Journal: Molecular cancer therapeutics

Article Title: GnRH-R targeted lytic peptide sensitizes BRCA wild-type ovarian cancer to PARP inhibition

doi: 10.1158/1535-7163.MCT-18-0770

Figure Lengend Snippet: (A) A network of downregulated/upregulated proteins in combination group compared to olaparib group was determined using Netwalker analysis. (B) Expression of PI3K/AKT, p-PI3K/p-AKT, PARP, Cleaved PARP, BRCA1, BRCA2, and RAD 51 after treatment. (C) Expression and localization of GnRH-R in BRCA1 mutant (BRCA1m) COV362 and MDA-MB-436 cells and BRCA2 mutant (BRCA2m) KURAMOCHI cells. The fixed cells without permeabilization (left panels) were visualized using confocal microscopy (TCS SP5 MP; Leica Microsystems, Buffalo Grove, IL). The fixed and permeabilized cells (right panels) were visualized using a laser-scanning microscope (Leica). (Scale bar: 50 μM). (D) cell viability of COV362, MDA-MB-436, and KURAMOCHI cells after treatment with EP-100, olaparib, or EP-100 plus olaparib for 72 h. (E) CI values are shown as in Fa-CI plots.

Article Snippet: The primary antibody dilution factors were as follows according to manufacturer’s instructions: anti-GnRH-R (1:1,000, Abcam, Cambridge, UK, Cat: ab183079), RAD 51 (1:10,000, Abcam, Cambridge, UK, Cat: ab133534), phosphorylated PI3K p85 (1:1,000, Cat: 4228s), PI3K (1:1,000, Cat: 4292s), phosphorylated AKT Ser473 (1:1,000, Cat: 9271s), AKT (1:1,000, Cat: 9272s), PARP (1:1,000, Cat: 9532s), BRCA1 (1:1,000, Cat: 9025s) and BRCA2 (1:1,000, Cat: 10741s) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Mutagenesis, Confocal Microscopy, Laser-Scanning Microscopy

Journal: Molecular Medicine Reports

Article Title: Effective elimination of chronic lymphocytic leukemia cells in the stromal microenvironment by a novel drug combination strategy using redox-mediated mechanisms

doi: 10.3892/mmr.2015.4364

Figure Lengend Snippet: Effect of SAHA on levels of GSH-associated enzyme and GSH in CLL cells co-cultured with stromal cells. (A) SAHA increased the expression of Nrf2 in CLL cells. CLL cells were treated with 2 µ M SAHA for 20 h, and cell lysates were assayed for Nrf2 using western blot analysis. Representative western blot results from three samples from patients with CLL are shown. The right panel shows the quantification of Nrf2 band density of eight CLL samples, with β-actin expression as an internal control (mean ± standard deviation; * P<0.05; Ctrl, control cells without treatment; S, SAHA treatment). (B) SAHA induced the translocation of Nrf2 between the cytosol and nucleus. SAHA (2 µ M) was added to the CLL cells for 20 h, and the cells were cytospun and immunostained with Nrf2 antibodies, and observed using a confocal laser scanning microscope. The nuclei were stained with 4,6-diamidino-2-phenylindole. (C) Upregulation of mRNA expression of GCLC following SAHA treatment. CLL cells were treated with 2 µ M SAHA for 22 h and the GCLC mRNA expression was examined using reverse transcription-quantitative polymerase chain reaction. (D) SAHA increases the expression of GCLC in CLL cells. CLL cells were treated with 2 µ M SAHA for 24 h, and the cell lysates were then assayed for the expression levels of GCLC by western blot analysis. The upper panel shows the representative western blot results from samples of four patients with CLL. The lower panel shows the quantification of GCLC band density of eight CLL samples, with β-actin as the internal control (mean ± standard deviation; ** P<0.01. (E) Treatment with SAHA enhanced stromal-mediated GSH upregulation in CLL cells. The CLL cells were treated with 2 µ M SAHA for 48 h in the presence or absence of HS5 cells. In another treatment group, CLL and HS5 cells in co-culture were incubated with cystine transporter inhibitor S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Values are presented as the mean ± standard deviation of three independent experiments using three CLL samples. (F) Sensitization of CLL cells to SAHA by inhibiting the cystine transporter with S-4-CPG. CLL and HS5 cells in co-culture were incubated with S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Cell viability was analyzed using an Annexin V/PI assay. Representative dot plots are shown. CLL, chronic lymphocytic leukemia; SAHA, suberoylanilide hydroxamic acid; GSH, glutathione; PI, propidium iodide; Nrf2, nuclear factor-E2-related factor 2; CPG, carboxyphenylglycine Ctrl, untreated control; S, SAHA treatment.

Article Snippet: The fixed CLL cells were incubated with rabbit anti-human polyclonal Nrf2 antibody (1:50; cat no. sc-13032; Santa Cruz Biotechnology, Inc.), at 4°C overnight, followed by incubation with Alexa-Fluor-594 goat anti-rabbit polyclonal antibody (1:400; cat. no. A11594; Molecular Probes at room temperature for 1 h. Finally, the slides were washed with phosphate-buffered saline, mounted and counterstained with mounting medium supplemented with DAPI prior to examination with a Nikon Eclipse TE2000 confocal microscope and analysis with Nikon EZ-C1 3.80 software (Nikon Corporation, Tokyo, Japan).

Techniques: Cell Culture, Expressing, Western Blot, Control, Standard Deviation, Translocation Assay, Laser-Scanning Microscopy, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Co-Culture Assay, Incubation

Journal: The FASEB Journal

Article Title: Shining a Light on Skeletal Muscle Regeneration: Red Photobiomodulation Boosts Myoblast Differentiation In Vitro

doi: 10.1096/fj.202502477R

Figure Lengend Snippet: Analyses of mitochondria and of peroxisome proliferator‐activated receptor‐gamma coactivator (PGC)‐1α expression. Myoblasts were cultured in proliferation medium (PM) or exposed or not (untreated) to PBM with 4 J/cm 2 energy density and then cultured in differentiation medium (DM) for 48 h. (A–C) Representative confocal laser scanning microscopy (CLSM)fluorescence images of myoblasts labeled with MitoTracker to mark mitochondria (red) and immunostained for PGC‐1α expression (green). Scale bar: 20 μm. (D, E) Densitometric analyses of the fluorescence intensity (F.I.) of mitochondria and PGC‐1α respectively, expressed in a.u. (arbitrary units). (F) WB analysis of PGC‐1α in the indicated conditions: representative blot and bar charts showing the densitometric analysis of the bands normalized to α‐tubulin. Molecular weight of full length of PGC‐1α, 115 kDa, molecular weight of N‐terminal truncated NT‐PGC‐1α: 37 kDa. a.u.: arbitrary units. Data are mean ± SD. ** p < 0.01, *** p < 0.001 vs. untreated PM; ° p < 0.05 vs. untreated DM 48 h (One‐way ANOVA with post hoc Tukey).

Article Snippet: After permeabilization with cold acetone for 3 min, fixed cells were blocked with 0.5% bovine serum albumin (BSA; Sigma Aldrich) and 3% glycerol in phosphate‐buffered saline (PBS) for 20 min and then incubated overnight at 4°C in a humidified chamber, with the following primary antibodies: rabbit polyclonal anti‐MyoD (M‐318) (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc‐760, RRID:AB_2148870), mouse monoclonal anti‐myogenin (F5D) (1:50; Santa Cruz, Cat# sc‐12732, RRID:AB_627980), mouse monoclonal anti‐peroxisome proliferator‐activated receptor‐gamma coactivator (PGC)‐1α (1:100; Santa Cruz, Cat# sc‐518025, RRID:AB_2890187).

Techniques: Expressing, Cell Culture, Confocal Laser Scanning Microscopy, Fluorescence, Labeling, Molecular Weight