Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Characterization of HSPB1 expression in the AMI infarction model (A) Myocardial infarction was induced in mice by ligating the left anterior descending artery (LAD), and TTC staining was performed 28 days post-surgery to visualize the infarcted area. Red tissue represents non-infarcted myocardium, while pale tissue indicates infarcted myocardium ( n = 9). (B) Quantification of the infarcted area was performed using ImageJ software. (C and D) Western blot analysis of HSPB1 and β-actin expression in cardiac tissue, with statistical data showing the grayscale ratio of HSPB1 to β-actin. (E) Immunohistochemical staining for HSPB1 expression in myocardial tissue (scale bars, 100 μm). (F) Quantification of HSPB1 expression area using ImageJ software. Data are presented as mean ± SD ( n = 6). p values shown in the graphs were calculated using unpaired two-tailed Student’s t test.

Article Snippet: Lentiviral vectors for HSPB1 modulation , GeneChem (Shanghai, China) , N/A.

Techniques: Expressing, Staining, Software, Western Blot, Immunohistochemical staining, Two Tailed Test

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Targeted silencing of HSPB1 in the heart alleviates myocardial fibrosis in MI mice (A) Tail vein injection of AAV9-HSPB1-RNAi or CON534 virus (with cTnT promoter) was performed at a concentration of 1.0×10 12 virus particles/ml and a volume of 150 μL for 4 weeks, followed by Western blot analysis to validate HSPB1 silencing. (B) Grayscale ratio of HSPB1 to β-actin. (C–H) Following AAV9-HSPB1-RNAi or CON534 virus injection and MI model construction, cardiac function and fibrosis were assessed 4 weeks post-surgery: (C) cardiac function parameters by small animal echocardiography, (D) left ventricular fractional shortening (LVFS%), (E) left ventricular ejection fraction (LVEF%), (F) left ventricular mass (LV mass), (G) H\&E and Masson staining for myocardial injury and fibrosis, and (H) collagen fiber area percentage in myocardial tissue (scale bars, 200 μm). Data are presented as mean ± SD ( n = 6). Exact p values are indicated in the graphs. Statistical analyses were performed using unpaired two-tailed Student’s t test.

Article Snippet: Lentiviral vectors for HSPB1 modulation , GeneChem (Shanghai, China) , N/A.

Techniques: Injection, Virus, Concentration Assay, Western Blot, Staining, Two Tailed Test

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: HSPB1 gene silencing exacerbates EndoMT in the heart of MI Mice (A) Immunohistochemical staining shows α-SMA expression in myocardial tissue (heart cross-sections). (B) Quantification of the α-SMA-positive area using ImageJ software. (C) Western blot analysis of CD31, α-SMA, and β-actin protein expression in different groups. (D) Statistical analysis shows the ratio of CD31 to β-actin. (E) Statistical analysis shows the ratio of α-SMA to β-actin. Data are presented as mean ± SD ( n = 6). p values shown in the graphs were calculated using unpaired two-tailed Student’s t test.

Article Snippet: Lentiviral vectors for HSPB1 modulation , GeneChem (Shanghai, China) , N/A.

Techniques: Immunohistochemical staining, Staining, Expressing, Software, Western Blot, Two Tailed Test

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Regulatory role of HSPB1 in endothelial cell EndoMT (A) Western blot shows HSPB1 expression in HUVECs following lentiviral-mediated overexpression (LV-HSPB1) or knockdown (LV-HSPB1-RNAi); β-actin served as a loading control. (B) Quantification of HSPB1/β-actin ratio shows significant differences between groups. (C) Representative images of Transwell migration assays evaluating the effect of HSPB1 on TGF-β1–induced endothelial migration (scale bars, 100 μm). (D) Quantification of migrated cells per field. (E) Representative tube formation images showing the effect of HSPB1 modulation on TGF-β1–induced angiogenic activity (scale bars, 200 μm). (F–H) Quantitative analysis of tube formation parameters, including the number of branches (F), loops (G), and total tube length (H), measured using ImageJ software. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Lentiviral vectors for HSPB1 modulation , GeneChem (Shanghai, China) , N/A.

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Control, Migration, Activity Assay, Software

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Effects of HSPB1 on signaling pathways and TGF-β secretion in HUVECs under hypoxic conditions (A and B) HUVECs were transfected with adenoviral vectors for HSPB1 overexpression (OE) or knockdown (KD) and cultured for 48 h before RNA extraction. Gene expression analysis was performed using RNA sequencing. Gene set enrichment analysis (GSEA) assessed the regulatory roles of HSPB1 in processes such as heart development, angiogenesis, and cell proliferation (A). Further analysis using Hallmark gene sets explored HSPB1 signaling pathway activation (B). (C–G) Following transfection, HUVECs were cultured for 24 h and subjected to hypoxic conditions (3% O 2 ) for 48 h. Western blot analysis of the indicated proteins was performed. (D) pSmad2/3/Smad2/3 ratio, (E) quantification of CD31 protein expression, (F) quantification of E-cadherin expression, (G) quantification of α-SMA expression, and (H) quantification of N-cadherin expression were measured relative to β-actin. (I) TGF-β levels were measured by ELISA in cell supernatants. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Lentiviral vectors for HSPB1 modulation , GeneChem (Shanghai, China) , N/A.

Techniques: Protein-Protein interactions, Transfection, Over Expression, Knockdown, Cell Culture, RNA Extraction, Gene Expression, RNA Sequencing, Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: HSPB1 regulation of Pro-TGF-β1 disulfide bond formation (A) HUVEC cells were transfected with adenoviral vectors for HSPB1 overexpression or silencing and cultured for 48 h, followed by an additional 48-h incubation under hypoxic conditions (3% O 2 ). After enzymatic digestion, protein samples were analyzed for peptide-level disulfide bond formation using high-resolution mass spectrometry. (B and C) HUVEC cells were transfected with adenoviral vectors for HSPB1 overexpression or silencing, cultured for 24 h, and the HSPB1-silenced group was subsequently transfected with the HSPB1C137S mutant. After 48 h, cells were subjected to 48-h hypoxic induction (3% O 2 ). The redox status of pro-TGF-β1 was analyzed by non-reducing SDS-PAGE (B), and the stability of pro-TGF-β1 disulfide bonds was assessed by electrochemical potential (Eh), calculated using the Nernst equation (C). Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Lentiviral vectors for HSPB1 modulation , GeneChem (Shanghai, China) , N/A.

Techniques: Transfection, Over Expression, Cell Culture, Incubation, Mass Spectrometry, Mutagenesis, SDS Page

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Proposed model of HSPB1-mediated redox regulation of TGF-β1 maturation during post-MI fibrosis. During myocardial fibrosis following myocardial infarction, the expression of HSPB1 is markedly upregulated in the peri-infarct region. Upon activation, HSPB1 exposes its reactive cysteine residue (Cys137), which may interact with critical cysteine sites within pre-pro-TGF-β1, thereby influencing its redox-dependent folding and disulfide bond formation. This interaction potentially interferes with the maturation and secretion of active TGF-β1 into the extracellular space. Reduced secretion of mature TGF-β1 limits Smad2/3 phosphorylation and endothelial-to-mesenchymal transition, ultimately alleviating myocardial fibrosis. The red dashed box highlights the hypothesized redox regulatory interaction between HSPB1 and pre-pro-TGF-β1, which requires further biochemical validation.

Article Snippet: Lentiviral vectors for HSPB1 modulation , GeneChem (Shanghai, China) , N/A.

Techniques: Expressing, Activation Assay, Residue, Phospho-proteomics, Biomarker Discovery

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Characterization of HSPB1 expression in the AMI infarction model (A) Myocardial infarction was induced in mice by ligating the left anterior descending artery (LAD), and TTC staining was performed 28 days post-surgery to visualize the infarcted area. Red tissue represents non-infarcted myocardium, while pale tissue indicates infarcted myocardium ( n = 9). (B) Quantification of the infarcted area was performed using ImageJ software. (C and D) Western blot analysis of HSPB1 and β-actin expression in cardiac tissue, with statistical data showing the grayscale ratio of HSPB1 to β-actin. (E) Immunohistochemical staining for HSPB1 expression in myocardial tissue (scale bars, 100 μm). (F) Quantification of HSPB1 expression area using ImageJ software. Data are presented as mean ± SD ( n = 6). p values shown in the graphs were calculated using unpaired two-tailed Student’s t test.

Article Snippet: HSPB1 , Santa Cruz Biotechnology , sc-13132.

Techniques: Expressing, Staining, Software, Western Blot, Immunohistochemical staining, Two Tailed Test

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Targeted silencing of HSPB1 in the heart alleviates myocardial fibrosis in MI mice (A) Tail vein injection of AAV9-HSPB1-RNAi or CON534 virus (with cTnT promoter) was performed at a concentration of 1.0×10 12 virus particles/ml and a volume of 150 μL for 4 weeks, followed by Western blot analysis to validate HSPB1 silencing. (B) Grayscale ratio of HSPB1 to β-actin. (C–H) Following AAV9-HSPB1-RNAi or CON534 virus injection and MI model construction, cardiac function and fibrosis were assessed 4 weeks post-surgery: (C) cardiac function parameters by small animal echocardiography, (D) left ventricular fractional shortening (LVFS%), (E) left ventricular ejection fraction (LVEF%), (F) left ventricular mass (LV mass), (G) H\&E and Masson staining for myocardial injury and fibrosis, and (H) collagen fiber area percentage in myocardial tissue (scale bars, 200 μm). Data are presented as mean ± SD ( n = 6). Exact p values are indicated in the graphs. Statistical analyses were performed using unpaired two-tailed Student’s t test.

Article Snippet: HSPB1 , Santa Cruz Biotechnology , sc-13132.

Techniques: Injection, Virus, Concentration Assay, Western Blot, Staining, Two Tailed Test

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: HSPB1 gene silencing exacerbates EndoMT in the heart of MI Mice (A) Immunohistochemical staining shows α-SMA expression in myocardial tissue (heart cross-sections). (B) Quantification of the α-SMA-positive area using ImageJ software. (C) Western blot analysis of CD31, α-SMA, and β-actin protein expression in different groups. (D) Statistical analysis shows the ratio of CD31 to β-actin. (E) Statistical analysis shows the ratio of α-SMA to β-actin. Data are presented as mean ± SD ( n = 6). p values shown in the graphs were calculated using unpaired two-tailed Student’s t test.

Article Snippet: HSPB1 , Santa Cruz Biotechnology , sc-13132.

Techniques: Immunohistochemical staining, Staining, Expressing, Software, Western Blot, Two Tailed Test

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Regulatory role of HSPB1 in endothelial cell EndoMT (A) Western blot shows HSPB1 expression in HUVECs following lentiviral-mediated overexpression (LV-HSPB1) or knockdown (LV-HSPB1-RNAi); β-actin served as a loading control. (B) Quantification of HSPB1/β-actin ratio shows significant differences between groups. (C) Representative images of Transwell migration assays evaluating the effect of HSPB1 on TGF-β1–induced endothelial migration (scale bars, 100 μm). (D) Quantification of migrated cells per field. (E) Representative tube formation images showing the effect of HSPB1 modulation on TGF-β1–induced angiogenic activity (scale bars, 200 μm). (F–H) Quantitative analysis of tube formation parameters, including the number of branches (F), loops (G), and total tube length (H), measured using ImageJ software. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: HSPB1 , Santa Cruz Biotechnology , sc-13132.

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Control, Migration, Activity Assay, Software

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Effects of HSPB1 on signaling pathways and TGF-β secretion in HUVECs under hypoxic conditions (A and B) HUVECs were transfected with adenoviral vectors for HSPB1 overexpression (OE) or knockdown (KD) and cultured for 48 h before RNA extraction. Gene expression analysis was performed using RNA sequencing. Gene set enrichment analysis (GSEA) assessed the regulatory roles of HSPB1 in processes such as heart development, angiogenesis, and cell proliferation (A). Further analysis using Hallmark gene sets explored HSPB1 signaling pathway activation (B). (C–G) Following transfection, HUVECs were cultured for 24 h and subjected to hypoxic conditions (3% O 2 ) for 48 h. Western blot analysis of the indicated proteins was performed. (D) pSmad2/3/Smad2/3 ratio, (E) quantification of CD31 protein expression, (F) quantification of E-cadherin expression, (G) quantification of α-SMA expression, and (H) quantification of N-cadherin expression were measured relative to β-actin. (I) TGF-β levels were measured by ELISA in cell supernatants. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: HSPB1 , Santa Cruz Biotechnology , sc-13132.

Techniques: Protein-Protein interactions, Transfection, Over Expression, Knockdown, Cell Culture, RNA Extraction, Gene Expression, RNA Sequencing, Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: HSPB1 regulation of Pro-TGF-β1 disulfide bond formation (A) HUVEC cells were transfected with adenoviral vectors for HSPB1 overexpression or silencing and cultured for 48 h, followed by an additional 48-h incubation under hypoxic conditions (3% O 2 ). After enzymatic digestion, protein samples were analyzed for peptide-level disulfide bond formation using high-resolution mass spectrometry. (B and C) HUVEC cells were transfected with adenoviral vectors for HSPB1 overexpression or silencing, cultured for 24 h, and the HSPB1-silenced group was subsequently transfected with the HSPB1C137S mutant. After 48 h, cells were subjected to 48-h hypoxic induction (3% O 2 ). The redox status of pro-TGF-β1 was analyzed by non-reducing SDS-PAGE (B), and the stability of pro-TGF-β1 disulfide bonds was assessed by electrochemical potential (Eh), calculated using the Nernst equation (C). Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: HSPB1 , Santa Cruz Biotechnology , sc-13132.

Techniques: Transfection, Over Expression, Cell Culture, Incubation, Mass Spectrometry, Mutagenesis, SDS Page

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Proposed model of HSPB1-mediated redox regulation of TGF-β1 maturation during post-MI fibrosis. During myocardial fibrosis following myocardial infarction, the expression of HSPB1 is markedly upregulated in the peri-infarct region. Upon activation, HSPB1 exposes its reactive cysteine residue (Cys137), which may interact with critical cysteine sites within pre-pro-TGF-β1, thereby influencing its redox-dependent folding and disulfide bond formation. This interaction potentially interferes with the maturation and secretion of active TGF-β1 into the extracellular space. Reduced secretion of mature TGF-β1 limits Smad2/3 phosphorylation and endothelial-to-mesenchymal transition, ultimately alleviating myocardial fibrosis. The red dashed box highlights the hypothesized redox regulatory interaction between HSPB1 and pre-pro-TGF-β1, which requires further biochemical validation.

Article Snippet: HSPB1 , Santa Cruz Biotechnology , sc-13132.

Techniques: Expressing, Activation Assay, Residue, Phospho-proteomics, Biomarker Discovery

Journal: Cancer Genomics & Proteomics

Article Title: Timescale-dependent Phosphoproteomic Remodeling and Motility-associated Adaptation under Chronic Cabozantinib Exposure in Renal Cell Carcinoma

doi: 10.21873/cgp.20576

Figure Lengend Snippet: Migration, invasion, and signaling adaptations in 786-O cells under chronic cabozantinib exposure. (A) Transwell migration assay of Par and Chr 786-O cells. Migrated cells were fixed, crystal-violet stained, and quantified using ImageJ. (B) Matrigel invasion assay performed using the same cell model and quantification workflow as in (A). Invasion was assessed following 6 h incubation through Matrigel-coated inserts. (C) Immunoblot analysis of selected signaling nodes in Par and Chr 786-O cells, including MET, phospho-MET (Y1234/1235), ERK1/2, phospho-ERK, c-Jun, phospho-c-Jun (S63), HSPB1, and phospho-HSPB1 (S82). β-actin served as loading control. (D) Network representation of Chr-associated phosphosites mapped to adhesion- and stress-associated signaling modules based on the annotation-enrichment analysis. Data are shown as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined using an unpaired two-tailed Student’s t-test; #p<0.05; *p<0.01.

Article Snippet: The following primary antibodies were used: β-actin (GTX109639; GeneTex, Hsinchu, Taiwan, ROC), HSPB1 (ab5579; Abcam, Cambridge, UK), ERK1 (sc-93; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-ERK (sc-7383; Santa Cruz), c-Jun (sc-1694; Santa Cruz), MET (#4560; Cell Signaling Technology, Danvers, MA, USA), phospho-MET (#3077; Cell Signaling Technology), phospho-c-Jun S63 (#9810; Cell Signaling Technology), and phospho-HSPB1 S82 (#2401; Cell Signaling Technology).

Techniques: Migration, Transwell Migration Assay, Staining, Invasion Assay, Incubation, Western Blot, Control, Standard Deviation, Two Tailed Test

Journal: NAR Cancer

Article Title: Human DEAH-box helicase 8 regulates HSF1-mediated stress response and cancer-associated pre-mRNA splicing in tumour cells

doi: 10.1093/narcan/zcag008

Figure Lengend Snippet: An siRNA screen in U2OS human osteosarcoma cells identifies regulators of HSF1-dependent HSPA1A mRNA induction. ( A ) Schematic overview of the primary siRNA screening workflow. ( B ) Control condition results from the screen, showing HSPA1A mRNA induction following transfection with control siRNAs and treatment with either DMSO vehicle control or 250 nM of HSP90 inhibitor 17-AAG to activate HSF1. Each data point represents HSPA1A mRNA induction for individual siRNA treatments ( n = 2 independent repeats). AllStars Negative Control and HSF1 siRNA were included as negative and positive controls, respectively. The dotted line shows the 60% inhibition cut-off used to define candidate hits for followup. ( C ) Genes silenced in the screen ranked based on their mean percentage induction of HSPA1A mRNA. Red-shaded highlights gene silencing resulting in HSPA1A mRNA altered by >2-fold ( n = 2 independent repeats). The inset plot shows data for the top 10 ranked hits (error bars = data range for n = 2 independent repeats). ( D ) Induction of HSPA1A mRNA following 250 nM 17-AAG treatment after silencing of candidate hits from the siRNA screen. Each point represents the mean percentage induction of HSPA1A from a single siRNA ( n = 3 independent replicates), horizontal bars indicate the overall mean values for the four different siRNA for each target. The grey-shaded region denotes hits that reduced HSPA1A mRNA induction by >60%. Red data points denote hits where ≥2 out of 4 siRNA reduced induction by >60%. ( E ) Effect of silencing the nine validated hits using four individual siRNAs each measured by RT-qPCR ( HSPA1A mRNA) or immunofluorescence (HSP72 protein) following treatment with 250 nM 17-AAG (mean, n = 4). The pink-shaded area indicates siRNAs that reduced HSPA1A mRNA levels by >60%. Induction of ( F ) HSPA1A mRNA or ( G ) HSPB1 mRNA following 250 nM 17-AAG treatment after 72-h knockdown of HSF1 or DHX8 using eight distinct siRNAs (mean ± SEM, n ≥ 3). Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test (ns = not significant, * P < .05, ** P < .01, and *** P < .005). ( B–G ) Induction was calculated as a percentage of mean mRNA or protein relative to treatment with the 250 nM 17-AAG and transfection with AllStars negative control siRNA condition (indicated by the black line). ( H ) Fold decrease in basal HSPA1A mRNA levels following 72-h silencing of DHX8 with two coding sequence (CDS) targeting siRNA (DHX8-O1 and -O3) and HSF1 relative to negative control siRNA (mean ± SEM, n ≥ 3). Only significant differences are indicated on the plots.

Article Snippet: TaqMan assay primers were obtained from Applied Biosystems: HSPA1A (Hs00359163_s1), HSPB1 (Hs03044127_g1), HSF1 (Hs00232134_m1), un-spliced HSF1 (Hs03673241_cn), spliced NOXA (Hs00560402_m1), un-spliced NOXA (Hs00906409_cn), spliced MYC (Hs0153408_m1), CDC37 (Hs01003386_g1), and control RPLP0 (4326314E).

Techniques: Control, Transfection, Negative Control, Inhibition, Quantitative RT-PCR, Immunofluorescence, Knockdown, Sequencing

Journal: NAR Cancer

Article Title: Human DEAH-box helicase 8 regulates HSF1-mediated stress response and cancer-associated pre-mRNA splicing in tumour cells

doi: 10.1093/narcan/zcag008

Figure Lengend Snippet: DHX8 knockdown inhibits heat shock gene induction and alters HSF1 splicing in U2OS human osteosarcoma cells. Quantification of heat shock gene induction following HSP90 inhibition with 250 nM 17-AAG treatment. Bar graphs show the percentage induction of ( A ) HSPA1A mRNA or ( B ) HSPB1 mRNA. Percentage induction was calculated as a percentage of mean mRNA relative to the treatment with 250 nM 17-AAG and transfection with AllStars negative control siRNA condition. ( C ) Fold change in basal HSF1 mRNA levels following silencing of HSF1 or DHX8 (mean ± SEM, n ≥ 3) relative to negative control siRNA. ( D ) Immunoblot analysis of heat shock protein expression over time following siRNA-mediated knockdown of DHX8 or HSF1, following 250 nM 17-AAG treatment. GAPDH served as a loading control. AS = AllStars negative control siRNA, O1 = DHX8 siRNA 1, O3 = DHX8 siRNA 3, HSF = HSF1 siRNA. Immunoblots shown are exemplars of two independent repeats. Effects of HSF1 or DHX8 knockdown on transcript levels on non-heat shock-regulated control mRNAs: ( E ) NOXA , ( F ) MYC , and ( G ) CDC37 were quantified using RT-qPCR TaqMan (mean ± SD, n = 3). Impact of HSF1 or DHX8 knockdown for 72 h on RNA processing was assessed by RT-qPCR using TaqMan probes targeting intron 1 regions of ( H ) HSF1 or ( I ) NOXA (mean ± SEM, n ≥ 3, ** P < .01 determined using a two-tailed t -test comparing pre-mRNA levels between AllStars negative control and DHX8 siRNA conditions. ( J ) Data from RIP microarray analysis showing association of HSF1 or NOXA RNA with DHX8 (mean ± SEM, n = 5 *** P < .001 determined using a two-tailed t -test comparing pull-down with DHX8 antibody compared to the IgG control). Only significant differences are indicated on the plots.

Article Snippet: TaqMan assay primers were obtained from Applied Biosystems: HSPA1A (Hs00359163_s1), HSPB1 (Hs03044127_g1), HSF1 (Hs00232134_m1), un-spliced HSF1 (Hs03673241_cn), spliced NOXA (Hs00560402_m1), un-spliced NOXA (Hs00906409_cn), spliced MYC (Hs0153408_m1), CDC37 (Hs01003386_g1), and control RPLP0 (4326314E).

Techniques: Knockdown, Inhibition, Transfection, Negative Control, Western Blot, Expressing, Control, Quantitative RT-PCR, Two Tailed Test, Microarray

Journal: NAR Cancer

Article Title: Human DEAH-box helicase 8 regulates HSF1-mediated stress response and cancer-associated pre-mRNA splicing in tumour cells

doi: 10.1093/narcan/zcag008

Figure Lengend Snippet: DHX8 knockdown impairs HSF1-dependent heat shock protein induction and proliferation and survival of cancer cell lines. ( A ) Cell cycle distribution of U2OS human osteosarcoma cells 72 h post-transfection with DHX8 or AllStars control siRNA. Bar graph quantifies the proportion of cells in G1, S, and G2/M phases, revealing G2/M phase accumulation following DHX8 knockdown (mean ± SD, n = 3). ( B ) Immunoblot showing increased levels of cleaved PARP and caspase-3 following DHX8 knockdown, indicative of apoptosis. ( C ) Immunoblot analysis of HSF. 1, HSP72, and HSP27 protein levels before and after treatment with 250 nM HSP90-inhibitor 17-AAG to activate HSF1, following DHX8 knockdown. Tumorigenic: U2OS (osteosarcoma), MDA-MB-231 (breast), and HCT116 (colorectal). Non-tumorigenic: MCF10A (human mammary epithelial) and CCD-18Co (human colon fibroblast). The immunoblots shown in panels (B) and (C) are representative of two independent repeats, with GAPDH as a loading control. ( D ) Concentration-responsiveness of HSPA1A mRNA quantified by TaqMan qPCR following 6-h treatment with 17-AAG (mean ± SEM, n = 3; dotted line indicates 250 nM 17-AAG used in HSF1-induction experiments). ( E ) Induction of HSPA1A mRNA by 250 nM 17-AAG (6-h treatment) across tumorigenic and non-tumorigenic cell lines. Induction was calculated as a percentage of mean mRNA relative to treatment with the 250 nM 17-AAG and transfection with the AllStars negative control siRNA (indicated by the dotted black line). ( F ) Cell viability measured by CellTiter-Glo assay following siRNA-mediated knockdown of DHX8 or ( G ) HSF1 for 3 or 7 days. Apoptosis measured by Caspase 3/7-Glo assay following knockdown of ( H ) DHX8 or ( I ) HSF1 for 3 or 7 days. (F–I) Data are plotted relative to AllStars negative control siRNA treatment condition (indicated by the dotted black line). Bar graphs display mean ± SEM ( n ≥ 3 independent repeats). Statistical significance was determined using a two-tailed t -test comparing paired tumorigenic and non-tumorigenic cell lines (ns = not significant; * P < .05; ** P < .01; *** P < .001). Only significant differences are indicated on the plots.

Article Snippet: TaqMan assay primers were obtained from Applied Biosystems: HSPA1A (Hs00359163_s1), HSPB1 (Hs03044127_g1), HSF1 (Hs00232134_m1), un-spliced HSF1 (Hs03673241_cn), spliced NOXA (Hs00560402_m1), un-spliced NOXA (Hs00906409_cn), spliced MYC (Hs0153408_m1), CDC37 (Hs01003386_g1), and control RPLP0 (4326314E).

Techniques: Knockdown, Transfection, Control, Western Blot, Concentration Assay, Negative Control, Glo Assay, Two Tailed Test