Supplementary MaterialsSupplementary Figure 1: P62, an autophagic receptor, regulates autophagic flux

Supplementary MaterialsSupplementary Figure 1: P62, an autophagic receptor, regulates autophagic flux. non-small cell lung tumor cells with EGFR mutation (L885R/T790 M). (A) H1975 cells had been transfected with AQTGTGKT peptide (10 M) for 24 h accompanied by immunoblot. (B) Identical to (A) except that immunoprecipitation was performed. (C) H1975 cells had been transfected with AQTGTGKT peptide (10 M) for 24 h accompanied by treatment with erlotinib (20 M) or osimertinib (5 M) for different time intervals. Immunoblot was performed then. (D) H1975 cells had been transfected with AQTGTGKT peptide (10 M) for 48 h accompanied by migration and invasion assays. * 0.05; ** 0.005. (E) H1975 cells had been transfected with AQTGTGKT peptide (10 M) for 24 h accompanied by Cetrimonium Bromide(CTAB) treatment with various concentrations of erlotinib or osimertinib for 24 h. MTT assays were then performed. (F) Same as (A) except that immunofluorescence staining was performed. *** 0.0005. (G) H1975 cells were transfected with the indicated peptide (10 M) for 24 h followed by immunoblot. Image_4.JPEG (280K) GUID:?53974972-A7D8-4B0C-B79A-364BAF4CEDE0 Supplementary Figure 5: AQTGTGKT peptide binds to CAGE and inhibits the binding of CAGE to Beclin1. (A) H1975 cells were transfected with FITC-AQTGTGKT peptide (10 M) or unlabeled AQTGTGKT peptide (10 M). At each time point after transfection, Cetrimonium Bromide(CTAB) fluorescence microscopic observation was performed. Scale bar represents 10 m. (B) H1975 cells were transfected with wild type or mutant CAGE-derived peptide (each at 10 M). At 24 h after transfection, immunoprecipitation was performed. (C) H1975 cells were transfected with the indicated peptide (10 M). At 24 h after transfection, immunoprecipitation was performed. Image_5.JPEG (155K) GUID:?CB44876E-EDAD-47F0-92EF-F853C793240F Supplementary Physique 6: AQTGTGKT peptide shows co-localization with CAGE and enhances cleavage of PARP in response to erlotinib and osimertinib. (A) FITC-labeled AQTGTGKT peptide (10 M) or unlabeled AQTGTGKT peptide (10 M) was transfected into PC-9/ER cells. At 24 h after Cetrimonium Bromide(CTAB) transfection, co-localization of AQTGTGKT peptide with CAGE was examined. Scale bar represents 10 m. (B) H1975 cells were transfected with the indicated peptide (each at 10 M). At 24 h after transfection, cells were then treated without or RXRG with erlotinib (20 M) or osimertiniub (5 M) for 24 h. Image_6.JPEG (112K) GUID:?8EB3EC88-5E67-4A68-BA26-56ADDCF39220 Supplementary Figure 7: CAGE regulates autophagic flux and anti-cancer drug-resistance. (A) PC-9/ER cell were transfected with the indicated siRNA (each at 10 nM). At 48 h after transfection, immunoblot, and immunoprecipitation were performed. (B) PC-9/ER cell were transfected with the indicated siRNA (each at 10 nM). On the next day, cells Cetrimonium Bromide(CTAB) were then treated with erlotinib (20 M) or osimertinib (5 M) for Cetrimonium Bromide(CTAB) various time intervals followed by immunoblot. (C) H1975 cells were transfected with the indicated siRNA (each at 10 nM). At 48 h after transfection, immunoblot, and immunoprecipitation were performed. (D) H1975 cells were transfected with the indicated siRNA (each at 10 nM). On the next day, cells were then treated with erlotinib (20 M) or osimertinib (5 M) for various time intervals followed by immunoblot. (E) H1975 cells were transfected with the indicated siRNA (each at 10 nM). At 48 h after transfection, migration, and invasion assays were performed. ** 0.005; *** 0.0005. Image_7.JPEG (265K) GUID:?5856BE89-E6D3-4877-A9FA-6395D49C1C83 Supplementary Figure 8: AQTGTGKT peptide decreases tumorigenic potential of H1975 non-small cell lung cancer cells. (A) H1975 (1 106) cells were injected into dorsal flanks of athymic nude mice. Following the establishment of sizeable tumor (~50 mm3), peptide (100, 200 g/mouse) was injected into the tail vein five times in a total of 31 days..