Journal: Cell Death Discovery

Article Title: c-Myb protects cochlear hair cells from cisplatin-induced damage via the PI3K/Akt signaling pathway

doi: 10.1038/s41420-022-00879-9

Figure Lengend Snippet: A Western blot showed a significantly decreased ratio of P-Akt/Akt and decreased level of P-PI3K (p85α) in the cultured HCs treated with cisplatin + AAV-ie for 48 h. The levels of P-PI3K (p85α) and P-Akt were significantly reduced in HCs co-treated with cisplatin, AAV-ie and LY294002 compared to the cisplatin + AAV-ie group, while they were significantly increased in HCs co-treated with AAV-c-Myb and cisplatin. The treatment with LY294002 decreased the above effects of c-Myb on p85α level and the ratio of P-Akt/Akt in the cisplatin + AAV-c-Myb + LY294002 group compared to the cisplatin + AAV-c-Myb group. B – F Immunostaining and cell counting showed that the overexpression of c-Myb reduced HC loss after cisplatin injury, but the treatment with LY294002 significantly increased the HC loss in the cisplatin + AAV-c-Myb + LY294002 group compared to the cisplatin + AAV-c-Myb group. Scale bar = 20 μm. *** p < 0.001, n = 6 for each group.

Article Snippet: The primary antibodies were as follows: anti-c-Myb antibody (1:1000 dilution, Millipore, USA), anti-P-Akt (1:1000 dilution; Cell Signaling Technology), anti-Akt (1:1000 dilution; Cell Signaling Technology), anti-P-PI3K (1:1000 dilution; Cell Signaling Technology), and anti-β-actin (1:2000 dilution; ZSGB-BIO).

Techniques: Western Blot, Cell Culture, Immunostaining, Cell Counting, Over Expression

Journal: bioRxiv

Article Title: Senescent cell survival relies on upregulation of lysosomal quality control mechanisms

doi: 10.1101/2025.03.31.646397

Figure Lengend Snippet: (A) Representative immunoblot analysis of dose-dependent activation of mTORC1 by LDL in proliferative or senescent cells. IMR90 ER:RasV12 primary fibroblasts were cultured in the presence of EtOH (Prol; proliferative) or 100 nM 4OHT for 6–8 days to induce RasV12 expression and oncogene-induced senescence (Sen; senescent). Cells were depleted of sterol with methyl-β cyclodextrin (MCD, 0.5% w/v) for 2 hours and then stimulated for 2 hours with increasing concentrations (0 to 100 μg/ml) of low-density lipoprotein (LDL). Cell lysates were analysed for phosphorylation of S6 (Ser235/236) and ULK1 (Ser757). (B) Quantification represents mean ± SEM; n = 3 independent experiments; two-way ANOVA followed by Dunnett’s multiple comparison. (C) Representative immunoblot analysis of activation of mTORC1 signalling by cholesterol. Proliferative or senescent cells were treated as stated in (A), however stimulated for 2 hours with increasing concentrations (0 to 100 μM) of MCD:cholesterol. (D) Phosphorylation of S6 (Ser235/236) was quantified as in (B). (E) Proliferating or senescent cells were fixed, permeabilised using a liquid-nitrogen pulse and subjected to cholesterol labelling by GST-D4H*–mCherry. Scale bar, 10 μm. The quantification of D4H*–mCherry signal intensity is shown in (F). The quantification represents the mean intensity ± SEM compared by Student’s t test (unpaired); n = 3 independent experiments with at least 5 fields of view per experimental repeat. (G) Proliferative or senescent cells were fixed and immunostained for antibodies against LDLR (green) and LAMP2 (magenta), scale bar, 20 μm. (H) Colocalisation analysis of endogenous LDLR and LAMP2. The graph represents means ± SEM. Pearson’s correlation; n = 3 independent experiments with ≥ 6 fields of view per experimental repeat. Student’s t test (unpaired). (I) Proliferating and senescent cells were surface biotinylated, and streptavidin agarose was used to capture biotinylated membrane proteins. Surface abundance of the indicated proteins was assessed by immunoblotting. (J) The quantification shows the mean ± SEM surface levels in senescent cells presented relative to proliferating cells (dashed line); n = 2 independent experiments for LDLR n = 4 independent experiments for CD36. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibodies raised against Lamp1 (H4A3, DSHB, 1:2000), PI4K2A (sc-390026, Santa Cruz, 1:2000), β-Actin (8H10D10, 3700, Cell Signalling Technology, 1:5000), GAPDH (D4C6R, 97166, Cell Signalling Technology, 1:2000), integrin beta 1/CD29 (610467, BD Biosciences, 1:2000), transferrin receptor/CD71 (3B8 2A1, sc-32272, Santa Cruz, 1:1000), GFP (7.1/13.1, Roche, 11814460001, 1:2000), rabbit monoclonal antibodies raised against cathepsin B (D1C7Y, 31718, Cell Signalling Technology, 1:2000), CI-MPR (EPR6599, 124767, Abcam, 1:2000), Lamp1 (D2D11 9091, Cell Signalling Technology, 1:2000), phospho-S6 Ser235/236 (4856, Cell Signaling Technology, 1:2000), S6 (2217, Cell Signaling Technology, 1:1000), phospho-ULK1 Ser757 (D7O6U, 14202, Cell Signaling Technology, 1:1000), ULK1 (D8H5, 8054, Cell Signaling Technology, 1:1000), integrin alpha 5 (EPR7854, ab150361, Abcam, 1:2000), rabbit polyclonal antibodies raised against PI4K2A (15318-1-AP, Proteintech, 1:1000), LDLR (10785-1-AP, Proteintech, 1:2000), CD36 (18836-1-AP, Proteintech, 1:1000), VAPA (15275-1-AP, Proteintech, 1:2000).

Techniques: Western Blot, Activation Assay, Cell Culture, Expressing, Comparison, Membrane

Journal: bioRxiv

Article Title: Senescent cell survival relies on upregulation of lysosomal quality control mechanisms

doi: 10.1101/2025.03.31.646397

Figure Lengend Snippet: (A) IMR90 ER:RasV12 primary fibroblasts expressing GFP-OSBP were cultured in the presence of EtOH (Prol; proliferative) or 100 nM 4OHT for 6–8 days to induce RasV12 expression and oncogene-induced senescence (Sen; senescent). Cells were fixed and immunostained with an antibody against LAMP2 (magenta), scale bar, 10 μm. (B) The number of GFP-OSBP puncta (defined as 0.5-0.05 μm 3 ) per cell were plotted as mean ± SEM, n = 2 independent experiments with at least 14 cells per experimental repeat. (C) Proliferating and senescent cells were serum starved overnight, then treated with DMSO or OSW-1 for 4 hours and incubated with EBSS or stimulated with complete media (DMEM) for the last hour of treatment. Cell lysate were analysed for phosphorylation of S6 (Ser235/236) (D) Quantification represents mean ± SEM; n = 3 independent experiments; two-way ANOVA followed by Dunnett’s multiple comparison. (E) and (F) DRAQ7 intensity measured by flow cytometry reported the death of proliferating (E) and senescent (F) following 4 hours treatment with DMSO or 50 nM OSW-1. (G) Proliferating and senescent cell viability was measured (% DRAQ7 negative cells) following 4 hours treatment with increasing concentrations (0 to 800 nM) of OSW-1. (H) Cell viability after 4 hours treatment with 50 nM OSW-1 was analysed by two-way ANOVA followed by Šídák’s multiple comparison. Quantification represents mean ± SEM; n = 3 independent experiments. *, P < 0.05; ***, P < 0.001.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibodies raised against Lamp1 (H4A3, DSHB, 1:2000), PI4K2A (sc-390026, Santa Cruz, 1:2000), β-Actin (8H10D10, 3700, Cell Signalling Technology, 1:5000), GAPDH (D4C6R, 97166, Cell Signalling Technology, 1:2000), integrin beta 1/CD29 (610467, BD Biosciences, 1:2000), transferrin receptor/CD71 (3B8 2A1, sc-32272, Santa Cruz, 1:1000), GFP (7.1/13.1, Roche, 11814460001, 1:2000), rabbit monoclonal antibodies raised against cathepsin B (D1C7Y, 31718, Cell Signalling Technology, 1:2000), CI-MPR (EPR6599, 124767, Abcam, 1:2000), Lamp1 (D2D11 9091, Cell Signalling Technology, 1:2000), phospho-S6 Ser235/236 (4856, Cell Signaling Technology, 1:2000), S6 (2217, Cell Signaling Technology, 1:1000), phospho-ULK1 Ser757 (D7O6U, 14202, Cell Signaling Technology, 1:1000), ULK1 (D8H5, 8054, Cell Signaling Technology, 1:1000), integrin alpha 5 (EPR7854, ab150361, Abcam, 1:2000), rabbit polyclonal antibodies raised against PI4K2A (15318-1-AP, Proteintech, 1:1000), LDLR (10785-1-AP, Proteintech, 1:2000), CD36 (18836-1-AP, Proteintech, 1:1000), VAPA (15275-1-AP, Proteintech, 1:2000).

Techniques: Expressing, Cell Culture, Incubation, Comparison, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-18 up-regulates amino acid transporters and facilitates amino acid–induced mTORC1 activation in natural killer cells

doi: 10.1074/jbc.RA118.005892

Figure Lengend Snippet: IL-18 can induce mTORC1 activation by leucine treatment. Ex vivo expanded NK cells were stimulated with IL-2 or IL-2/IL-18 for 18 h, starved for 3 h in amino acid–depleted medium, then replenished with l-glutamine for 1 h before being treated with l-leucine for 30 min. A, phosphorylation of ribosomal protein S6 upon addition of leucine in NK cells was measured by flow cytometry. B, NK cells were treated with leucine, and the effect of rapamycin on mTORC1 activation was measured. C, phosphorylation of p70 S6 kinase upon addition of leucine was measured by Western blotting. D, IL-2/18–stimulated NK cells were treated with leucine for the indicated time before measuring pS6. E, starved NK cells were preloaded with unlabeled l-glutamine for 1 h and treated with 3H-labeled l-leucine for the indicated time up to 30 min. The intracellular levels of 3H-labeled l-leucine are shown. F, starved NK cells were preloaded with unlabeled glutamine and 3H-labeled glutamine for 1 h and treated with unlabeled leucine for the indicated time up to 30 min. The levels of 3H-labeled glutamine in the culture medium are shown. G, NK cells were treated with leucine without preloading with glutamine. H–K, NK cells stimulated with IL-2 or IL-2/IL-18 were treated with leucine, and the effects of BCH (H), MeAIB (I), GPNA (J), and NALA (K) on mTORC1 activation were measured. Data are from one experiment representative of three to four independent experiments, with two to three replicates per group. Data represent mean ± S.D. (error bars). aa, amino acid; NS, nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The intracellular staining of anti-phospho-S6 (Ser-235/Ser-236) was performed using BD Cytofix/Cytoperm protocols (BD Biosciences).

Techniques: Activation Assay, Ex Vivo, Flow Cytometry, Western Blot, Labeling