Journal: BioMed Research International

Article Title: Chloroquine Protects Human Corneal Epithelial Cells from Desiccation Stress Induced Inflammation without Altering the Autophagy Flux

doi: 10.1155/2018/7627329

Figure Lengend Snippet: Phosphorylation levels of MAP kinase, AKT/p70S6kinase and AMPK proteins in HCE-T cells under desiccation stress . Immunoblot shows phosphorylated ERK1/2, p38, AKT and P70S6kinase, and AMPK . Densitometric analysis of the blots showed the ratios of phosphorylated AKT, p70s6kinase, ERK1/2 and p38 to AKT, p70s6kinase, ERK1/2, and p38. Data are the mean ± SD values, n = 3, statistical significance denoted ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ns- nonsignificant as compared to desiccated cells). Note that --- is control, +-- HCE-T cells exposed to desiccation (Des), -+- HCE-T cells treated with chloroquine (CQ), ++- desiccated HCE-T cells treated with chloroquine (CQ), --+ HCE-T cells treated with cyclosporine (CsA), and -++ desiccated HCE-T cells treated with cyclosporine (CsA).

Article Snippet: All primary antibodies were incubated overnight in dark at 4 degree: p65 [1:1000, Cell Signaling-C20], P-p65 (Ser536) [1:1000, Cell Signaling-(93H1)], I κ B α [1:1000, Cell Signaling (L35A5)], LAMP1 [1:1000, Cell Signaling (D2D11) XP], LC3A/B [1:1000, Cell Signaling, (#4108)], SQSTM1/p62 [1:1000,Cell Signaling (#5114)], p38 [1:1000, Cell signalling (#9202)], P-p38 (Thr180/Thy182) [1:1000, Cell Signaling (3D7)], p70S6Kinase [1:1000, Cell Signaling (9202)], P-p70S6Kinase (Thy389) [1:1000, Cell Signaling (9205)], ERK1/2 [1:1000, Cell signalling (137F5)], P-ERK1/2 (Thr180/Thy204) [1:1000, Cell signalling (D13.14.4E)], Akt [1:1000, Cell Signaling (4691)], P-Akt (Ser473) [1:1000, Cell Signaling (9271)], Beclin-1 [1:1000, Cell signalling (D40C5)] β - actin [1:3000, Santa Cruz, C-4], and GAPDH [1:2000, Abgenex, Clone:ABM22C5].

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0097016

Figure Lengend Snippet: (A, C & E): The effect of IGF-1R suppression on AKT/PI3K signaling was examined in pancreatic cancer cells. PANC-1 and HPAC cells were treated with IGF-1R siRNA for 48 h. The cells were harvested and the expression of phospho-AKT, AKT, phospho-PI3K, PI3K, phospho-PTEN, phospho-mTOR, mTOR, phospho-p70s6kinase, p70s6kinase and the internal control β-actin was measured by Western blotting. (B, D & F): Densitometric analysis is also shown to the right of each representative image.

Article Snippet: The following primary antibodies were used in this study: pAKT(sc-101629), AKT(5298), Bcl-2(sc-783), pERK, (sc-101760), ERK (sc-94) and STAT3(H-190)(sc-7179) (Santa Cruz,CA,USA); IGF-1R(3027), Notch 2 (4530P), Snail (3879), E-cadherin (3195), N-cadherin (4061), Zeb (3396), Vimentin (5741), Slug (9585), Bax (2772), Caspase3 (9661), PARP (9542), pPI3K p85(4228), PI3K p85(4292), IR-β (3024), pIRS-1(2388), IRS-1(2382), pSTAT3 (ser727) (4113), COX-2 (4842), pPTEN (9549), pmTOR (2974), mTOR(4517), p-p70s6kinase (9206) and p70s6kinase (9202) (Cell Signaling Technology, (Boston, MA); Caspase8 (ab 25901) (Abcam, Cambridge, MA, USA); β-actin (Sigma Aldrich, (St.Louis, MO, USA).

Techniques: Expressing, Western Blot

Journal: GeroScience

Article Title: SMAD4 mutations and cross-talk between TGF-β/IFNγ signaling accelerate rates of DNA damage and cellular senescence, resulting in a segmental progeroid syndrome—the Myhre syndrome

doi: 10.1007/s11357-020-00318-6

Figure Lengend Snippet: TGF-β accelerates senescence in cultures of preadipocytes via regulation of the mTOR pathway. a Representative images of untreated or control cells or cells stimulated with TGF-β 10 ng/ml or 50 ng/ml for 7 days. b Quantification of mRNA of p21, p16, Ki67, and GLB1 in control cells or cells stimulated with TGF-β (50 ng/ml) for 7 days by real-time PCR. Data represent mean of three independent experiments. c Western blotting analysis of phosphorylated mTOR in cells treated with TGF-β (20 ng/ml) for indicated times. d Cells pre-treated with the mTOR inhibitor rapamycin (10 μm) 30 min prior to stimulation with TGF-β (50 ng/ml) for 7 days. Protein extracts were analyzed for phosphorylated mTOR, phosphorylated p70S6Kinase, and p21. β-actin was used as loading control. e Real-time PCR analysis of control cells or cells treated with rapamycin (10 μm) and TGF-β (50 ng/ml) or TGF-β (50 ng/ml) alone for 7 days. p16, Ki67, and CDK1 were normalized to GADPH, which was used as an internal control. Data represent mean of three independent experiments. f Immunofluorescence staining of γH2AX foci with nuclear DAPI staining in control or treated with TGF-β (50 ng/ml), or cells treated with rapamycin (10 μm) and TGF-β (50 ng/ml) for 7 days. Scale bar 10 μm. g Percentage of γH2AX foci in control or cells treated with TGF-β (50 ng/ml), or cells treated with combination of rapamycin (10 μm) and TGF-β (50 ng/ml) for 7 days. *denotes statistical significance (*p < 0.05), n.s. denotes not significant. Error bars represent SEM

Article Snippet: Western blotting analyses were performed using a commercial anti-SMAD antibody (1:1000 dilution, #MA5-15682, Thermo Fisher Scientific, Waltham, MA), anti-V5 antibody (1:10000 dilution, #R960-25, Thermo Fisher), anti-phospho-mTOR (Ser2448) (1:1000, #2971, Cell Signaling Technology, Danvers, MA), anti-phospho-p70S6kinase (Thr389) (1:1000, #9206, Cell Signaling), anti-p21 (1:1000, #2947, Cell Signaling), anti-β-actin (1:4000, #A1978; Sigma), and biotinylated anti-mouse IgG antibody (#BA-9200, Vector Laboratories, Burlingame, CA), as described previously ( 26 ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining