Journal: eLife

Article Title: Heat shock factor 1 (HSF1) cooperates with estrogen receptor α (ERα) in the regulation of estrogen action in breast cancer cells

doi: 10.7554/eLife.69843

Figure Lengend Snippet: ( A ) Overlap of genes stimulated or repressed after the E2 treatment (RNA-seq analyses) in HSF1+ and HSF1− cells (model created as described in ). The bottom panel compares the degree of response to E2 of overlapping genes. ( B ) Heatmap with hierarchical clustering of normalized read counts from RNA-seq (row z-score) for selected genes (identified as similarly responding in all MCF7 cell models; see ) stimulated or repressed after the E2 treatment. ( C ) The response to E2 stimulation presented as a mean fold change E2 versus Ctr (dots; scale on the top) as well as changes in the expression level between Ctr and E2 (arrows begin at the level of mean expression in untreated cells and end at the level of mean expression in treated cells; normalized RNA-seq read counts with a scale on the bottom). Genes are sorted according to the hierarchical clustering shown in the heatmap. Upregulation, fold change >1.0; downregulation, fold change <1.0. Statistically significant differences between untreated HSF1+ and HSF1− cells are marked: ***p<0.0001, **p<0.001, *p<0.05. ( D ) Nascent RNA gene expression analyses by RT-qPCR in wild-type (WT), HSF1+, and HSF1− cells. The upper panel shows E2-stimulated changes (E2 versus Ctr fold change; E2 treatment: 10 nM, 4 hr), bottom panel shows basal expression level represented as fold differences between untreated wild-type control (WT), HSF1+, and HSF1− cells. Corresponding total RNA analyses are shown in . ***p<0.0001, **p<0.001, *p<0.05 (significance of differences versus the corresponding control – above the bar, or between cell variants). ( E ) Analyses at the protein level (western blot) after 48 hr treatment with E2. HSPA8 was used as a protein loading control. The graph below shows the results of densitometric analyses (n = 3).

Article Snippet: Proteins (20–30 μg) were separated on 10% SDS-PAGE gels and blotted to a 0.45 μm pore nitrocellulose filter (GE Healthcare, Europe GmbH, Freiburg, Germany) using Trans Blot Turbo system (Bio-Rad, Hercules, CA) for 10 min. Primary antibodies against HSF1 (1:4000, ADI-SPA-901), HSP90 (1:2000, ADI-SPA-836), and HSP70 (1:2000, ADI-SPA-810), all from Enzo Life Sciences (Farmingdale, NY), HSP105 (1:600, #3390-100, BioVision, Milpitas, CA), ERα (1:2000, #8644), phosphoERα (S118) (1:2000, #2511), HSPB8 (1:1000, #3059), all from Cell Signaling Technology (Danvers, MA), PHLDA1 (1:1000, #sc-23866), EGR3 (1:1000, #sc-390967), HSPA8/HSC70 (1:5000, #sc-7298), all from Santa Cruz Biotechnology (Dallas, TX), and ACTB (1:25,000, #A3854, Merck KGaA) were used.

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: Heat shock factor 1 (HSF1) cooperates with estrogen receptor α (ERα) in the regulation of estrogen action in breast cancer cells

doi: 10.7554/eLife.69843

Figure Lengend Snippet:

Article Snippet: Proteins (20–30 μg) were separated on 10% SDS-PAGE gels and blotted to a 0.45 μm pore nitrocellulose filter (GE Healthcare, Europe GmbH, Freiburg, Germany) using Trans Blot Turbo system (Bio-Rad, Hercules, CA) for 10 min. Primary antibodies against HSF1 (1:4000, ADI-SPA-901), HSP90 (1:2000, ADI-SPA-836), and HSP70 (1:2000, ADI-SPA-810), all from Enzo Life Sciences (Farmingdale, NY), HSP105 (1:600, #3390-100, BioVision, Milpitas, CA), ERα (1:2000, #8644), phosphoERα (S118) (1:2000, #2511), HSPB8 (1:1000, #3059), all from Cell Signaling Technology (Danvers, MA), PHLDA1 (1:1000, #sc-23866), EGR3 (1:1000, #sc-390967), HSPA8/HSC70 (1:5000, #sc-7298), all from Santa Cruz Biotechnology (Dallas, TX), and ACTB (1:25,000, #A3854, Merck KGaA) were used.

Techniques: Transfection, Construct, Recombinant, Plasmid Preparation, shRNA, Expressing, Sequencing, In Situ, SYBR Green Assay, Software, Staining, CRISPR, Protease Inhibitor

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of 1α,25-dihydroxyvitamin D 3 and tacalcitol on cell signaling and anchorage-independent growth in T98G and U251 glioblastoma cells

doi: 10.1016/j.bbrep.2022.101313

Figure Lengend Snippet: ( A ) Western blot analysis of pY783-PLCγ, PLCγ, pT389-p70-S6 kinase, p70-S6 kinase, pY705-STAT3 and STAT3 after treating T98G cells with 1 μM of 1α,25-dihydroxyvitamin D 3 (1α,25), 1 μM of tacalcitol (Tac) or vehicle (EtOH) for 24 h. ( B ), ( C ) and ( D ) Quantitative analysis of the levels of pY783-PLCγ, pT389-p70-S6 kinase and pY705-STAT3 inT98G cell lysates in the treatment groups as compared to vehicle. β-Actin was used as loading control for pY783-PLCγ and pT389-p70-S6. pY705-STAT3 was normalized to total STAT3. The values are presented as mean ± standard deviation of at least four independent determinations and shown as fold difference compared to ethanol-treated cells. ( E ) Analysis of STAT3 mRNA in T98G cells treated with tacalcitol (1 μM, 24h) compared to vehicle-treated cells. The graph shows the means ± standard deviation of four independent experiments. ( F ) Determination of STAT3 activation in T98G cells using a STAT3 assay kit. STAT3 activation was measured in cells cultured for 24 h in the absence (EtOH) and presence of 1α,25-dihydroxyvitamin D 3 (1α,25) or tacalcitol (Tac) in the indicated concentrations. The values are presented as mean ± standard deviation of five independent determinations. * = p < 0.05.

Article Snippet: The following primary antibodies were used: rabbit anti-pY783-PLCγ1 (#2821, 1:1000, Cell Signaling Technology), mouse anti-PLCγ1 (H00005335-M01, 1:1000, Novus Biologicals), mouse anti-pThr389-p70-S6 Kinase (#7053, 1:1000, Cell Signaling Technology), rabbit anti-p70-S6 Kinase (#7053, 1:1000, Cell Signaling Technology), rabbit anti-pY705-STAT3 (#9145, 1:500, Cell Signaling Technology), mouse anti-STAT3 (#9139, 1:1000, Cell Signaling Technology), rabbit anti-pS473-AKT (#4060, 1:500, Cell Signaling Technologies), mouse anti-AKT (#2920, 1:500 Cell Signaling Technologies), rabbit anti-pT202/Y204-ERK1/2 (#9101, 1:1000, Cell Signaling Technology), rabbit anti-ERK1/2 (#4695, 1:1000, Cell Signaling Technology), mouse anti-β-Actin (sc-47778, 1:1000, Santa Cruz Biotechnology), mouse anti-pT180/182-p38 (#9216, 1:1000, Cell Signaling Technology), rabbit anti-p38 (#9212, 1:1000, Cell Signaling Technology) and rabbit anti-β-Actin (ab8227, 1:1000, Abcam).

Techniques: Western Blot, Control, Standard Deviation, Activation Assay, Cell Culture