Journal: Journal of Biological Chemistry

Article Title: Lysosomal Two-pore Channel Subtype 2 (TPC2) Regulates Skeletal Muscle Autophagic Signaling

doi: 10.1074/jbc.m114.608471

Figure Lengend Snippet: FIGURE 2. ReducedenduranceandalteredautophagicsignalinginskeletalmuscleofTpcn2/mice.A,mice(male;4–5monthsold)weretestedonatreadmill underdietaryconditionsofregularchowor3-dayfasting.Thedistancestraveled(inmeters)untilexhaustionwererecordedandanalyzed(n6mice/group).Dataare means S.E. (*, p 0.05; **, p 0.01). B, autophagy flux studies under regular diet or extended starvation. Mice (male; 4–5 months old) were fed with regular chow or fasted for 3 days. The TA muscles were then harvested and homogenized for immunoblotting with p-mTOR, total mTOR (t-mTOR), p-AKT, total AKT (t-AKT), p62, LAMP1, and -tubulin. C–F, densitometry quantification of mTOR activity (p-mTOR/total mTOR; C), AKT activity (p-AKT/total AKT; D), normalized (over -tubulin) p62 (E), and LAMP1 (F). Error bars represent S.E. (n 6 mice/group; *, p 0.05; **, p 0.01; NS, not significant; analysis of variance).

Article Snippet: Antibodies for Western blotting were as follows: -tubulin (Sigma), LC3 (clone 5F10, Nanotools), lysosome-associated membrane protein-1 (LAMP1) (Santa Cruz Biotechnology), HA (clone12CA5, Roche Applied Science), dystrophin (Spring Bioscience), p62/ sequestosome-1 (SQSTM1) (Novus Biological), S6K, phosphoS6K (phospho-Thr-389), S6RP (ribosomal protein), phosphoS6RP (phospho-Ser-235/236), AKT and phospho-AKT, and mTOR and phospho-mTOR (phospho-Ser-2448) (all from Cell Signaling Technology).

Techniques: Muscles, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Enhancing the efficiency of oncolytic vaccinia virus for ovarian cancer treatment

doi: 10.1101/2025.02.04.636413

Figure Lengend Snippet: A. Representative phase contrast images of ID8 Trp53 -/- cells after 16 hours treatment with DMSO or vinorelbine (1 μM). Scale bar = 150 μm. B. Immunofluorescent images of microtubule cytoskeleton (Tubulin - green) in ID8 Trp53 -/- cells treated with DMSO or vinorelbine with or without infection with ΔVFTK (Vaccinia - red). DAPI (blue) was used to stain nuclei and cytoplasmic viral factories. Yellow arrows indicate fragmented nuclei. Scale bar = 10 μM. C. Immunoblot of the indicated viral proteins following infection for 4 or 8 hours with ΔVFTK-NG following 16 hours pre-treatment with DMSO or vinorelbine. Vinculin is the cell loading control. D. Immunoblot examining the levels of LC3 lipidation (LC3-II) and p62 in ID8 Tr53 -/- cells pretreated with vinorelbine or DMSO for 8 hours and infected with ΔVFTK-NG for 24 hours. Chloroquine (Clq) treatment for 3 hours represents the positive control. E. Immunoblot examining the levels pf MLKL phosphorylation and PARP cleavage in ID8 Tr53 -/- cells pretreated with vinorelbine or DMSO for 8 hours and infected with ΔVFTK-NG for 24 hours. Staurosporine (S) (8 hours) and 0.1% hydrogen peroxide (H2O2) combined with Z-VAD (two hours) represent positive controls for apoptosis and necroptosis respectively. F. Immunoblot examining the levels of cleaved caspase 8, 3 and PARP in ID8 Tr53 -/- cells treated with vinorelbine 8 hours before or after infection with ΔVFTK-NG for 24 hours. In D, E and F H5 and GAPDH repsent the viral and cell loading controls, respectively. For all experiments, uninfected DMSO-treated cells (UI) were the negative control. Asterisks (*) indicate non-specific bands. Immunoblot experiments were repeated three times and representative examples are shown. G. Median percentage cell confluency over time for the indicated conditions. Cells were treated with the indicated compounds or ΔVFTK-NG at 16 hours post seeding (dashed line numbered 1). The dashed line numbered 2 (24 hours post cell seeding) represents the time of addition of either vinorelbine or ΔVFTK-NG for the combination groups.

Article Snippet: Primary antibodies used were: F12 (1:4,000, ), F13 (1:6,000, ), H5 (1:10,000, ), GRB2 (1:1,000, Santa Cruz, Dallas, Texas, #sc-255), Vinculin (1:10,000, Sigma-Aldrich, #V9264), GAPDH (1:1,000, Santa Cruz, #sc-32233), PARP (1:1,000, Cell Signalling, Danvers, Massachusetts, #9542), cleaved caspase 8 (1:1,000, Cell Signalling, #8592), cleaved caspase 3 (1:1,000, Cell Signalling, #9664), LC3-B (1:1,000, Abcam [#ab48394], Cambridge, UK) and p62/SQSTM1 (1:1,000, Novus Biologicals, Centennial, Colorado, #NBP1-42822).

Techniques: Infection, Staining, Western Blot, Control, Positive Control, Negative Control

Journal: Molecules

Article Title: Identification of Novel Vacuolin-1 Analogues as Autophagy Inhibitors by Virtual Drug Screening and Chemical Synthesis

doi: 10.3390/molecules22060891

Figure Lengend Snippet: Figure 1. Vacuolin-1 analogues identified via virtual screening induced the accumulation of both LC3B-II and SQSTM1 in HeLa cells in a dose dependent manner after a 6 h treatment.

Article Snippet: Antibodies and reagents—LC3B (Novus, Littleton, CO, USA, NB100-2220; 1:1000 for the Western blotting analysis (WB)), SQSTM1 (Novus, NBP1-48320; 1:1000 WB), EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-03; 1:250 WB), LAMP1 (Cell Signaling Technology, 9091; 1:500 immunofluorescence analysis), GAPDH antibodies (Sigma, Saint Louis, MO, USA, G8795; 1:5000 WB).

Techniques: Analogues

Journal: Molecules

Article Title: Identification of Novel Vacuolin-1 Analogues as Autophagy Inhibitors by Virtual Drug Screening and Chemical Synthesis

doi: 10.3390/molecules22060891

Figure Lengend Snippet: Figure 2. Vacuolin-1 analogues identified via virtual screening inhibited autophagic flux in HeLa cells. (A) Treatment of HeLa cells with vacuolin-1 analogues (10 µM) and BAF (100 nM) failed to further increase the accumulation of both LC3B-II and SQSTM1 as compared to either drug alone. (B) Vacuolin-1 (10 µM) or VS6 (10 µM) induced the accumulation of yellow LC3B-II puncta in RFP-GFP-LC3B expressing HeLa cells. Scale bar = 10 µm.

Article Snippet: Antibodies and reagents—LC3B (Novus, Littleton, CO, USA, NB100-2220; 1:1000 for the Western blotting analysis (WB)), SQSTM1 (Novus, NBP1-48320; 1:1000 WB), EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-03; 1:250 WB), LAMP1 (Cell Signaling Technology, 9091; 1:500 immunofluorescence analysis), GAPDH antibodies (Sigma, Saint Louis, MO, USA, G8795; 1:5000 WB).

Techniques: Analogues, Expressing

Journal: Molecules

Article Title: Identification of Novel Vacuolin-1 Analogues as Autophagy Inhibitors by Virtual Drug Screening and Chemical Synthesis

doi: 10.3390/molecules22060891

Figure Lengend Snippet: Figure 4. Synthesized vacuolin-1 analogues induced the accumulation of both LC3B-II and SQSTM1 in HeLa cells in a dose dependent manner after a 6 h treatment.

Article Snippet: Antibodies and reagents—LC3B (Novus, Littleton, CO, USA, NB100-2220; 1:1000 for the Western blotting analysis (WB)), SQSTM1 (Novus, NBP1-48320; 1:1000 WB), EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-03; 1:250 WB), LAMP1 (Cell Signaling Technology, 9091; 1:500 immunofluorescence analysis), GAPDH antibodies (Sigma, Saint Louis, MO, USA, G8795; 1:5000 WB).

Techniques: Synthesized, Analogues

Journal: Molecules

Article Title: Identification of Novel Vacuolin-1 Analogues as Autophagy Inhibitors by Virtual Drug Screening and Chemical Synthesis

doi: 10.3390/molecules22060891

Figure Lengend Snippet: Figure 5. Synthesized vacuolin-1 analogues inhibited autophagic flux in HeLa cells. (A) A5 (1 µM), not C5 (1 µM), induced the accumulation of yellow LC3B-II puncta in RFP-GFP-LC3B expressing HeLa cells. Scale bar = 10 µm. (B) Treatment of HeLa cells with vacuolin-1 analogues (1 µM) and BAF (100 nM) failed to further increase the accumulation of both LC3B-II and SQSTM1 as compared to either drug alone.

Article Snippet: Antibodies and reagents—LC3B (Novus, Littleton, CO, USA, NB100-2220; 1:1000 for the Western blotting analysis (WB)), SQSTM1 (Novus, NBP1-48320; 1:1000 WB), EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-03; 1:250 WB), LAMP1 (Cell Signaling Technology, 9091; 1:500 immunofluorescence analysis), GAPDH antibodies (Sigma, Saint Louis, MO, USA, G8795; 1:5000 WB).

Techniques: Synthesized, Analogues, Expressing

Journal: Cancer cell international

Article Title: Inhibition of autophagy induced by tetrandrine promotes the accumulation of reactive oxygen species and sensitizes efficacy of tetrandrine in pancreatic cancer.

doi: 10.1186/s12935-024-03410-5

Figure Lengend Snippet: Fig. 1 Tetrandrine (TET) induces autophagy in pancreatic cancer cells. a PANC-1 cells were treated with or without TET for 6 and 24 h, and transmission electron microscopy was used to observe autophagosomes. b, c PANC-1, SW1990 and BxPC-3 cells were treated with TET in gradient concentrations for 24 h. PANC-1, SW1990 and BxPC-3 cells were treated with TET for indicated time. LC3 and p62 protein expression was detected by western blot. d PANC-1, SW1990 and BxPC-3 cells were pretreated with 10mM 3-MA for 24 h and then treated with 10µM TET for another 24 h. Acidic vesicular organelles (AVOs) were detected with acridine staining, and the number of cells with AVOs was quantified. Scale bars indicate 200 μm. e SW1990 cells were transfected with GFP-mRFP-LC3 and then treated with TET and Bafilomycin A1 for 24 h. Cells were observed using a fluorescence microscope and the number of autopha gosomes (yellow dots) and autolysosomes (red dots) was counted. Scale bars indicate 50 μm.c Data was presented as mean ± SD of three independent experiments.* indicates p<0.05, *** indicates p<0.005, **** indicates p<0.001

Article Snippet: Primary antibodies against LC3B (3868), P62 (23,214), AKT(4691), phosphor-AKT(Ser473) (4060), mTOR (2972), phosphor-mTOR(Ser2448) (2971), Cleaved-caspase3 (9664), Cleaved-caspase9 (20,750) and Cleaved-caspase PARP (5625) were purchased from CST(USA).

Techniques: Transmission Assay, Electron Microscopy, Expressing, Western Blot, Staining, Transfection, Fluorescence, Microscopy

Journal: Cancer cell international

Article Title: Inhibition of autophagy induced by tetrandrine promotes the accumulation of reactive oxygen species and sensitizes efficacy of tetrandrine in pancreatic cancer.

doi: 10.1186/s12935-024-03410-5

Figure Lengend Snippet: Fig. 8 Inhibition of autophagy enhances TET-mediated cytotoxicity against pancreatic cancer in vivo. a Schematic illustration for the nude mouse experi mental design. b Changes in tumor volume. c Xenograft tumors (n = 4). d Tumor weight(n = 4). e Immunohistochemical (IHC) assay of Ki-67, P62, cleaved caspase-3, SOD2 in tumor specimens. Scale bars indicate 200 μm. f Quantification of IHC staining of Ki-67(n = 5), P62 and cleaved caspase-3. g ROS ac cumulation in tumor specimens were detected using DHE staining. h Quantification of DHE fluorescent intensity (n = 5). Scale bars indicate 200 μm. Data was presented as mean ± SD. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.005, **** indicates p<0.001

Article Snippet: Primary antibodies against LC3B (3868), P62 (23,214), AKT(4691), phosphor-AKT(Ser473) (4060), mTOR (2972), phosphor-mTOR(Ser2448) (2971), Cleaved-caspase3 (9664), Cleaved-caspase9 (20,750) and Cleaved-caspase PARP (5625) were purchased from CST(USA).

Techniques: Inhibition, In Vivo, Immunohistochemical staining, Immunohistochemistry, Staining