fip200 (1:300)  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Transient receptor potential ion channel TRPM2 promotes AML proliferation and survival through modulation of mitochondrial function, ROS, and autophagy

doi: 10.1038/s41419-020-2454-8

Figure Lengend Snippet: a Western blotting was performed to determine expression of HIF-1α, HIF-2α, FOXO3a, IQGAP1, Nrf2, CREB, and CREB phosphorylation in TRPM2-depleted U937 cells. In these experiments, two knockout (KO-1-2) and two scrambled clones (Scr-2-3) were selected randomly for western blots with or without treatment with 0.3 µM doxorubicin. b Expression of autophagy proteins ULK1, Atg7, Atg5, Atg13, FIP200, Atg101, and transcription factor ATF4 was examined by western blotting. p62, Tom20, and LC3B-I and II, which are modified in autophagy, were also studied. Actin was probed to confirm equivalent loading. In a and b , western blots performed for each protein are shown on the left. One blot for each protein is shown here and the others in Supplemental Information Figs. , . Densitometry measurements from two to three experiments for each protein were standardized to results for each experiment’s average untreated scrambled control and means ± s.e.m. calculated and shown on the right ( n = 4-6, Tom20 n = 8). * p ≤ 0.001, ** p < 0.03, group effect, Scr vs KO, two-way ANOVA. c RT-PCR was used to measure TRPM2, HIF-1α, HIF-2α, CREB, ULK1, and Atg7 mRNA in TRPM2-depleted leukemia cells. Results summarizing two (TRPM2, ULK1), three (Atg7) or four (HIF-1/2α, CREB) experiments are shown (mean ± s.e.m., n = 8-16). * p ≤ 0.0001, ** p < 0.015, one-way ANOVA. d U937 cell were incubated with or without bafilomycin A1, and conversion of LCB-I to II was examined with western blotting. Four experiments were performed. One blot is shown here and the others in Supplemental Information Fig. . Densitometry measurements were obtained. Relative autophagic flux was calculated as: (O.D. LC3B-II + Bafilomycin/O.D. Actin) - (O.D. LC3B-II-Bafilomycin/O.D. Actin) for each band. Means ± s.e.m. for the four experiments for each cell line are shown below the figure ( n = 8). * p < 0.05, unpaired, two-tailed t -test. e Western blotting was performed to measure TRPM2, CREB, ATF4, ULK1, Atg7, Atg5, and LC3B-I and II expression in four experiments after TRPM2-L reconstitution. One blot is shown here and the others in Supplemental Information Fig. . Densitometry measurements were normalized to each blots’ untreated scrambled control, and mean densitometry measurements ± s.e.m. for experiments with each protein are shown on the right. * p < 0.04 ( n = 4) one-way ANOVA.

Article Snippet: Blots were probed with anti-TRPM2-C (1:300; Bethyl Laboratories, Montgomery, TX, USA); with antibodies to ATF4 (1:400), Atg7 (1:2000), Atg13 (1:500), Atg101 (1:500), pCREB1 and CREB1 (1:250), FIP200 (1:300), forkhead box transcription factor 3a (FOXO3a; 1:400), HIF-1α (1:250), Nrf2 (1:400), and ULK1 (1:1000) from Cell Signaling Technology (Boston, MA, USA); Atg5 (1:1000) from Medical Biological Lab (Japan); COX6B1 (1:500), MT-CO2 (1:500), MT-ND2 (1:500), and NDUFA13 (1:300) from LSBio (Seattle, WA, USA); HIF-2α (1:500) and LC3B (1:2000) from Novus (Littleton, CO, USA); IQGAP1 (1:5000) from Abcam (Cambridge, MA, USA); p62 (1:5000) from American Research Products (Waltham, MA, USA); Tom20 (1:5000) from Santa Cruz Biotech (Dallas, TX, USA); and actin (1:5000) from Sigma (St. Louis, MO, USA).

Techniques: Western Blot, Expressing, Knock-Out, Clone Assay, Modification, Reverse Transcription Polymerase Chain Reaction, Incubation, Two Tailed Test