Journal: Human Mutation

Article Title: A Regulator SNP Affecting the Transcription Factor SP1 Binding Site in the PIK3R3 Gene Contributes to Noise‐Induced Hearing Loss Risk

doi: 10.1155/humu/7010704

Figure Lengend Snippet: SP 1 regulated autophagy, apoptosis, and cell viability in HEI‐OC1 cells through PIK 3 R 3. (a) Western blotting was conducted to evaluate the efficiency of SP 1 knockdown in HEI‐OC1 cells transfected with three specific siRNAs. (b) Quantitative analysis of the expression level of SP 1 protein. (c) Western blotting was performed to detect the expression levels of autophagy‐ and apoptosis‐related proteins in HEI‐OC1 cells following SP 1 knockdown and PIK 3 R 3 overexpression. (d) Quantitative analysis of the expression levels of autophagy‐ and apoptosis‐related proteins. (e) Cell viability of HEI‐OC1 was measured by CCK‐8 assay at 0‐, 24‐, 48‐, and 72‐h posttransfection after SP 1 knockdown and PIK 3 R 3 overexpression. PIK 3 R 3 OE represents PIK 3 R 3 overexpression. Data are presented as mean ± SD from three independent experiments, each performed in triplicate ( n = 3). ∗∗ p < 0.01, and ∗∗∗ p < 0.001, compared with the si‐NC group. # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the si − S P 1 + P I K 3 R 3 − OE group.

Article Snippet: HEI‐OC1 cell viability was assessed using a Cell Counting Kit‐8 (CCK‐8) (Vazyme, Nanjing, China).

Techniques: Western Blot, Knockdown, Transfection, Analysis, Expressing, Over Expression, CCK-8 Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Tumor Treating Fields Enhance Temozolomide‐Associated Growth Suppression in Glioblastoma Cells and Patient‐Derived Organoids Through Cell‐Cycle Blockade, Apoptosis, and SMAD ‐Associated Signaling

doi: 10.1002/cns.71195

Figure Lengend Snippet: Combined TMZ+TTF treatment produces greater growth suppression, clonogenic inhibition, and reduction in migratory capacity in glioblastoma cells. (A, B) ATP‐based luminescence viability assays showing relative ATP activity in U251 and SHG140 cells at 24, 48, and 72 h under different treatment conditions. (C, D) CCK‐8 viability assays of U251 and SHG140 cells at 72 h. (E) Representative colony formation images of cells subjected to different treatments; scale bar = 5 mm. (F) Quantitative analysis of colony numbers across groups. (G) Transwell migration assays showing migrated cells under different treatment conditions using crystal violet staining; scale bar = 500 μm. (H) Quantification of migrated cells. Vehicle control indicates complete culture medium containing 0.2% DMSO, matching the final DMSO concentration in TMZ‐containing groups. Pairwise comparisons among treatment groups were performed for the quantitative analyses, including TMZ versus TMZ+TTF and TTF versus TMZ+TTF comparisons in the Transwell migration assay. For ATP‐based and CCK‐8 assays, five replicate wells were averaged within each independent experiment. For Transwell analysis, three random fields per insert were averaged to generate one value per experiment. Data are presented as mean ± SD ( n = 3 independent experiments). Statistical analysis for ATP‐based time‐course viability assays was performed using two‐way ANOVA followed by Tukey's multiple comparisons test. Statistical analysis for CCK‐8, colony formation, and Transwell migration assays was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For CCK‐8 assays, cells were seeded in 96‐well plates at 8 × 10 3 –1 × 10 4 cells/well, treated as indicated, and incubated with 10 μL CCK‐8 reagent (Targetmol, USA; Cat. No. C0005) for 1–2 h at 37°C.

Techniques: Inhibition, Activity Assay, CCK-8 Assay, Colony Assay, Analysis, Migration, Staining, Control, Concentration Assay, Transwell Migration Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Tumor Treating Fields Enhance Temozolomide‐Associated Growth Suppression in Glioblastoma Cells and Patient‐Derived Organoids Through Cell‐Cycle Blockade, Apoptosis, and SMAD ‐Associated Signaling

doi: 10.1002/cns.71195

Figure Lengend Snippet: ALK5/TGFβRI inhibition partially attenuates TTF‐associated SMAD phosphorylation, growth suppression, and cleaved caspase‐3 induction. (A) Western blot analysis of phosphorylated SMAD2 (p‐SMAD2), phosphorylated SMAD3 (p‐SMAD3), and total SMAD2 in U251 and SHG140 cells treated with vehicle control, TTF, SB525334, or TTF+SB525334. (B) Quantification of p‐SMAD2, p‐SMAD3, and total SMAD2 protein expression levels. Densitometric values for p‐SMAD2, p‐SMAD3, and SMAD2 were normalized to GAPDH. (C, D) CCK‐8 assays measuring cell viability in U251 and SHG140 cells treated with vehicle control, TTF, SB525334, or TTF+SB525334 under the same 72 h assay conditions used in Figure . SB525334 was used at 10 μM as a selective ALK5/TGFβRI inhibitor. (E) Immunofluorescence staining of cleaved caspase‐3 (red) and nuclei (blue, DAPI) in U251 and SHG140 cells under the indicated treatment conditions; merged images are shown. (F) Quantification of cleaved caspase‐3 fluorescence intensity. For cleaved caspase‐3 quantification, three random 20× fields were averaged within each independent experiment. Data are presented as mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For CCK‐8 assays, cells were seeded in 96‐well plates at 8 × 10 3 –1 × 10 4 cells/well, treated as indicated, and incubated with 10 μL CCK‐8 reagent (Targetmol, USA; Cat. No. C0005) for 1–2 h at 37°C.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Analysis, Control, Expressing, CCK-8 Assay, Immunofluorescence, Staining, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: ARHGAP40 suppresses clear cell renal cell carcinoma progression and is associated with altered glutamine-related metabolism and redox balance

doi: 10.1080/15384047.2026.2746137

Figure Lengend Snippet: ARHGAP40 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT)-associated changes in ccRCC cells. (A) CCK-8 proliferation curves for 786-O and ACHN cells with ARHGAP40 overexpression and A498 cells transfected with siARHGAP40-1. (B) Representative Annexin V/PI flow-cytometry plots and quantification of Annexin V-positive cells. (C) Representative Transwell migration images and quantification. Scale bar, 50 μm. (D) Representative Transwell invasion images and quantification. Scale bar, 50 μm. (E) Representative western blots and densitometric analyzes of E-cadherin, N -cadherin, and vimentin. n = 3 independent biological replicates; data are presented as mean ± SEM. Time-course CCK-8 data were analyzed by two-way repeated-measures ANOVA with Geisser-Greenhouse correction, followed by Šídák-adjusted multiple comparisons between groups at each time point. For 786-O cells, significant effects were observed for ARHGAP40 status ( P < 0.0001), time ( P < 0.0001), and the ARHGAP40 × time interaction ( P = 0.00011). For ACHN cells, the corresponding P values were < 0.0001, < 0.0001, and 0.0043, respectively; for A498 cells, they were 0.0037, < 0.0001, and 0.0012, respectively. Other two-group comparisons were analyzed using two-tailed Student's t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8) assays (Cat. No. C0038, Beyotime Biotechnology, Shanghai, China).

Techniques: Migration, CCK-8 Assay, Over Expression, Transfection, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Cancer Biology & Therapy

Article Title: ARHGAP40 suppresses clear cell renal cell carcinoma progression and is associated with altered glutamine-related metabolism and redox balance

doi: 10.1080/15384047.2026.2746137

Figure Lengend Snippet: AKT activation with SC79 partially rescues ARHGAP40-associated growth and redox phenotypes. (A) Representative p -AKT, AKT, and β -actin western blots and p -AKT/AKT quantification. (B) CCK-8 proliferation curves. (C) Annexin V/PI flow cytometry and quantification. (D) ROS histograms and mean fluorescence intensity. (E) GSH/GSSG ratios. n = 3 independent biological replicates; data are presented as mean ± SEM. Single-time-point panels were analyzed by two-way factorial ANOVA (ARHGAP40 status × SC79 treatment) followed by Tukey-adjusted multiple comparisons. CCK-8 data were analyzed by a three-way mixed-design ANOVA with ARHGAP40 status and SC79 treatment as factorial variables and time as the repeated-measures factor; Geisser-Greenhouse correction was applied to effects involving time, followed by prespecified Šídák-adjusted multiple comparisons. In 786-O cells, the ARHGAP40 × SC79 interaction was significant (F(1, 8) = 15.69, P = 0.0042), as was the ARHGAP40 × SC79 × time interaction (F(1.89,15.15) = 4.17, P = 0.0378). In ACHN cells, the corresponding interactions were also significant (F(1, 8) = 15.07, P = 0.0047; and F(2.48,19.87) = 10.62, P = 0.00039, respectively). ** P < 0.01, *** P < 0.001.

Article Snippet: Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8) assays (Cat. No. C0038, Beyotime Biotechnology, Shanghai, China).

Techniques: Activation Assay, Western Blot, CCK-8 Assay, Flow Cytometry, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: ARHGAP40 suppresses clear cell renal cell carcinoma progression and is associated with altered glutamine-related metabolism and redox balance

doi: 10.1080/15384047.2026.2746137

Figure Lengend Snippet: Constitutively active RhoA partially restores AKT signaling and reverses ARHGAP40-associated growth and redox phenotypes. (A) Representative GTP-RhoA, total RhoA, p -AKT, AKT, and β -actin western blots. (B) p -AKT/AKT ratios. (C) GTP-RhoA/total RhoA ratios. (D) CCK-8 proliferation curves obtained after reseeding transfected cells. (E) Annexin V/PI flow cytometry and quantification. (F) ROS histograms and mean fluorescence intensity. (G) GSH/GSSG ratios. n = 3 independent biological replicates; data are presented as mean ± SEM. Single-time-point panels were analyzed by two-way factorial ANOVA (ARHGAP40 status × RhoA-Q63L expression), followed by Tukey-adjusted multiple comparisons. CCK-8 data were analyzed by a three-way mixed-design ANOVA with ARHGAP40 status and RhoA-Q63L expression as between-subject factors and time as the repeated-measures factor. Geisser-Greenhouse correction was applied to effects involving time, followed by prespecified Šídák-adjusted multiple comparisons. In 786-O cells, the ARHGAP40 × RhoA-Q63L interaction was significant (F(1, 8) = 27.22, P = 0.00081), whereas the ARHGAP40 × RhoA-Q63L × time interaction did not reach statistical significance after Geisser-Greenhouse correction (F(2.18,17.41) = 3.39, P = 0.0536). In ACHN cells, both the ARHGAP40 × RhoA-Q63L interaction (F(1, 8) = 26.35, P = 0.00089) and the ARHGAP40 × RhoA-Q63L × time interaction (F(1.01,8.09) = 72.82, P < 0.0001) were significant. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8) assays (Cat. No. C0038, Beyotime Biotechnology, Shanghai, China).

Techniques: Western Blot, CCK-8 Assay, Transfection, Flow Cytometry, Fluorescence, Expressing

Journal: Journal of Inflammation Research

Article Title: Time-Stable Ferroptosis-Related Candidate Genes in Myocardial Ischemia/Reperfusion Injury Identified by Multi-Time-Point Transcriptomic Analysis: Prioritization of NCF2 Through Public Datasets and AC16 Cell Validation

doi: 10.2147/JIR.S627947

Figure Lengend Snippet: Experimental assessment in AC16 H/R cells. ( A ) CCK-8 cell viability assay. ( B ) MDA levels. ( C and D ) FerroOrange staining and mean fluorescence intensity (MFI) quantification of intracellular Fe 2 ⁺. ( E and F ) BODIPY 581/591 C11 staining and oxidized green fluorescence MFI quantification of lipid ROS. ( G ) Representative Western blot bands for GPX4, NCF2 and β-actin. ( H and I ) Quantification of GPX4 and NCF2 protein levels. ( J – P ) RT-qPCR assessment of candidate-gene mRNA expression. Data are presented as mean ± SD, n = 3 independent biological replicates. Between-group differences were assessed using one-way ANOVA followed by LSD post hoc tests ( A – I ) or independent-samples t tests ( J – P ). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay (Beyotime, Shanghai, China).

Techniques: CCK-8 Assay, Cell Viability Assay, Staining, Fluorescence, Western Blot, Quantitative RT-PCR, Expressing