Fig 2: CRL4 knockdown induces autophagy in cisplatin resistance OCCs. a Immunoblotting analysis of LC3B, ATG5, and p62 expression in (left panel) A2780CP or (right panel) COC1/DDP cells with DDB1 or CUL4A knockdown. b Representative images of the transmission electron microscope in A2780CP cells treated as in (a). c Quantification of the LC3 puncta number in (b) (***p < 0.001, ****p < 0.0001, t-test). d Immunofluorescence analysis of LC3 in (upper panel) A2780CP and (lower panel) COC1/DDP cells with or without CRL4 knockdown. Scale Bar: 10 µm. e Quantification of autophagic vesicles in (d). Data represent mean ± SEM with three replicates, **p < 0.01, ***p < 0.001, ****p < 0.0001. f, g The interaction between BECN1 and BCL2, ATG14L was detected by Co-IP assay in f A2780CP and g COC1/DDP cells with CRL4 knockdown. h Immunofluorescence analysis of CRL4-knockdown A2780CP cells transiently transfected with tandem mRFP-GFP-tagged LC3B, followed by treatment with 10 µM chloroquine for another 24 h. Scale Bar: 10 µm. i Quantification of the ratio of autolysosome (AL, red puncta) versus autophagosome (AP, yellow puncta). *p < 0.05, **p < 0.01; ***p < 0.001, t-test. j Colocalization of mitochondria and autolysosomes analyzed by staining A2780CP cells with Lyso-Tracker Red and MitoTracker Green. Red indicates staining with Lyso-Tracker Red, green represents staining with MitoTracker Green, and yellow stands for the two colors merged together. Yellow puncta were counted as mitochondria having autolysosomes. White arrows indicate colocalization points. Scale bar: 10 µm. k Immunoblotting of mitochondrial outer membrane protein Tomm20 and inner membrane protein Tim23 in A2780CP or COC1/DDP cells transfected with lentivirus carrying shRNA targeting CUL4A (right panel) or DDB1 (left panel). GAPDH was used as a loading control for Tomm20, Tim23, DDB1, and CUL4A. Representative images were selected from three independent experiments. *p < 0.05, **p < 0.01; ***p < 0.001, t-test. l Representative images of CRL4 knockdown OCCs incubated with BODIPY-conjugated bovine serum (DQ-BSA, red) for 1 h, or with serum- and glucose-free medium (starvation). Scale bar: 20 µm

Fig 3: STYK1 inhibitsEGFR-Beclin1interaction and reverses its efforts to the assembly of Beclin1 interactome. (A) Interaction between HA-Beclin1 and EGFR in HEK293T cells treated with or without EGF. Cell lysates were used for IP and western blotting with indicated antibodies. (B) Interaction between endogenous Beclin1 and EGFR after STYK1 knockdown in HEK293T cells treated with or without EGF. Cell lysates were used for IP and western blotting with the indicated antibodies. (C and D) Interaction between activated EGFR L858R/?746-750 and HA-Beclin1 after STYK1 overexpression in HEK293T cells treated with or without EGF, erlotinib (1 µM) or gefitinib (1 µM). Cell lysates were used for IP and western blotting with the indicated antibodies. (E) Level of total tyrosine phosphorylation of HA-Beclin1 after STYK1 overexpression in HEK293T cells with EGF treatment. Cell lysates were used for IP and western blotting with the indicated antibodies. (F) Level of Beclin1 dimerization after STYK1 knockdown in EGFR L858R transfected HEK293T cells. Cell lysates were used for IP and western blotting with the indicated antibodies. (G and H) Interaction between Beclin1 and Bcl2, Rubicon and VPS34 after STYK1 overexpression in HEK293T cells treated with or without EGF. Cell lysates were used for IP and western blotting with the indicated antibodies. (I) Interaction between HA-Beclin1 and FLAG-Bcl2 after STYK1 overexpression in HCC827 cells transfected with or without EGFR siRNAs. Cell lysates were used for IP and western blotting with the indicated antibodies. (J) Different PtdIns3K-C1 complex components from the control or STYK1 overexpressed HEK293T cells co-transfected with EGFR L858R or ?746-750 mutant were immunoprecipitated by the ATG14 antibody. PIK3C3 activity was measured by analyzing PtdIns3P production using the ELISA assay described in the Materials and Methods. The PtdIns3P fold change was calculated based on the concentration of PtdIns3P and was normalized to the amount of ATG14 used in the assay. Data were represented as mean ?± ?SD, *P ?< ?0.05; **P ?< ?0.01; ***P ?< ?0.001.

Fig 4: STYK1 abrogatesEGF-mediatedautophagy inhibition. (A) Western blotting analysis were used to analyze the protein expression levels of LC3-II in A549 cells overexpressing FLAG-STYK1 with or without EGF treatment. (B) Representative confocal images of GFP-ATG14, GFP-WIPI1 and GFP-ZFYVE1 distribution in FLAG-STYK1 transfected HeLa cells with or without EGF treatment. (C) The numbers of GFP-ATG14, GFP-WIPI1 and GFP-ZFYVE1 puncta were quantified (n = 10). Scale bars, 10 µm. (D and E) Representative confocal microscopy images of the red-only puncta and the yellow puncta in A549 cells after co-transfection with FLAG-STYK1 with or without EGF treatment for indicated times. The numbers of the autolysosomes (GFP-negative/RFP-positive, red puncta) and autophagosomes (GFP-positive/RFP-positive, yellow puncta) were quantified (n = 10). Scale bars, 10 µm. (F and G) Representative images of autophagosomes or autolysosomes of the A549 cells transfected with FLAG-STYK1 with or without EGF treated conditions. Both low- and high-power (zoom) images are displayed. Red arrows indicate autophagic structures. The puncta number of autophagic structures per area were quantified (n = 10). (H and I) Western blotting analysis were used to analyze the protein expression levels of LC3-II in MEF cells co-overexpressing FLAG-STYK1 and EGFR-L858R mutant with or without erlotinib (1 µM) treatment. (J) Representative immunohistochemical images of p62 and LC3 in excised HCC827 tumor tissues. Data were represented as mean ?± ?SD, *P ?< ?0.05; **P ?< ?0.01; ***P ?< ?0.001.

Fig 5: Phosphorylation of STYK1 S304 collaborated with Y356 to enhance autophagy activation. (A) Interaction between EGFR and STYK1 or its mutants as indicated in HEK293T cells treated with EGF. Cell lysates were used for IP and western blotting with the indicated antibodies. (B) HCC827 cells transfected with either MYC-STYK1 or its mutants Y356A/S304A expression plasmid for 48 h treated with erlotinib (1 µM) were harvest and cell lysates were subjected to co-IP and western blotting with the indicated antibodies. (C) The assembly of Beclin1 interactomes after STYK1 or its mutants overexpressing as indicated in HEK293T cells transfected with EGFR L858R. Cell lysates were used for IP and western blotting with the indicated antibodies. (D) Different PtdIns3K-C1 complex components from the control or STYK1 overexpressed HCC827 cells under starvation or gefitinib (1 µM) treatment conditions were immunoprecipitated by the ATG14 antibody. PIK3C3 activity was measured by analyzing PtdIns3P production using the ELISA assay described in the Materials and Methods. The PtdIns3P fold change was calculated based on the concentration of PtdIns3P and was normalized to the amount of ATG14 used in the assay. (E and F) Western blotting analysis were used to analyze the protein expression levels of LC3-II in HCC827 cells overexpressing MYC-STYK1 and its mutants with or without gefitinib (1 µM), erlotinib (1 µM) and CQ (10 µM) treatment. (G) Western blotting analysis were used to analyze the protein expression levels of LC3-II in A549 cells co-expressing EGFR ?746-750 and MYC-STYK1 mutants. (H) Western blotting analysis were used to analyze the protein expression levels of LC3-II in MEF cells with ATG7 knockout after EGFR ?746-750 and MYC-STYK1 mutants co-expressing. (I and J) Representative confocal images of GFP-ATG14, GFP-LC3 and GFP-ZFYVE1 distribution in MYC-STYK1 and its mutants expressing HCC827 cells treated with or without gefitinib (1 µM). The number of GFP-ATG14, GFP-LC3 and GFP-ZFYVE1 puncta was quantified (n ?= ?10). Scale bars, 10 ?µm. Data were represented as mean ?± ?SD, *P ?< ?0.05; **P ?< ?0.01; ***P ?< ?0.001.