Journal: Cell Death & Disease

Article Title: PURPL represses autophagic cell death to promote cutaneous melanoma by modulating ULK1 phosphorylation

doi: 10.1038/s41419-021-04362-8

Figure Lengend Snippet: a Visualization of PURPL in A375 cells by RNA fluorescence in situ hybridization (FISH). b Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the proteins interacting with PURPL by subtracting the proteins non-specific binding to EGFP RNA after identified by HPLC-MS. “mTOR signaling pathway” is highlighted. c Biotin-labeled PURPL transcript was used to retrieve interacting protein partners by RNA pulldown with beads only and EGFP as controls. The resulting protein mix was applied to detect ULK1, mTOR, and AMPK by western blot. d RNA immunoprecipitation (RIP) assay was performed using antibodies against ULK1, mTOR, and AMPK while IgG was used as control. The retrieved PURPL RNA was detected by qPCR. e Co-localization of PURPL with ULK1 was detected using FISH and IF staining and observed by confocal microscope. f , g LC3B and p62 proteins were detected after PURPL knockdown or overexpression by western blot, GAPDH was used as loading control. h The autophagy levels in melanoma cells following PURPL knockdown by ASO mix (ASO1:ASO2 in 1:1 ratio) was evaluated by LC3 and LAMP1 staining. PBS-treated melanoma cells were used as positive control for autophagy induction. Scale bar, 50 μM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies and dilutions were used: ULK1 (Cell Signaling Technology, 8054, 1:2000), Phospho-ULK1 (Ser757) (Cell Signaling Technology, 14202, 1:2000), Phospho-ULK1 (Ser317) (Cell Signaling Technology, 37762, 1:2000), Phospho-ULK1 (Ser555) (Cell Signaling Technology, 5869, 1:2000), LC3B (SellckChem, A5202, 1:2000), mTOR (Santa Cruz Biotechnology, sc-517464, 1:2000), P62/SQSTM1 (SellckChem, A5180, 1:2000), P53 (Santa Cruz Biotechnology, sc-47698, 1:2000), p-P53 (Ser315) (WanLeiBio, WLP1333, 1:2000), and GAPDH (Santa Cruz Biotechnology, sc-25778,1:5000).

Techniques: Fluorescence, In Situ Hybridization, Binding Assay, Labeling, Western Blot, Immunoprecipitation, Staining, Microscopy, Over Expression, Positive Control

Journal: Cell Death & Disease

Article Title: PURPL represses autophagic cell death to promote cutaneous melanoma by modulating ULK1 phosphorylation

doi: 10.1038/s41419-021-04362-8

Figure Lengend Snippet: a , b The PURPL-regulated autophagy was validated in autophagy inhibitor 3-MA-treated (5 mM for 5 h) melanoma cells following PURPL knockdown by ASO mix (ASO1:ASO2 in 1:1 ratio) using LC3 staining. c LC3B and P62 protein was detected after PURPL knockdown and 3-MA treatment by western blot, GAPDH was used as loading control. d , e The PURPL-regulated autophagy levels in melanoma cells following PURPL overexpression was evaluated by LC3 staining. PBS-treated melanoma cells were used as positive control for autophagy induction. f LC3B and P62 protein was detected after PURPL overexpression and PBS treatment by western blot, GAPDH was used as loading control. g LC3B and P62 protein was detected after PURPL overexpression and Rapamycin treatment by western blot, GAPDH was used as loading control. Scale bar, 100 μM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies and dilutions were used: ULK1 (Cell Signaling Technology, 8054, 1:2000), Phospho-ULK1 (Ser757) (Cell Signaling Technology, 14202, 1:2000), Phospho-ULK1 (Ser317) (Cell Signaling Technology, 37762, 1:2000), Phospho-ULK1 (Ser555) (Cell Signaling Technology, 5869, 1:2000), LC3B (SellckChem, A5202, 1:2000), mTOR (Santa Cruz Biotechnology, sc-517464, 1:2000), P62/SQSTM1 (SellckChem, A5180, 1:2000), P53 (Santa Cruz Biotechnology, sc-47698, 1:2000), p-P53 (Ser315) (WanLeiBio, WLP1333, 1:2000), and GAPDH (Santa Cruz Biotechnology, sc-25778,1:5000).

Techniques: Staining, Western Blot, Over Expression, Positive Control

Journal: Cell Death & Disease

Article Title: PURPL represses autophagic cell death to promote cutaneous melanoma by modulating ULK1 phosphorylation

doi: 10.1038/s41419-021-04362-8

Figure Lengend Snippet: a Loss of PURPL inhibits subcutaneous melanoma growth in a mouse xenograft model. Tumor volumes (mm 3 ) were plotted according to day. The statistical data represent the average of three independent experiments ±s.d, respectively. b The mice were sacrificed at the end of the experiment and the dissected tumors from three representative mice are shown. black arrows indicate the NC ASO-treated xenografts whereas white arrows indicate PURPL ASO-treated xenografts. c PURPL expression was measured in the dissected tumors by qPCR. The statistical data represent the average of three independent experiments ±s.d. d The expression levels of PURPL in tumor sections were evaluated using in situ hybridization. Scale bar, 100 µm. e Protein amount of total ULK1, phosphorylated ULK1, LC3B, and p62 was detected in paired control and PURPL-depleted groups by western blot, GAPDH was used as loading control. f A model depicts PURPL interacts with mTOR and ULK1 to differentially regulate ULK1 phosphorylation to repress autophagic cell death and promote melanoma development.

Article Snippet: The following primary antibodies and dilutions were used: ULK1 (Cell Signaling Technology, 8054, 1:2000), Phospho-ULK1 (Ser757) (Cell Signaling Technology, 14202, 1:2000), Phospho-ULK1 (Ser317) (Cell Signaling Technology, 37762, 1:2000), Phospho-ULK1 (Ser555) (Cell Signaling Technology, 5869, 1:2000), LC3B (SellckChem, A5202, 1:2000), mTOR (Santa Cruz Biotechnology, sc-517464, 1:2000), P62/SQSTM1 (SellckChem, A5180, 1:2000), P53 (Santa Cruz Biotechnology, sc-47698, 1:2000), p-P53 (Ser315) (WanLeiBio, WLP1333, 1:2000), and GAPDH (Santa Cruz Biotechnology, sc-25778,1:5000).

Techniques: Expressing, In Situ Hybridization, Western Blot

Journal: Oncotarget

Article Title: Resistance to glucose starvation as metabolic trait of platinum-resistant human epithelial ovarian cancer cells

doi: 10.18632/oncotarget.14118

Figure Lengend Snippet: ( A ) Flow cytometry evaluation of membrane GLUT1. Data expressed as mean percent values in tumor cells from GA and GNA patients (± SD). * P < 0.05. ( B ) qRT-PCR analysis of key enzymes of the glucose metabolic chain in FACS sorted CD45 neg /CD44 pos tumor cells from GA ( n = 6) and GNA ( n = 6) patients. * P < 0.01. ( C ) ECAR analysis in CD45 neg /CD44 pos tumor cells from GA and GNA patients. The left histogram shows the mean values of the basal ECAR in 6 GA vs 6 GNA patients (± SD). * P < 0.05. One representative experiment is shown on the right; the first three points of the graph indicate the basal ECAR ratio in glucose starvation. At different time points (arrows) the indicated inhibitors of OXPHOS (oligomycin) or glycolysis (2DG) were added. ( D ) WB analysis of MCT4 lactate transporter in pooled CD45 neg /CD44 pos tumor cells from GA or GNA patients (pools #1) and xenotransplants (pools #2). Each pool consisted of 3 GA or 3 GNA samples. After normalization against actin, expression ratios were calculated by dividing GNA signal intensity values by those of GA cells. On the left a representative blot, on the right graph depicts mean expression ratios in 8 different pools (± SD). * P < 0.05. ( E ) WB analysis of Beclin1and LC3-II proteins in CD45 neg /CD44 pos tumor cell pools of GA or GNA patients. One representative blot is shown on the left, while the right histogram shows the mean values of 4 different experiments (three samples/pool; ± SD). * P < 0.05. ( F ) Left panel: flow cytometry analysis of cell morphology and viability by AnnexinV/PI staining in two representative GA and GNA samples, treated in vitro for 72 h with 20 μM CQ or 100 nM BafA1. Right panel: histogram displaying the mean fold change (± SD) in cell viability, calculated by dividing the percentage of live cells after treatment by untreated values of 4 GA and 5 GNA samples. * P < 0.05.

Article Snippet: The membranes were hybridized using antibodies against GLUT1 (1:2000, Novus Biological, Abingdon, U.K.), PARP (1:1000, Cell Signaling, Danvers, MA), MCT4 (1:1000, Santa Cruz, Dallas, TX), LC3 (1:1000, Cell Signaling), actin (1:500, Sigma-Aldrich), Cyclin D1 (1:1000, Cell Signaling), Beclin1 (1:1000, Abcam) and α-tubulin (1:4000, Sigma-Aldrich) followed by the appropriate secondary HRP-conjugated antibody (Amersham-Pharmacia, Amersham, U.K.).

Techniques: Flow Cytometry, Membrane, Quantitative RT-PCR, Expressing, Staining, In Vitro