C . TPA without IL15IgK ( mock ) was also included as control . Experiments were repeated twice and shown are the results from a representative experiment . Data are expressed as mean + SEM , one - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(C) Targeted (RGD4C) and non-targeted (NT) TPA particles carrying IL15IgK were produced and used to transduce B16.F1 or CT26.CL25 cells at 0.5x106 or 1x106 TU/cell. Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post-transduction. RGD4C.TPA without IL15IgK (mock) was also included as control. Experiments were repeated twice and shown are the results from a representative experiment. Data are expressed as mean + SEM, one-way ANOVA with Tukey's HSD test was used for data analysis.


( D ) Evaluation of CTLL - 2 proliferation in the presence of media collected from B16 . F1 or CT26 . CL25 tumor cells after transduction with TPA particles carrying IL15IgK . Cells treated with a recombinant IL2 cytokine were included as a positive control . Data are representative of one experiment , expressed as mean + SEM and shown as change in CTLL - 2 number relative to treatment initiation day . Experiments were repeated twice and in triplicates , one - way ANOVA with Tukey ' s HSD test was used to analyse the data .

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(D) Evaluation of CTLL-2 proliferation in the presence of media collected from B16.F1 or CT26.CL25 tumor cells after transduction with TPA particles carrying IL15IgK. Cells treated with a recombinant IL2 cytokine were included as a positive control. Data are representative of one experiment, expressed as mean + SEM and shown as change in CTLL-2 number relative to treatment initiation day. Experiments were repeated twice and in triplicates, one-way ANOVA with Tukey's HSD test was used to analyse the data.


( A ) Tumor - bearing BALB / c mice with established subcutaneous CT26 . CL25 tumors were intravenously injected with a single dose , 5x1010 TU / mouse , of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration . Data are representative of one experiment , n = 3 , and expressed as mean + SEM . Two - way ANOVA test was used for data analysis .

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(A) Tumor-bearing BALB/c mice with established subcutaneous CT26.CL25 tumors were intravenously injected with a single dose, 5x1010 TU/mouse, of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration. Data are representative of one experiment, n = 3, and expressed as mean + SEM. Two-way ANOVA test was used for data analysis.


( B ) Safety of dose escalation regimens assessed at day 5 following TPA delivery , by evaluating the serum levels of LDH from n = 2 mice / TPA dose of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Serums from untreated mice were also analysed .

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(B) Safety of dose escalation regimens assessed at day 5 following TPA delivery, by evaluating the serum levels of LDH from n = 2 mice/TPA dose of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Serums from untreated mice were also analysed.


( C ) Representative tumor - bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C . TPA . IL15IgK ( RGD4C ) or non - targeted ( NT ) particles . The CT26 . CL25 tumor cells stably express a luciferase Luc reporter gene . Untreated mice were also included as controls ( CTRL ) .

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(C) Representative tumor-bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C.TPA.IL15IgK (RGD4C) or non-targeted (NT) particles. The CT26.CL25 tumor cells stably express a luciferase Luc reporter gene. Untreated mice were also included as controls (CTRL).


( D ) Changes in tumor viability in mice ( n = 4 ) from day 0 to day 5 post TPA administration . Tumor viability was measured by bioluminescence activity . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Kruskal - Wallis test was used for data analysis .

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(D) Changes in tumor viability in mice (n = 4) from day 0 to day 5 post TPA administration. Tumor viability was measured by bioluminescence activity. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Kruskal-Wallis test was used for data analysis.


( E ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from all experimental groups .

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(E) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from all experimental groups.


( F ) Animal weights ( n = 4 ) were monitored during therapy experiments ad expressed as mean + SEM .

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(F) Animal weights (n = 4) were monitored during therapy experiments ad expressed as mean + SEM.


( A ) The immune profile of CT26 . CL25 tumors was investigated using RT - qPCR at day 5 after TPA . IL15IgK administration . A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8 + T cell population ; NKp46 for NK cell population . Markers for cytotoxic cell - killing by CD8 + T cells ( GZMa , GZMb ) and by NK cells ( Prf1 ) were also analysed . Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma ( IFN - γ ) , and FoxP3 for Treg cells . Data shown are representative of n = 5 mice and expressed as mean + SEM . Non - parametric Mann - Whitney test was used for GZMb and TBX2 data analysis , and independent t - test to analyse data of the other markers .

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

(A) The immune profile of CT26.CL25 tumors was investigated using RT-qPCR at day 5 after TPA.IL15IgK administration. A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8+ T cell population; NKp46 for NK cell population. Markers for cytotoxic cell-killing by CD8+ T cells (GZMa, GZMb) and by NK cells (Prf1) were also analysed. Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma (IFN-γ), and FoxP3 for Treg cells. Data shown are representative of n = 5 mice and expressed as mean + SEM. Non-parametric Mann-Whitney test was used for GZMb and TBX2 data analysis, and independent t-test to analyse data of the other markers.


( B ) TPA particles encoding IL12 were used to treat immunocompetent BALB / c mice bearing subcutaneous CT26 . CL25 tumors labelled with a Luc reporter gene . Representative tumor - bearing mice at day 5 post - treatment with targeted RGD4C . TPA . IL12 ( RGD4C ) or non - targeted TPA . IL12 ( NT ) .

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(B) TPA particles encoding IL12 were used to treat immunocompetent BALB/c mice bearing subcutaneous CT26.CL25 tumors labelled with a Luc reporter gene. Representative tumor-bearing mice at day 5 post-treatment with targeted RGD4C.TPA.IL12 (RGD4C) or non-targeted TPA.IL12 (NT).


( C ) Analysis of total flux change in tumors , n = 4 mice , after IL12 immunotherapy . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Mann - Whitney test was used for data analysis .

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(C) Analysis of total flux change in tumors, n = 4 mice, after IL12 immunotherapy. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Mann-Whitney test was used for data analysis.


( D ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from two experimental groups .

(D) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from two experimental groups.


( E ) Serial quantification of IL12 in media of B16 . F10 cells treated with RGD4C . TPA . IL12 or controls . Data are representative of one experiment , n = 3 biological repeats , and shown as mean + SD Experiments were repeated three times . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(E) Serial quantification of IL12 in media of B16.F10 cells treated with RGD4C.TPA.IL12 or controls. Data are representative of one experiment, n = 3 biological repeats, and shown as mean + SD Experiments were repeated three times. One-way ANOVA with Tukey's HSD test was used for data analysis.


( F ) RGD4C . TPA . IL12 immunotherapy of subcutaneous B16 . F10 tumors in C57BL / 6 mice ( n = 4 ) . Data are shown as mean + SEM independent t - test was used for data analysis . Tumor volume changes are shown over time .

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(F) RGD4C.TPA.IL12 immunotherapy of subcutaneous B16.F10 tumors in C57BL/6 mice (n = 4). Data are shown as mean + SEM independent t-test was used for data analysis. Tumor volume changes are shown over time.


Kaplan - Meier survival curves stratified by the patient MICA - 129 genotype for all patients ( n = 446 ) . The survival curve is displayed for the first 66 months ; 7 . 6 % of the patients were followed longer . Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2 . The HR indicates the risk per MICA - 129Met allele carried by the patients ( additive risk model ) . The numbers of patients carrying the three genotypes and the number of events ( in brackets ) are indicated .

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Kaplan-Meier survival curves stratified by the patient MICA-129 genotype for all patients (n = 446). The survival curve is displayed for the first 66 months; 7.6% of the patients were followed longer. Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2. The HR indicates the risk per MICA-129Met allele carried by the patients (additive risk model). The numbers of patients carrying the three genotypes and the number of events (in brackets) are indicated.


Kaplan - Meier survival curves for patients receiving a graft matched for the MICA - 129 genotype ( n = 404 ) .

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Kaplan-Meier survival curves for patients receiving a graft matched for the MICA-129 genotype (n = 404).


Kaplan - Meier survival curves for patients who experienced aGVDH ( any grade , n = 244 ) .

Kaplan-Meier survival curves for patients who experienced aGVDH (any grade, n = 244).


Kaplan - Meier survival curves for patients not experiencing aGVHD ( n = 189 ) . The HR indicates the risk of patients carrying two MICA - 129Met alleles ( recessive risk model ) .

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Kaplan-Meier survival curves for patients not experiencing aGVHD (n = 189). The HR indicates the risk of patients carrying two MICA-129Met alleles (recessive risk model).


Kaplan - Meier survival curves for patients who did not receive a T - cell - depleting treatment with ATG ( n = 193 ) . Kaplan - Meier survival curves for patients treated with ATG ( n = 250 ) .

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Kaplan-Meier survival curves for patients who did not receive a T-cell-depleting treatment with ATG (n = 193).Kaplan-Meier survival curves for patients treated with ATG (n = 250).


Kaplan - Meier survival curves for patients with the MICA - 129Val / Val genotype ( n = 229 ) stratified by treatment with ATG .

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Kaplan-Meier survival curves for patients with the MICA-129Val/Val genotype (n = 229) stratified by treatment with ATG.


Kaplan - Meier survival curves for patients carrying one or two MICA - 129Met alleles ( n = 214 ) stratified by treatment with ATG .

Kaplan-Meier survival curves for patients carrying one or two MICA-129Met alleles (n = 214) stratified by treatment with ATG.


The linear regression of MICA expression intensity and binding of a recombinant NKG2D - Fc fusion protein both determined as MFI by flow cytometry is displayed for L - MICA - 129Met ( n = 79 , left panel ) and L - MICA - 129Val clones ( n = 81 , right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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The linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met (n = 79, left panel) and L-MICA-129Val clones (n = 81, right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


Purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells ( 106 ) were stimulated with immobilized MICA - 129Met - mIgG2a - Fc or MICA - 129Val - mIgG2a - Fc or OVA - mIgG2a - Fc fusion proteins ( 10 μg / ml ) for 3 , 10 , or 30 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb , an anti - phospho - SRC family ( Tyr419 ) kinases Ab , and an anti - β - actin mAb as a loading control . The arrow points toward phosphorylated SRC family kinases . Blots obtained from three independent experiments were analyzed by densitometry , and the means plus SD of the ratio between phospho - SRC family kinase and β - actin signals is displayed . The difference between NK cells stimulated for 10 min by MICA - 129Met - Fc or MICA - 129Val - Fc proteins was assessed by t - 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 B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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

Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (106) were stimulated with immobilized MICA-129Met-mIgG2a-Fc or MICA-129Val-mIgG2a-Fc or OVA-mIgG2a-Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases.Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t-test.


Purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days , 106 ) were incubated with the SRC kinase inhibitor PP2 ( 25 μM ) , the vehicle DMSO , or medium only ( Ø ) for 30 min before being added to immobilized MICA - 129Met - Fc , MICA - 129Val - Fc , or OVA - Fc fusion proteins ( 10 μg / ml ) for 10 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb and an anti - β - actin mAb as a loading control . The blot is representative for two independent experiments .

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Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.


In parallel , degranulation of the NK cells was measured by anti - CD107a staining in flow cytometry . The difference between DMSO - and PP2 - treated cells with respect to CD107a + cells and the MFI of CD107a is indicated in the histograms . The results are representative for two independent experiments .

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In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.


The degranulation ( CD107a expression ) of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells in response to immobilized MICA - 129Met - Fc and MICA - 129Val - Fc , and as negative control , OVA - Fc fusion proteins was determined by flow cytometry after 1 h ( n = 3 ) . Displayed are means and SD of the MFI ( left panel ) and the percentage of CD107a + cells ( right panel ) . Differences between MICA - 129Met - Fc - and MICA - 129Val - Fc - induced NK - cell degranulation were analyzed by two - way ANCOVA adjusted for MICA protein concentration , and the respective P - values are indicated .

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The degranulation (CD107a expression) of purified IL-2-stimulated (100 U/ml for 4 days) NK cells in response to immobilized MICA-129Met-Fc and MICA-129Val-Fc, and as negative control, OVA-Fc fusion proteins was determined by flow cytometry after 1 h (n = 3). Displayed are means and SD of the MFI (left panel) and the percentage of CD107a+ cells (right panel). Differences between MICA-129Met-Fc- and MICA-129Val-Fc-induced NK-cell degranulation were analyzed by two-way ANCOVA adjusted for MICA protein concentration, and the respective P-values are indicated.


The degranulation of IL - 2 - stimulated LAK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 27 ) or L - MICA - 129Val clones ( n = 27 ) for 1 h was determined by flow cytometry . CD107a cell surface expression was analyzed after gating on CD56 + NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of CD107a on NK cells ( upper panels ) or the proportion of CD107a + NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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The degranulation of IL-2-stimulated LAK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 27) or L-MICA-129Val clones (n = 27) for 1 h was determined by flow cytometry. CD107a cell surface expression was analyzed after gating on CD56+ NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of CD107a on NK cells (upper panels) or the proportion of CD107a+ NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.