Figure 7A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT - qPCR . ( N , Normal tissue ; T , Tumor tissue ) * * p < 0 . 01 ( non - parametric Mann - Whitney t - test ) B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues . NAT10 - specific immuno - complex was detected with DAB as shown as brown color . Quantification of NAT10 - positive or NAT10 - negative CRC cases is shown in the lower table . C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top . Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53 . D CRISPR - Cas9 system mediated NAT10 gene editing . The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures . The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins . E HCT116 Ctrl or c2 - 4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points . Cell cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test ) F HCT116 Ctrl cells or c2 - 4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed . The total proteins were analyzed by western blot using the indicated antibodies . G HCT116 Ctrl or c2 - 4 cells were irradiated with 80 J / m2 UV . Forty - eight hours later , apoptotic cells were determined . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test ) H Colony formation assay were performed with HCT116 cells ( Ctrl , c1 - 1 and c2 - 4 ) . The right panel shows the quantification of formed colonies . ( two - tailed Student ' s t test ) I HCT116 c1 - 1 and c2 - 4 cell lines were plated and cell growth curves were determined as described in Fig 6F . Error bars represent the SEM of the triplicate experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT-qPCR. (N, Normal tissue; T, Tumor tissue) **p<0.01 (non-parametric Mann-Whitney t-test)B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues. NAT10-specific immuno-complex was detected with DAB as shown as brown color. Quantification of NAT10-positive or NAT10-negative CRC cases is shown in the lower table.C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top. Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53.D CRISPR-Cas9 system mediated NAT10 gene editing. The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures. The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins.E HCT116 Ctrl or c2-4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)F HCT116 Ctrl cells or c2-4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed. The total proteins were analyzed by western blot using the indicated antibodies.G HCT116 Ctrl or c2-4 cells were irradiated with 80 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)H Colony formation assay were performed with HCT116 cells (Ctrl, c1-1 and c2-4). The right panel shows the quantification of formed colonies. (two-tailed Student's t test)I HCT116 c1-1 and c2-4 cell lines were plated and cell growth curves were determined as described in Fig 6F. Error bars represent the SEM of the triplicate experiments.


Figure 2Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR - GFP and H2B - RFP were treated as described above and images were acquired once per hour for 12 h ( A ) . For one representative experiment among three , the mean ± SEM of the average area of high CALR dots ( normalized to the control at each timepoint ) of quadruplicates is shown ( B ) . Values are depicted as the area under the curve ± SD of triplicatesHuman osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1 - GFP and H2B - RFP images were acquired every hour for 24 h ( D ) . For one representative experiment among three , the mean ± SEM of the green fluorescence intensity in the nucleus ( normalized to the control at each timepoint ) of quadruplicates is depicted ( E ) . For each cell , the speed of nuclear release ( difference of HMGB1 nuclear green fluorescence intensity between two time points ) was calculated . Values are depicted as the average speed of the nuclear release ± SD of quadruplicatesHuman osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS cells were treated for 6 , 12 or 24 h and ATP was stained with quinacrine ( G ) . The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions . For one representative experiment among three the mean ± SD of quadruplicate assessments is shownHuman osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS wild - type cells were treated with MTX or DACT as described above for 6 h . Then medium was refreshed and 24 h later , type I interferon response was assessed by transferring the supernatant on HT29 MX1 - GFP reporter cells lines cells for additional 48 h . Human type 1α interferon ( IFNα1 ) was also added on the cells as an additional positive control . Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions ( I ) . The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is depictedHuman osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS wild - type cells were treated as mentioned above for 6 h and then medium was refreshed . Twenty - four hours later , cells were collected and surface - exposed calreticulin ( CALR ) was stained with an antibody specific for CALR . DAPI was used as an exclusion dye and cells were acquired by flow cytometry ( K ) . The percentage of CALR + cells among viable ( DAPI - ) ones are depicted . The mean ± SEM of six independent experiments is depictedHuman osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit , then normalized to control . The mean ± SEM of four independent experiments is shown . Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h . Concentration of secreted ATP in the supernatant was quantified with a luciferase - based bioluminescence kit . The mean ± SD of quadruplicates from one representative among three experiments is depicted .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR-GFP and H2B-RFP were treated as described above and images were acquired once per hour for 12 h (A). For one representative experiment among three, the mean ± SEM of the average area of high CALR dots (normalized to the control at each timepoint) of quadruplicates is shown (B). Values are depicted as the area under the curve ± SD of triplicatesHuman osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1-GFP and H2B-RFP images were acquired every hour for 24 h (D). For one representative experiment among three, the mean ± SEM of the green fluorescence intensity in the nucleus (normalized to the control at each timepoint) of quadruplicates is depicted (E). For each cell, the speed of nuclear release (difference of HMGB1 nuclear green fluorescence intensity between two time points) was calculated. Values are depicted as the average speed of the nuclear release ± SD of quadruplicatesHuman osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated for 6, 12 or 24 h and ATP was stained with quinacrine (G). The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions. For one representative experiment among three the mean ± SD of quadruplicate assessments is shownHuman osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated with MTX or DACT as described above for 6 h. Then medium was refreshed and 24 h later, type I interferon response was assessed by transferring the supernatant on HT29 MX1-GFP reporter cells lines cells for additional 48 h. Human type 1α interferon (IFNα1) was also added on the cells as an additional positive control. Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions (I). The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is depictedHuman osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated as mentioned above for 6 h and then medium was refreshed. Twenty-four hours later, cells were collected and surface-exposed calreticulin (CALR) was stained with an antibody specific for CALR. DAPI was used as an exclusion dye and cells were acquired by flow cytometry (K). The percentage of CALR+ cells among viable (DAPI-) ones are depicted. The mean ± SEM of six independent experiments is depictedHuman osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit, then normalized to control. The mean ± SEM of four independent experiments is shown.Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h. Concentration of secreted ATP in the supernatant was quantified with a luciferase-based bioluminescence kit. The mean ± SD of quadruplicates from one representative among three experiments is depicted.


Figure 3Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin ( DACT ) ( 0 . 25 , 0 . 5 or 1 µM ) for 6 h . Thapsigargin ( THAPS ) at 3 µM was used as a positive control . After fixation , cells were stained with phospho - eIF2α ( Ser51 ) specific antibody followed by an AlexaFluor - 647 secondary antibody , nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy . Images were segmented , analyzed and the red cytoplasmic fluorescence intensity was measured . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1 µMU2OS cells stably expressing ATF4 - reporter were treated as described above for 12 h . The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1 µMU2OS stably expressing ATF6 - GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1 µMU2OS cells stably expressing venus in frame with alternatively spliced XBP1 ( sXBP1 ) were treated as described above for 12 h . De novo expressed venus was measured intracellular . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1 µMU2OS wild - type and knock out for eIF2α kinases 1 , 2 , 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3 - mediated eIF2α phosphorylation or with 1 µM DACT for 6 h . After fixation , cells were stained for peIF2α as described above and cytoplasmic intensity was quantified . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1 µMU2OS CALR - RFP were injected subcutaneously ( s . c ) in the flank of nu / nu mice . Tumors were further injected with PBS ( Ctr ) ( n = 5 ) , 0 . 5 mg / kg tunicamycin ( TM ) for 6 h ( n = 3 ) or 0 . 5 mg / kg DACT for 6 h ( n = 4 ) or 24 h ( n = 3 ) . Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor - 647 secondary antibody and counterstained with Hoechst 33342 . Two slices per tumor were imaged for their DAPI , RFP and Cy5 signals . Out - of - focused images were removed from the dataset leading to the analysis of the following conditions : 8 x Ctr , 5 x TM , 8 x DACT 6 h and 6 x for DACT 24 h . PeIF2α was quantified measuring Cy5 intensity in the cytoplasm ( K , L ) and CALR translocation by measuring the coefficient variation ( CV ) of the RFP signal in the cytoplasm ( M , N ) . Representative images of peIF2α are shown for Ctr , TM and DACT at 6 h ( K ) , whereas images of CALR are shown for Ctr and DACT at 24

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin (DACT) (0.25, 0.5 or 1 µM) for 6 h. Thapsigargin (THAPS) at 3 µM was used as a positive control. After fixation, cells were stained with phospho-eIF2α (Ser51) specific antibody followed by an AlexaFluor-647 secondary antibody, nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy. Images were segmented, analyzed and the red cytoplasmic fluorescence intensity was measured. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µMU2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µMU2OS stably expressing ATF6-GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µMU2OS cells stably expressing venus in frame with alternatively spliced XBP1 (sXBP1) were treated as described above for 12 h. De novo expressed venus was measured intracellular. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µMU2OS wild-type and knock out for eIF2α kinases 1, 2, 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3-mediated eIF2α phosphorylation or with 1 µM DACT for 6 h. After fixation, cells were stained for peIF2α as described above and cytoplasmic intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µMU2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h


Figure 4Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange ( CMTMR ) and treated for 24 h with 1 µM dactinomycin ( DACT ) or 500 µM oxaliplatin ( OXA ) as a positive control . Then , untreated or dying MCA205 were co - cultured with differentiated bone marrow - derived dendritic cells ( BMDCs ) for 4 h at 37 ° C or at 4 ° C . Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry The percentage of CMTMR and CD11c double positive cells among all CD11c + cells are indicatedMCA205 cells were treated for 24 h with 500 µM oxaliplatin ( OXA ) or with 0 . 25 , 0 . 5 or 1 µM dactinomycin ( DACT ) . Then , untreated or dying MCA205 were co - cultured with BMDCs for 24 h at 37 ° C . As a positive control , BMDCs were co - cultured with 1 μg / mL LPS and 100 ng / mL IFNγ . The increase in the percentages of MHCII + and CD86 + cells among CD11c + cells was quantified with respect to untreated controls . 1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin ( DACT ) . Next , tumor size was measured regularly and individual tumor growths of DACT - vaccinated versus Ctr mice are depicted1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin ( DACT ) . Overall survival is depicted and p - values ( * * * p < 0 . 001 . ) were calculated with a Log - Rank

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange (CMTMR) and treated for 24 h with 1 µM dactinomycin (DACT) or 500 µM oxaliplatin (OXA) as a positive control. Then, untreated or dying MCA205 were co-cultured with differentiated bone marrow-derived dendritic cells (BMDCs) for 4 h at 37 °C or at 4 °C. Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry The percentage of CMTMR and CD11c double positive cells among all CD11c+ cells are indicatedMCA205 cells were treated for 24 h with 500 µM oxaliplatin (OXA) or with 0.25, 0.5 or 1 µM dactinomycin (DACT). Then, untreated or dying MCA205 were co-cultured with BMDCs for 24 h at 37 °C. As a positive control, BMDCs were co-cultured with 1 μg/mL LPS and 100 ng/mL IFNγ. The increase in the percentages of MHCII+ and CD86+ cells among CD11c+ cells was quantified with respect to untreated controls.1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Next, tumor size was measured regularly and individual tumor growths of DACT-vaccinated versus Ctr mice are depicted1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Overall survival is depicted and p-values (*** p<0.001.) were calculated with a Log-Rank test


Figure 53 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . Tumor size was assessed regularly and individual tumor growth curves of DACT versus Ctr3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . DACT + anti - CD4 / CD8 versus anti - CD - 4 / anti - CD - 8 Mean tumor area for each group was calculated and significances were tested using a type II ANOVA test Overall survival is depicted and p - values were calculated with a Log - Rank test Stars indicate the p - values of each treatment versus Ctr and hashes indicate the p - values of the DACT + anti - CD - 4 / anti - CD - 8 versus DACT alone3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . DACT + anti - PD - 1 versus anti - PD - 1 are depicted . Overall survival is depicted and p - values were calculated with a Log - Rank test Stars indicate the p - values of each treatment versus Ctr and of DACT + anti - PD - 1 versus anti - PD - 1 alone3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . Five naïve mice or the eleven mice that were cured by treatment with DACT alone or in combination with PD - 1 blockade were ( re ) challenged with WEHI 164 cells , and individual tumor growths ( I , J ) , as well as overall survival ( * * * p < 0 . 001 , Log - Rank test ) ( K ) , were monitored .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 53 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Tumor size was assessed regularly and individual tumor growth curves of DACT versus Ctr3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-CD4/CD8 versus anti-CD-4/anti-CD-8 Mean tumor area for each group was calculated and significances were tested using a type II ANOVA test Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and hashes indicate the p-values of the DACT + anti-CD-4/anti-CD-8 versus DACT alone3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-PD-1 versus anti-PD-1 are depicted. Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and of DACT + anti-PD-1 versus anti-PD-1 alone3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Five naïve mice or the eleven mice that were cured by treatment with DACT alone or in combination with PD-1 blockade were (re)challenged with WEHI 164 cells, and individual tumor growths (I, J), as well as overall survival (*** p<0.001, Log-Rank test) (K), were monitored.


Figure 6Mice bearing WEHI 164 sarcomas were treated by systemic ( intraperitoneal , i . p . ) injections of DACT or PBS as a control ( Ctr ) , and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in the Ctr group and 23 mice in the DACT - treated group , with each data point indicating one tumor . The cycle threshold of the qRT - PCR of IFNγ was normalized to the one of the housekeeping gene peptidylpropyl isomerase A ( Ppia ) in each mouse and results are shown normalized with respect to controls as a dot plot depicting mean ± SD . The p - value ( * p < 0 . 05 ) was calculated by means of a Student ' s t test ( B ) . Balb / c mice with palpable WEHI 164 sarcomas ( n = 7 mice in Ctr and anti - IFNγ groups ; n = 8 mice in DACT and DACT + anti - IFNγ groups ) received two injections of DACT - based chemotherapy ( 0 . 5 mg / kg ) as well as multiple injections ( 3 times per week ) of neutralizing IFNγ - specific antibody ( C ) . Tumor growth curves are shown for individual mice ( D ) and as means ( E ) . Statistical difference between DACT - treated tumors and respective controls , which was calculated with a type II ANOVA ( * p < 0 . 05 ) , is lost upon IFNγ neutralization ( E ) . Overall survival of mice is also indicated with statistics to respective controls calculated with a Log - Rank test ( * p < 0 . 05 ) ( F ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Mice bearing WEHI 164 sarcomas were treated by systemic (intraperitoneal, i.p.) injections of DACT or PBS as a control (Ctr), and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in the Ctr group and 23 mice in the DACT-treated group, with each data point indicating one tumor. The cycle threshold of the qRT-PCR of IFNγ was normalized to the one of the housekeeping gene peptidylpropyl isomerase A (Ppia) in each mouse and results are shown normalized with respect to controls as a dot plot depicting mean ± SD . The p-value (* p<0.05) was calculated by means of a Student's t test (B).Balb/c mice with palpable WEHI 164 sarcomas (n=7 mice in Ctr and anti-IFNγ groups; n=8 mice in DACT and DACT+anti-IFNγ groups) received two injections of DACT-based chemotherapy (0.5 mg/kg) as well as multiple injections (3 times per week) of neutralizing IFNγ-specific antibody (C). Tumor growth curves are shown for individual mice (D) and as means (E). Statistical difference between DACT-treated tumors and respective controls, which was calculated with a type II ANOVA (* p<0.05), is lost upon IFNγ neutralization (E). Overall survival of mice is also indicated with statistics to respective controls calculated with a Log-Rank test (* p<0.05) (F).


Figure 7Human osteosarcoma U2OS cells were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) and vincristine ( VC ) at 0 . 5 , 1 and 5 µM ; with cisplatin ( CDDP ) at 75 , 150 and 300 µM , with oxaliplatin ( OXA ) at 250 , 500 and 1000 µM and with crizotinib ( CRIZ ) at 10 , 20 and 40 µM for 1 . 5 to 2 . 5 h and followed by an additional hour of treatment in the presence of 100 mM 5 - ethynyl uridine ( EU ) . After fixation cells were permeabilized and EU was stained with an AlexaFluor - 488 - coupled azide . Representative images are shown for each treatment ( A ) . The EU intensity in the nucleus of each condition was ranked between the untreated control ( Ctr , 0 % transcription inhibition ) and the control that was not incubated with EU ( corresponding to 100 % transcription inhibition ) ( B ) . Representative images of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are shownHuman osteosarcoma U2OS cells were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) and vincristine ( VC ) at 0 . 5 , 1 and 5 µM ; with cisplatin ( CDDP ) at 75 , 150 and 300 µM , with oxaliplatin ( OXA ) at 250 , 500 and 1000 µM and with crizotinib ( CRIZ ) at 10 , 20 and 40 µM for 1 . 5 to 2 . 5 h Cells were treated for 2 . 5 h and before fixation and permeabilization . Then , cells were stained with a rabbit anti - fibrillarin antibody followed by a staining with an anti - rabbit AlexaFluor - 647 - or AlexaFluor - 546 - coupled secondary antibody as well as with a mouse anti - nucleolin antibody followed by a staining with an anti - mouse AlexaFluor - 488 - coupled secondary antibody . Then images were acquired and colocalization between both signals was assessed ( C ) . The surface overlap coefficient ( SOC ) was calculated and ranked between the untreated control ( Ctr ) and the positive control ( DACT ) Representative images of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are shownHuman osteosarcoma U2OS cells were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) and vincristine ( VC ) at 0 . 5 , 1 and 5 µM ; with cisplatin ( CDDP ) at 75 , 150 and 300 µM , with oxaliplatin ( OXA ) at 250 , 500 and 1000 µM and with crizotinib ( CRIZ ) at 10 , 20 and 40 µM for 1 . 5 to 2 . 5 h Cells were pre - treated overnight with the aforementioned compounds in complete medium followed by washout and treatment pursued in methionine - free medium for 30 min . Afterwards , the treatments were continued in methionine - free medium supplemented with 25 µM L - azidohomoalanine ( AHA ) for additional 1 . 5 h . AHA incorporation was detected after fixation , permeabilization and blocking by the addition of an AlexaFluor - 488 - coupled azide . Then images were acquired ( E ) and AHA intensity in the cells was ranked between the untreated control ( Ctr , 0 % translation inhibition ) and the untreated control without AHA ( corresponding to 100 % translation inhibition ) ( F ) . Representative images of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are shownCorrelation between the transcription measured by EU incorporation and measured by fibrillarin and nucleolin colocalization is depicted with Pearson correlation coefficient ( R ) and p - value ( p ) ( G ) . The same parameters are shown for the correlation between transcription measured by EU incorporation and translation measured with AHA

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h and followed by an additional hour of treatment in the presence of 100 mM 5-ethynyl uridine (EU). After fixation cells were permeabilized and EU was stained with an AlexaFluor-488-coupled azide. Representative images are shown for each treatment (A). The EU intensity in the nucleus of each condition was ranked between the untreated control (Ctr, 0 % transcription inhibition) and the control that was not incubated with EU (corresponding to 100 % transcription inhibition) (B). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shownHuman osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were treated for 2.5 h and before fixation and permeabilization. Then, cells were stained with a rabbit anti-fibrillarin antibody followed by a staining with an anti-rabbit AlexaFluor-647- or AlexaFluor-546-coupled secondary antibody as well as with a mouse anti-nucleolin antibody followed by a staining with an anti-mouse AlexaFluor-488-coupled secondary antibody. Then images were acquired and colocalization between both signals was assessed (C). The surface overlap coefficient (SOC) was calculated and ranked between the untreated control (Ctr) and the positive control (DACT) Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shownHuman osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were pre-treated overnight with the aforementioned compounds in complete medium followed by washout and treatment pursued in methionine-free medium for 30 min. Afterwards, the treatments were continued in methionine-free medium supplemented with 25 µM L-azidohomoalanine (AHA) for additional 1.5 h. AHA incorporation was detected after fixation, permeabilization and blocking by the addition of an AlexaFluor-488-coupled azide. Then images were acquired (E) and AHA intensity in the cells was ranked between the untreated control (Ctr, 0 % translation inhibition) and the untreated control without AHA (corresponding to 100 % translation inhibition) (F). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shownCorrelation between the transcription measured by EU incorporation and measured by fibrillarin and nucleolin colocalization is depicted with Pearson correlation coefficient (R) and p-value (p) (G). The same parameters are shown for the correlation between transcription measured by EU incorporation and translation measured with AHA incorporation


Figure 8U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and ICD prediction score Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR ) U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and peIF2α expression Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR ) U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and CALR exposure Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR ) U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and ATP decrease Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR ) U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and HMBG1 exodus Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR ) U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and biological ICD score ( F ) previously measured and expressed as z - scores ( except for ICD prediction score ) Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR ) U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . For assessing translation , cells were pre - treated with the library for 12 h followed by 30 min in methionine free - medium , before addition of azidohomoalanine ( AHA ) . Percentage of inhibition was calculated and transformed as z - scores . The correlations between transcription and translation inhibitions Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR ) The inhibition of transcription was assessed for the negative and positive ICD hits identified with the predictive algorithm ( Figure 1 ) . U2OS cells were treated with the agents at concentrations corresponding to their IC60 : 1 μM dactinomycin ( DACT ) , 50 μM topotecan , 1 μM becatecarin , 0 . 5 μM trabectedin , 5 μM UCN - 01 , 30 μM mycophenolate mofetil , 30 μM nonoxynol - 9 , 25 μM dactolisib , 2 . 5 μM β - lapachone , 5 μM 5 - fluorodeoxycytidine and 2 μM RH - 1 for 1 . 5 h followed by 1 h with EU . The percentage of transcription inhibition was calculated and the coefficient of correlation ( R ) and associated p - value ( p ) between the percentage of inhibition and the theoretical ICD score was calculated using the Pearson method .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ICD prediction score Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and peIF2α expression Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and CALR exposure Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ATP decrease Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and HMBG1 exodus Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and biological ICD score (F) previously measured and expressed as z-scores (except for ICD prediction score)  Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. For assessing translation, cells were pre-treated with the library for 12 h followed by 30 min in methionine free-medium, before addition of azidohomoalanine (AHA). Percentage of inhibition was calculated and transformed as z-scores. The correlations between transcription and translation inhibitions Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)The inhibition of transcription was assessed for the negative and positive ICD hits identified with the predictive algorithm (Figure 1). U2OS cells were treated with the agents at concentrations corresponding to their IC60: 1 μM dactinomycin (DACT), 50 μM topotecan, 1 μM becatecarin, 0.5 μM trabectedin, 5 μM UCN-01, 30 μM mycophenolate mofetil, 30 μM nonoxynol-9, 25 μM dactolisib, 2.5 μM β-lapachone, 5 μM 5-fluorodeoxycytidine and 2 μM RH-1 for 1.5 h followed by 1 h with EU. The percentage of transcription inhibition was calculated and the coefficient of correlation (R) and associated p-value (p) between the percentage of inhibition and the theoretical ICD score was calculated using the Pearson method.


Figure 1G Verify the mobility of GFP ( control ) mRNA and GFP - MdCAX3 mRNA by RT - PCR in Mx leaf , stem , and root . The recombinant vectors ( F ) were transformed into A . tumefaciens GV3101 and introduced into Mx leaves by vacuum infiltration . H GFP fluorescence image of the Mx roots under different treatment . Nuclei were labeled using DAPI . Scale Bar : 100 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 1G Verify the mobility of GFP (control) mRNA and GFP-MdCAX3 mRNA by RT-PCR in Mx leaf, stem, and root. The recombinant vectors (F) were transformed into A. tumefaciens GV3101 and introduced into Mx leaves by vacuum infiltration.H GFP fluorescence image of the Mx roots under different treatment. Nuclei were labeled using DAPI. Scale Bar: 100 μm.


Figure 3H Ratio ( 488 / 440 ) image of BCECF fluorescence in the roots of MdCAX3 - suppressed in leaves Md / Mx . Scale bars : 100 μm . I Rhizosphere acidification of MdCAX3 - suppressed in leaves Md / Mx . Bromocresol purple was used as a pH indicator for visualization . Scale bars : 1 cm .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3H Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed in leaves Md/Mx. Scale bars: 100 μm.I Rhizosphere acidification of MdCAX3-suppressed in leaves Md/Mx. Bromocresol purple was used as a pH indicator for visualization. Scale bars: 1 cm.


Figure 4F GUS staining in N . benthamiana leaves after transient co - expression of pro35S : : GFP with proMxCXIP1 : GUS or proMbCXIP1 : GUS , and co - expression with pBI101 : GUS as a control . G Interaction between MdCXIP1 and MdCAX3 tested by yeast two - hybrid ( Y2H ) assays . A dilution series of NMY51 yeast cells co - expressing MdCXIP1 and MdCAX3 cultured on SD / II and SDIV screening media . pPBT3 - N ( empty vector ) co - expressed with MdCXIP1 was used as a negative control . X - gal was used in SD / - Trp / - Leu / - His / - Ade screening medium to further test for possible interactions . I Interaction between MdCXIP1 and the MdCAX3 N - terminal autoinhibitory domain in an in vitro pull - down assay . The His - MdCXIP1 , GST and GST - N - MdCAX3 fusion proteins were expressed in the Escherichia coli BL21 strain , and the GST protein alone was used as a negative control . ' + ' indicates presence and " − " indicates absence . IB , immunoblot .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4F GUS staining in N. benthamiana leaves after transient co-expression of pro35S::GFP with proMxCXIP1:GUS or proMbCXIP1:GUS, and co-expression with pBI101:GUS as a control.G Interaction between MdCXIP1 and MdCAX3 tested by yeast two-hybrid (Y2H) assays. A dilution series of NMY51 yeast cells co-expressing MdCXIP1 and MdCAX3 cultured on SD/II and SDIV screening media. pPBT3-N (empty vector) co-expressed with MdCXIP1 was used as a negative control. X-gal was used in SD/-Trp/-Leu/-His/-Ade screening medium to further test for possible interactions.I Interaction between MdCXIP1 and the MdCAX3 N-terminal autoinhibitory domain in an in vitro pull-down assay. The His-MdCXIP1, GST and GST-N-MdCAX3 fusion proteins were expressed in the Escherichia coli BL21 strain, and the GST protein alone was used as a negative control. '+' indicates presence and "−" indicates absence. IB, immunoblot.


Figure 5C Fluorescence ratio ( 488 / 440 nm excitations ) of BCECF fluorescence in the roots of MdCAX3 and MdCXIP1 co - overexpressing Mb . The central band represents the median and the box ranges showing the interquartile range and covers the central 50 % of the data . The whiskers showing the minimum and maximum of the data . Three biological replicates ; 3 roots were quantified for each replicate . Asterisks indicate statistically significant differences ( * * * , P < 0 . 001 ; ANOVA , Tukey correction ) . D Ratio ( 488 / 440 ) image of BCECF fluorescence in MdCAX3 and MdCXIP1 co - overexpressing Mb . Scale Bar : 100 μm . E Rhizosphere acidification in MdCAX3 and MdCXIP1 co - overexpressing Mb . Bromocresol purple was used as a pH indicator for visualization . Scale Bar : 1 cm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C Fluorescence ratio (488/440 nm excitations) of BCECF fluorescence in the roots of MdCAX3 and MdCXIP1 co-overexpressing Mb. The central band represents the median and the box ranges showing the interquartile range and covers the central 50% of the data. The whiskers showing the minimum and maximum of the data. Three biological replicates; 3 roots were quantified for each replicate. Asterisks indicate statistically significant differences (***, P < 0.001; ANOVA, Tukey correction).D Ratio (488/440) image of BCECF fluorescence in MdCAX3 and MdCXIP1 co-overexpressing Mb. Scale Bar: 100 μm. E Rhizosphere acidification in MdCAX3 and MdCXIP1 co-overexpressing Mb. Bromocresol purple was used as a pH indicator for visualization. Scale Bar: 1 cm.


Figure 6D Cellular localization of Zn2 + ( blue ) in Mx and MdCAX3 - suppressed Mx roots under Fe deficient conditions . Arrowhead indicates the cytoplasm . Scale Bars : 20 μm . E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6D Cellular localization of Zn2+ (blue) in Mx and MdCAX3-suppressed Mx roots under Fe deficient conditions. Arrowhead indicates the cytoplasm. Scale Bars: 20 μm. E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D.


Figure 1HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( A ) Concentrations of IL - 6 were determined ( CTRL , n = 9 ; Bax / Bak , n = 6 ; Bcl - XL , n = 3 ; STING , n = 3 ) . HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( B ) Concentrations of IL - 8 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) . HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( C ) Concentrations of CXCL1 were determined ( CTRL , n = 8 ; Bax / Bak , n = 5 ) . HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( D ) Concentrations of FGF - 2 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) . HeLa cell lines were treated with the Mcl - 1 - inhibitor S63845 for 72 h . Cytokines were measured by ELISA . ( E ) HeLa CTRL cells and Bax / Bak - deficient cells were treated with the Mcl - 1 - inhibitor S63845 . Concentrations of IL - 6 in supernatants are shown ( CTRL , n = 8 ; Bax / Bak , n = 7 ) . F 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing Bcl - XL were treated with ABT - 737 . Concentrations of IL - 6 in the supernatants were measured after 48 h ( n = 5 ) . G 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing mouse Bcl - XL were treated with various concentrations of ABT - 737 . To some aliquots the caspase - inhibitor zVAD - fmk ( 50 μM ) was added . Relative numbers of apoptotic cells were determined as in EV 1B after 48 h . Data are means / SEM of at least three independent experiments . ( 1205Lu , n = 4 ; 1205Lu 5 µM ABT - 737 , n = 3 ; 1205Lu / Bcl - XL and 1205Lu + zVAD , n = 3 ) . H HeLa - UL12 . 5 cells untreated or treated with tamoxifen ( 100 nM , 24 h ) were stained with Picogreen ( 3 µl / ml ) to detect mitochondrial DNA . Scale bare , 10 µmI HeLa CTRL cells and HeLa - UL12 . 5 cells were treated with ABT - 737 ( 10µM ) . Concentration of IL - 6 in the supernatant were measured after 72h ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (A) Concentrations of IL-6 were determined (CTRL, n=9; Bax/Bak, n=6; Bcl-XL, n=3; STING, n=3).HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (B) Concentrations of IL-8 were determined (CTRL, n=7; Bax/Bak, n=4).HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (C) Concentrations of CXCL1 were determined (CTRL, n=8; Bax/Bak, n=5).HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (D) Concentrations of FGF-2 were determined (CTRL, n=7; Bax/Bak, n=4).HeLa cell lines were treated with the Mcl-1-inhibitor S63845 for 72 h. Cytokines were measured by ELISA. (E) HeLa CTRL cells and Bax/Bak-deficient cells were treated with the Mcl-1-inhibitor S63845. Concentrations of IL-6 in supernatants are shown (CTRL, n=8; Bax/Bak, n=7).F 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing Bcl-XL were treated with ABT-737. Concentrations of IL-6 in the supernatants were measured after 48 h (n=5).G 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing mouse Bcl-XL were treated with various concentrations of ABT-737. To some aliquots the caspase-inhibitor zVAD-fmk (50 μM) was added. Relative numbers of apoptotic cells were determined as in EV 1B after 48 h. Data are means/SEM of at least three independent experiments. (1205Lu, n=4; 1205Lu 5 µM ABT-737, n=3; 1205Lu/Bcl-XL and 1205Lu + zVAD, n=3).H HeLa-UL12.5 cells untreated or treated with tamoxifen (100 nM, 24 h) were stained with Picogreen (3 µl/ml) to detect mitochondrial DNA. Scale bare, 10 µmI HeLa CTRL cells and HeLa-UL12.5 cells were treated with ABT-737 (10µM). Concentration of IL-6 in the supernatant were measured after 72h (n=3).


Figure 2A , The caspase - 3 - reporter cell line ( labelled DEVD ) and the control cell line ( DEVG ; see methods ) were infected and analysed at various time points post - infection by flow cytometry ( Chlamydia trachomatis ( Ctr ; MOI = 1 , 30 h ) ; Salmonella Typhimurium ( STy ; MOI = 50 , 24h ) ; Toxoplasma gondii ( MOI = 5 , 16 h ) ; MVA ( MOI = 10 , 16h ) ; Influenza A virus ( IAV ; MOI = 1 , 16h ) ; Herpes simplex virus 1 ( HSV - 1 ; MOI = 1 , 16h ) . Staurosporine treated cells ( 1 μM , 3h ) were used as a positive control . Percentages of cells positive for caspase - 3 - activation ( FRET - loss ) were determined . Data are means / SEM of three independent experiments . ( n = 3 ) B , C Cells were infected with modified vaccinia virus Ankara ( MVA ) ( MOI = 10 ) . The caspase - inhibitor zVAD - fmk was used at 100µM ; cells were analysed 16 h post - infection . D , E Cells were infected with influenza A virus ( IAV ) ( MOI = 1 ; analysis was 16 h post - infection ) . D , detection of viral M2 protein was used as infection control . F , G Cells were infected with Herpes simplex virus 1 ( HSV - 1 ) ( MOI = 1 ; analysis was 16 h post - infection ) . H Cells were infected with Chlamydia trachomatis ( Ctr ) ( MOI = 1 ; analysis was 30 h post - infection ) . I Cells were infected with Salmonella Typhimurium ( Sty ) ( MOI = 50 ; analysis was 24 h post - infection ) . J Cells were infected with the protozoan parasite Toxoplasma gondii ( MOI = 5 ; analysis was 16 h post - infection ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, The caspase-3-reporter cell line (labelled DEVD) and the control cell line (DEVG; see methods) were infected and analysed at various time points post-infection by flow cytometry (Chlamydia trachomatis (Ctr; MOI=1, 30 h); Salmonella Typhimurium (STy; MOI=50, 24h); Toxoplasma gondii (MOI=5, 16 h); MVA (MOI=10, 16h); Influenza A virus (IAV; MOI=1, 16h); Herpes simplex virus 1 (HSV-1; MOI=1, 16h). Staurosporine treated cells (1 μM, 3h) were used as a positive control. Percentages of cells positive for caspase-3-activation (FRET-loss) were determined. Data are means/SEM of three independent experiments. (n=3)B, C Cells were infected with modified vaccinia virus Ankara (MVA) (MOI=10). The caspase-inhibitor zVAD-fmk was used at 100µM; cells were analysed 16 h post-infection.D, E Cells were infected with influenza A virus (IAV) (MOI=1; analysis was 16 h post-infection). D, detection of viral M2 protein was used as infection control.F, G Cells were infected with Herpes simplex virus 1 (HSV-1) (MOI=1; analysis was 16 h post-infection).H Cells were infected with Chlamydia trachomatis (Ctr) (MOI=1; analysis was 30 h post-infection).I Cells were infected with Salmonella Typhimurium (Sty) (MOI=50; analysis was 24 h post-infection).J Cells were infected with the protozoan parasite Toxoplasma gondii (MOI=5; analysis was 16 h post-infection).


Figure 3A Infection with MVA ( MOI = 10 for 16 h ; IL - 6 ) . B infection with HSV - 1 ( MOI = 1 for 16 h ; IL - 6 ) . Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; IL - 6Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; , IL - 8 ) . Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; IL - 6Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; CXCL - 1 ) . Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; IL - 6Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; , IL - 8Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; CXCL - 1Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; FGF - 2 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O

Figure 3A Infection with MVA (MOI=10 for 16 h; IL-6).B infection with HSV-1 (MOI=1 for 16 h; IL-6).Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; IL-6Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; , IL-8).Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; IL-6Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; CXCL-1).Infection with Toxoplasma gondii (MOI=5 for 16 h; IL-6Infection with Toxoplasma gondii (MOI=5 for 16 h; , IL-8Infection with Toxoplasma gondii (MOI=5 for 16 h; CXCL-1Infection with Toxoplasma gondii (MOI=5 for 16 h; FGF-2).


Figure 4 ( A ) HeLa cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of six experiments . ( A ) Growth of C . trachomatis was measured in HeLa cells as increase in GFP - fluorescence between 18 and 38 h . The data show means / SEM of six independent experiments ( genotype effect ( significance of the difference of GFP - expression between the cell lines overall , p = 0 . 08 ) . Red line indicates the fold change calculation of the individual different time points and cell lines ( significance tested ( one sample t - test ) for each time point for a difference between Bax / Bak vs CTRL ) . AGS cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of 4 - 5 experiments . ( B ) Growth of C . trachomatis was measured in AGS cells as increase in GFP - fluorescence . The data show means / SEM of 4 to 5 independent experiments ( genotype effect : p < 0 . 0001 ; time - genotype interaction : p < 0 . 0001 ) ( 12 h and 38 h , n = 4 ; 18 h , n = 5 ) . C Chlamydial genomes per cell culture ( expressed as chlamydial DNA in 50 ng of total DNA , determined by quantitative PCR ) . * , p < 0 . 05 between control and mutant cells . ( n = 3 ) D CTRL , Bax / Bak - deficient or Bcl - XL - over - expressing HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . Extracellular bacteria were killed using non cell - permeable antibiotic . At the indicated time points cells were lysed , and aliquots were plated on agar plates . Recovered colony forming units were calculated . Data are means / SEM of four independent experiments . Significant genotype effects were found between mutant and control cells ( Bax / Bak p < 0 . 0001 ; Bcl - XL p 0 . 0001 ) . E CTRL , Bax / Bak - deficient or Apaf - 1 - deficient HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . CFU / well were calculated as in ( D ) . n = 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) HeLa cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of six experiments. (A) Growth of C. trachomatis was measured in HeLa cells as increase in GFP-fluorescence between 18 and 38 h. The data show means/SEM of six independent experiments (genotype effect (significance of the difference of GFP-expression between the cell lines overall, p = 0.08). Red line indicates the fold change calculation of the individual different time points and cell lines (significance tested (one sample t-test) for each time point for a difference between Bax/Bak vs CTRL).AGS cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of 4-5 experiments. (B) Growth of C. trachomatis was measured in AGS cells as increase in GFP-fluorescence. The data show means/SEM of 4 to 5 independent experiments (genotype effect: p<0.0001; time-genotype interaction: p<0.0001) (12 h and 38 h, n=4; 18 h, n=5).C Chlamydial genomes per cell culture (expressed as chlamydial DNA in 50 ng of total DNA, determined by quantitative PCR). *, p<0.05 between control and mutant cells. (n=3)D CTRL, Bax/Bak-deficient or Bcl-XL-over-expressing HeLa cells were infected with Salmonella Typhimurium (MOI=50). Extracellular bacteria were killed using non cell-permeable antibiotic. At the indicated time points cells were lysed, and aliquots were plated on agar plates. Recovered colony forming units were calculated. Data are means/SEM of four independent experiments. Significant genotype effects were found between mutant and control cells (Bax/Bak p<0.0001; Bcl-XL p 0.0001).E CTRL, Bax/Bak-deficient or Apaf-1-deficient HeLa cells were infected with Salmonella Typhimurium (MOI=50). CFU/well were calculated as in (D). n=3.


Figure 1B Yeast two - hybrid analysis for interaction between the above - shown Rabaptin5 segments ( Rbpt5 # , fused to LexA on the bait plasmid ) and residues 257 - 444 of FIP200 ( FIP , fused to the Gal4 activation domain on the prey plasmid ) to drive HIS3 expression . Three different clones each were replica - plated on medium with His or without His , but containing 3 - amino - 1 , 2 , 4 - triazole ( 3AT ; an inhibitor of His synthesis to increase stringency ) and grown in the absence of Trp and leucine as a control . As negative controls , empty bait or prey plasmids were used . The asterisk indicates a clone invalidated by recombination . D FIP200 was immunoprecipitated ( IP ) from lysates of HeLa or HEK293A cells and probed for FIP200 , Rabaptin5 ( Rbpt5 ) , and EEA1 ( early endosome antigen 1 ) by immunoblotting . Input lysate ( 10 % ) was immunoblotted blotted parallel . As a negative control , the immunoprecipitation was performed using an anti - GAPDH antibody . E - H Lysates of HeLa cells transiently transfected with full - length FIP200 - mCherry ( FIP200 - mCh ) or a deletion mutant lacking the segment interacting with Rabaptin5 ( ∆ 280 - 440 ) were immuprecipitated with anti - mCherry ( IP FIP200 - mCh ) or , as a control , with anti - FLAG antibodies ( IP FLAG ) . Immunoprecipitates and input lysates ( 10 % ) were immunoblotted for mCherry and Rabaptin5 ( E ) , ATG13 ( F ) , or ULK1 ( G ) . Co - immunoprecipitation of Rabaptin5 , ATG13 , and ULK1 with FIP200 ∆ 280 - 440 ( FIP ∆ ) was quantified in comparison to that with wild - type FIP200 ( H ; signals normalized to that of the immunoprecipitated protein ;

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257-444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination.D FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti-GAPDH antibody. E-H Lysates of HeLa cells transiently transfected with full-length FIP200-mCherry (FIP200-mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280-440) were immuprecipitated with anti-mCherry (IP FIP200-mCh) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co-immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280-440 (FIP∆) was quantified in comparison to that with wild-type FIP200 (H; signals normalized to that of the immunoprecipitated protein;


Figure 2To more easily visualize Rabaptin5 , a stable HEK293A cell line overexpressing Rabaptin5 ( HEK + Rbpt5 ) was generated . Immunofluorescence microscopy of Rabaptin5 and transferrin receptor ( TfR ) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min ( + CQ , panel A ) compared to untreated cells ( - CQ , A ' HEK + Rbpt5 cells , untransfected or 24 h after transfection with mCherry - galectin3 ( mCh - Gal3 ) , mRuby3 - galectin8 ( mRuby - Gal8 ) , mCherry - FIP200 , or - ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry - galectin3 or mRuby3 - galectin8 ( C ) , ubiquitin ( Ub ; D ) , p62 ( E ) , mCherry - FIP200 ( F ) , WIPI2 ( G ) , mCherry - ATG16L1 ( H ) , or LC3B ( I ) . Scale bar , 10 µm . In the enlarged insets , arrowheads point out chloroquine - induced enlarged , ring - like early endosomes . Rabaptin5 - positive enlarged endosomes positive for mCherry - galectin3 ( Gal3 ) or mRuby3 - galectin8 ( Gal8 ) were quantified ( C ' ; mean ± S . D . J , K HEK + Rbpt5 cells , untransfected or 24 h after transfection with mCherry - ATG16L1 , were treated with 60 µM chloroquine for 0 , 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry - ATG16L1 . Manders ' colocalization coefficients were determined , M1 showing the fraction of Rabaptin5 - positive structures also positive for WIPI2 ( J ) or mCherry - ATG16L1 ( K ) , and M2 showing the respective inverse ( mean ± S . D . of three independent experiments ;

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared to untreated cells (-CQ, A'HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin3 (mCh-Gal3), mRuby3-galectin8 (mRuby-Gal8), mCherry-FIP200, or -ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin3 or mRuby3-galectin8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin3 (Gal3) or mRuby3-galectin8 (Gal8) were quantified (C'; mean ± S.D.J, K HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± S.D. of three independent experiments;


Figure 3A HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h and treated without ( - ) or with 60 µM chloroquine ( + CQ ) or 250 nM Torin1 for 150 min . Scale bar , 10 µm . Below , the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin ( Tub ) as a loading control . B WIPI2 or LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * P < 0 . 01 ) . CC HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy . Cells were fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . D WIPI2 or LC3B puncta per cell were quantified ( mean ± S . D . of three independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (-) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. B WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01). CC HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. D WIPI2 or LC3B puncta per cell were quantified (mean ± S.D. of three independent experiments).


Figure 4A HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ATG13 ( siATG13 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , and fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls . B WIPI2 of LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * * P < 0 . 01 ) . CHEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ULK1 ( siULK1 ) , ULK2 ( siULK2 ) , or both ( siULK1 + 2 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , and fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT - PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent experimentsE HEK293A cells were incubated without ( - ) , with 60 µM chloroquine ( + CQ ) , or with chloroquine and 5 µM MRT68921 ( + CQ + MRT ) for 150 min and fixed and stained for WIPI2 and LC3B . F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B ( mean ± S . D . of three independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ATG13 (siATG13) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls. B WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: **P<0.01). CHEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ULK1 (siULK1), ULK2 (siULK2), or both (siULK1+2) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT-PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experimentsE HEK293A cells were incubated without (-), with 60 µM chloroquine (+CQ), or with chloroquine and 5 µM MRT68921 (+CQ +MRT) for 150 min and fixed and stained for WIPI2 and LC3B. F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B (mean ± S.D. of three independent experiments).


Figure 5B Co - immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR / Cas9 knockout cells lacking FIP200 ( FIP - KO ) . Anti - HA antibodies were used as a control ( IP HA ) . On the right , HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin ( Tub ) . Signals were quantified and the ratios of mCherry - ATG16L1 / Rabaptin5 normalized to that of HEK293A cells without chloroquine treatmentLysates of HEK293A or HeLa cells transiently transfected with full - length mCherry - ATG16L1 ( wt ) or a mutant lacking the WD domain ( ∆ WD ; residues 1 - 319 of ATG16L1 , precisely deleting only the WD40 repeats residues 320 - 607 ) were immunoprecipitated with anti - Rabaptin5 or anti - FLAG antibodies , and immunoblotted for Rabaptin5 and mCherry - ATG16L1 , or immunoprecipitated with anti - FIP200 or anti - HA antibodies Co - immunoprecipitation of ATG16L1 ∆ WD with Rabaptin5 ( C ) was reduced to 6 . 6 ± 2 . 1 % and 3 . 4 ± 2 . 1 % in HEK293A and HeLa cells , respectively , relative to that of full - length ATG16L1 ( signals normalized to the immunoprecipitated protein ; mean ± S . D . deviation of three independent experiments each ) . F Lysates of HeLa cells transiently transfected with myc - tagged wild - type Rabaptin5 ( wt ) or triple - alanine mutant ( AAA ) were immunoprecipitated with anti - myc ( IP myc ) or anti - FLAG antibodies ( IP FLAG ) , and immunoblotted for myc and ATG16L1 . Co - immunoprecipitation of ATG16L1 with Rabaptin5 - AAA triple mutant was reduced to 1 . 5 ± 1 . 2 % relative to that with wild - type Rabaptin5 ( signals normalized to that of the immunoprecipitated protein ; mean ± S . D . of three independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatmentLysates of HEK293A or HeLa cells transiently transfected with full-length mCherry-ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1-319 of ATG16L1, precisely deleting only the WD40 repeats residues 320-607) were immunoprecipitated with anti-Rabaptin5 or anti-FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry-ATG16L1 , or immunoprecipitated with anti-FIP200 or anti-HA antibodies Co-immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6±2.1% and 3.4±2.1% in HEK293A and HeLa cells, respectively, relative to that of full-length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± S.D. deviation of three independent experiments each).F Lysates of HeLa cells transiently transfected with myc-tagged wild-type Rabaptin5 (wt) or triple-alanine mutant (AAA) were immunoprecipitated with anti-myc (IP myc) or anti-FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co-immunoprecipitation of ATG16L1 with Rabaptin5-AAA triple mutant was reduced to 1.5±1.2% relative to that with wild-type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± S.D. of three independent experiments).