Journal: Molecular Cell

Article Title: The Cargo Receptor NDP52 Initiates Selective Autophagy by Recruiting the ULK Complex to Cytosol-Invading Bacteria

doi: 10.1016/j.molcel.2019.01.041

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-FIP200 , ProteinTech , Cat# 10043-2-AP; RRID: AB_2253571.

Techniques: Luciferase, Transduction, Virus, Recombinant, Plasmid Preparation, Protease Inhibitor, Reverse Transcription, Negative Control, Software, Chromatography, Molecular Weight

Journal: International journal of oncology

Article Title: HMGB1 translocation is involved in the transformation of autophagy complexes and promotes chemoresistance in leukaemia.

doi: 10.3892/ijo.2015.2985

Figure Lengend Snippet: Figure 4. The ULK1-mAtg13-FIP200 complex functions upstream of the HMGB1-Beclin1 complex. (A) Jurkat cells were transfected with control or HMGB1 shRNA. The mRNA and protein levels of HMGB1 were assessed via RT-qPCR and western blot analysis, respectively (n=3, *p<0.05). (B) Jurkat cells were transfected with control or HMGB1 shRNA and treated with or without DNR (0.4 µM) for 24 h. The resulting immunocomplexes were analysed via immuno- precipitation and western blot analysis using specific antibodies. Tubulin was used as a loading control. (C and D) Jurkat cells were transfected with control, FIP200 or HMGB1 shRNA and then treated with or without DNR (0.4 µM) for 24 h. The cell lysates were subjected to immunoprecipitation followed by western blot analysis. GAPDH and actin were used as loading controls.

Article Snippet: The antibodies to FIP200, ULK1, mAtg13, PI3KC3, Phospho-ULK1 (Ser555) and Rabbit (DA1E) mAb IgG XPTM Isotype Control were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Control, shRNA, Quantitative RT-PCR, Western Blot, Immunoprecipitation

Journal: International journal of oncology

Article Title: HMGB1 translocation is involved in the transformation of autophagy complexes and promotes chemoresistance in leukaemia.

doi: 10.3892/ijo.2015.2985

Figure Lengend Snippet: Figure 6. Schematic representation of the potential autophagy-related drug resistance process in leukaemia. Under normal conditions, the ULK1-mAtg13-FIP200 complex is suppressed by mTOR, and Bcl-2 binds to Beclin1 to reduce its pro-autophagic activity. Under stress conditions, HMGB1 translocates to the cytosol, which is promoted by the activation of the upstream complex ULK1-mAtg13-FIP200. Consequently, the Bcl-2-Beclin1 interaction is disrupted by the phos- phorylation of Bcl-2 and by competitive displacement of Bcl-2 with HMGB1. The assembly of the HMGB1-Beclin1 complex facilitates the formation of the PI3KC3-Beclin1 complex. These dissociations and recouplings likely enhance the anti-apoptotic state, promote autophagy, and ultimately protect the tumour cells during chemotherapy.

Article Snippet: The antibodies to FIP200, ULK1, mAtg13, PI3KC3, Phospho-ULK1 (Ser555) and Rabbit (DA1E) mAb IgG XPTM Isotype Control were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Activation Assay

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