anti casp8  (Proteintech)

Journal: International Journal of Oncology

Article Title: Regulation and reversal of paclitaxel resistance via the STAT1-mediated apoptotic pathway in ovarian cancer

doi: 10.3892/ijo.2025.5832

Figure Lengend Snippet: Association of STAT1 with PTX resistance. (A) Analysis of the biological functions of STAT1. High STAT1 expression (expression level >80th percentile) based on pan-cancer transcriptomics data was associated with enriched pathways. Horizontal coordinates indicate the different types of cancer in The Cancer Genome Atlas and vertical coordinates indicate the names of cell biological functions. The significance of each enrichment result was determined using the default setting. (B) Gene Set Enrichment Analysis of 'HALLMARK_APOPTOSIS' between STAT1 high and low expression samples of ovarian cancer (expression level >80th and <20th percentile, respectively). (C) Effect of apoptosis inhibitor Z-VAD-FMK in STAT1-overexpressing PTX-resistant cells. SK3R-PTX, OV3R-PTX and A2780-PTX cells were stably infected with empty vector, STAT1α or STAT1β plasmids in the presence of PTX, followed by 20 μ M Z-VAD-FMK or solvent DMSO treatment for 48 h. Cell viability was detected using a Cell Counting Kit-8 assay. Data were normalized using reads from the empty vector group. Differences among multiple groups were analyzed by one-way ANOVA followed by Tukey's Honestly Significant Difference test. Data are presented as the mean ± SD (n=5). ** P<0.01; *** P<0.001 (oe-STAT1α/β vs. Vector). (D) Analysis of genes closely related to apoptotic pathways obtained after TFoTF prediction and screening of the apoptosis gene set ('HALLMARK_APOPTOSIS'). Information about these genes is shown in the table on the right. The scatterplot on the left shows the visualization of the TFoTF prediction outcomes for these genes. The horizontal coordinate indicates the R score based on TFoTF and the vertical coordinate indicates the PWM score ( k max1 ). The red circles denote genes in the extrinsic apoptosis pathway, and the blue circles denote genes in the intrinsic apoptosis pathway. (E) Correlation of CASP8 and FAS expression with cellular PTX resistance. The significance of each regression coefficient was determined using the Wald test with a t-distribution using the SciPy Python package. (F) Analysis and screening of STAT1-targeted PTX resistance-associated apoptotic genes. In the bubble plot, the horizontal coordinate indicates the Pearson correlation coefficient between the target gene of STAT1 and PTX resistance. The vertical coordinate indicates the statistical significance of the correlation. The red horizontal dashed line indicates the position of the regression P-value of 0.05. The size of each bubble indicates the R score based on TFoTF. The color depth of each bubble indicates the PWM score based on TFoTF ( k max1 ). CASP8, caspase-8; FAS, Fas cell surface death receptor; NES, normalized enrichment score; NOM_p_val, normalized P-value; ns, not significant; OD, optical density; oe, overexpression vector; PTX, paclitaxel; PWM, position weight matrix; r, R-value; TFoTF, Target Finder of Transcription Factor.

Article Snippet: Cells were incubated with either rabbit anti-FAS antibody (cat. no. 13098-1-AP; dilution, 1:400; Proteintech Group, Inc.) or mouse anti-CASP8 antibody (cat. no. 66093-1-Ig; dilution, 1:400; Proteintech Group, Inc.) at 4°C for 12 h, followed by incubation with rabbit anti-STAT1 antibody (cat. no. 42H3; dilution, 1:4,000; Cell Signaling Technology, Inc.) at 4°C for another 12 h. Subsequently, the cells were treated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (cat. no. 8760S; dilution, 1:1,000) or Alexa Fluor 647-conjugated goat anti-mouse IgG (cat. no. 4410S; dilution, 1:1,000) secondary antibody (Cell Signaling Technology, Inc.) for 2 h at room temperature in the dark.

Techniques: Expressing, Stable Transfection, Infection, Plasmid Preparation, Solvent, Cell Counting, Over Expression

Journal: International Journal of Oncology

Article Title: Regulation and reversal of paclitaxel resistance via the STAT1-mediated apoptotic pathway in ovarian cancer

doi: 10.3892/ijo.2025.5832

Figure Lengend Snippet: Effect of PTX on CASP8, FAS and STAT1 expression in time-course and dose-dependent experiments, and effect of STAT1 on apoptosis. mRNA expression levels of CASP8, FAS, t-STAT1, STAT1α and STAT1β were detected by reverse transcription-quantitative PCR in (A) OVACR-3 and (B) A2780 cells after treatment with 0.001, 0.01 and 0.1 μ M PTX for 6, 12, 18 and 24 h. Expression values for each group were normalized using β-actin as an internal reference. Differences among multiple groups were analyzed using one-way ANOVA followed by Tukey's Honestly Significant Difference test. Data are presented as the mean ± SD (n=3). (C) Results of linear trend test for changes in (A). The vertical coordinate indicates the concentration of PTX, and the horizontal coordinate indicates the genes. The color of the bubble represents the slope of the change trend, while the size of the bubble represents the statistical significance. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. (D) Results of linear trend test for changes in (B). The vertical coordinate indicates the concentration of PTX, and the horizontal coordinate indicates the genes. The color of the bubble represents the slope of the change trend, while the size represents the statistical significance. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Detection of apoptosis in (E) OV3R-PTX and (F) A2780-PTX cells using flow cytometry. OV3R-PTX and A2780-PTX cells were infected with either oe-STAT1α or oe-STAT1β in the presence or absence of 0.1, 1 and 10 μ M PTX. Quantification of apoptotic cells based on flow cytometry in STAT1α/β-overexpressing (G) OV3R-PTX and (H) A2780-PTX cells in the presence or absence of PTX. Data were evaluated and analyzed using ModFit software and are presented as the mean ± SD (n=3). * P<0.05; ** P<0.01; *** P<0.001 vs. 6 h in (A) and (B) and vs. oe-NC in (G) and (H). CASP8, caspase-8; FAS, Fas cell surface death receptor; NC, negative control; ns, not significant; oe, overexpression vector; PTX, paclitaxel; t-STAT1, total STAT1.

Article Snippet: Cells were incubated with either rabbit anti-FAS antibody (cat. no. 13098-1-AP; dilution, 1:400; Proteintech Group, Inc.) or mouse anti-CASP8 antibody (cat. no. 66093-1-Ig; dilution, 1:400; Proteintech Group, Inc.) at 4°C for 12 h, followed by incubation with rabbit anti-STAT1 antibody (cat. no. 42H3; dilution, 1:4,000; Cell Signaling Technology, Inc.) at 4°C for another 12 h. Subsequently, the cells were treated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (cat. no. 8760S; dilution, 1:1,000) or Alexa Fluor 647-conjugated goat anti-mouse IgG (cat. no. 4410S; dilution, 1:1,000) secondary antibody (Cell Signaling Technology, Inc.) for 2 h at room temperature in the dark.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Concentration Assay, Flow Cytometry, Infection, Software, Negative Control, Over Expression, Plasmid Preparation

Journal: International Journal of Oncology

Article Title: Regulation and reversal of paclitaxel resistance via the STAT1-mediated apoptotic pathway in ovarian cancer

doi: 10.3892/ijo.2025.5832

Figure Lengend Snippet: Effect of STAT1 on CASP8 and FAS expression in PTX-resistant cells. Detection of (A) CASP8 and (B) FAS mRNA expression in oe-STAT1α- and oe-STAT1β-lentivirus-infected SK3R-PTX, OV3R-PTX and A2780-PTX cells by reverse transcription-quantitative PCR. Expression values for each group were normalized using β-actin as an internal reference. Differences between the two groups were analyzed using Student's t-test. Data are presented as the mean ± SD (n=3). * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Detection of STAT1, (C) CASP8 and (D) FAS protein expression in oe-STAT1α- and oe-STAT1β-lentivirus-infected SK3R-PTX, OV3R-PTX and A2780-PTX cells by western blotting. The specific antibodies recognized a specific target. STAT1α, 91 kDa; STAT1β, 84 kDa; pre-CASP8, 57 kDa; cleaved-CASP8, 43 kDa; FAS, 40-50 kDa; β-actin, 42 kDa. The numbers under the bands indicate the densitometric value of the protein (total CASP3 and FAS) after normalization. Detection of STAT1, (E) CASP8 and (F) FAS protein expression in oe-STAT1α- and oe-STAT1β-lentivirus-infected SK3R-PTX, OV3R-PTX and A2780-PTX cells by immunofluorescence staining. The immunofluorescent signals were detected after the induction of STAT1α and STAT1β using doxycycline. DAPI was used to stain the nucleus. Red scale bar, 100 μ m. CASP8, caspase-8; FAS, Fas cell surface death receptor; ns, not significant; oe, overexpression vector; pre-CASP8, caspase-8 precursor; cleaved-CASP8, cleaved caspase-8; PTX, paclitaxel.

Article Snippet: Cells were incubated with either rabbit anti-FAS antibody (cat. no. 13098-1-AP; dilution, 1:400; Proteintech Group, Inc.) or mouse anti-CASP8 antibody (cat. no. 66093-1-Ig; dilution, 1:400; Proteintech Group, Inc.) at 4°C for 12 h, followed by incubation with rabbit anti-STAT1 antibody (cat. no. 42H3; dilution, 1:4,000; Cell Signaling Technology, Inc.) at 4°C for another 12 h. Subsequently, the cells were treated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (cat. no. 8760S; dilution, 1:1,000) or Alexa Fluor 647-conjugated goat anti-mouse IgG (cat. no. 4410S; dilution, 1:1,000) secondary antibody (Cell Signaling Technology, Inc.) for 2 h at room temperature in the dark.

Techniques: Expressing, Infection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Over Expression, Plasmid Preparation

Journal: International Journal of Oncology

Article Title: Regulation and reversal of paclitaxel resistance via the STAT1-mediated apoptotic pathway in ovarian cancer

doi: 10.3892/ijo.2025.5832

Figure Lengend Snippet: Detection of the interaction of STAT1 with CASP8 and FAS. (A) Prediction of STAT1 binding site sequence. The PWM of STAT1 was used to predict the binding site in the promoter region. The visualized sequence logo of the PWM is illustrated at the top, and the specific values of the PWM are shown in the table at the bottom. The x-axis represents the position in the sequence alignment. The y-axis represents the information content in bits. A higher value means that position is highly conserved. (B) Prediction of STAT1 binding sites in the CASP8 and FAS promoter regions using PWM analysis. Blue wireframes and arrows indicate high-scoring binding sites (four sites on the CASP8 promoter and two sites on the FAS promoter). (C) The correlation regression results of CASP8 and FAS with STAT1 in OC. The significance of each regression was determined using the Wald test with the t-distribution using the SciPy Python package. (D) Schematic diagram showing the full-length promoter (top) and three truncated fragments of CASP8 with colored squares indicating predicted binding sites and sequences. (E) Detection of STAT1 binding to CASP8 promoter DNA using dual-luciferase reporter gene assays. (F) Schematic diagram showing the full-length promoter (top) and one truncated fragment of FAS (bottom) with colored squares indicating predicted binding sites and sequences. (G) Detection of STAT1 directly binding to FAS promoter DNA using dual-luciferase reporter gene assays. 293T cells were co-transfected with a dual-luciferase reporter gene plasmid and oe-STAT1α, oe-STAT1β or empty vector. Differences among multiple groups were analyzed using one-way ANOVA followed by Tukey's Honestly Significant Difference test. Data are presented as the mean ± standard error of the mean (n=3). * P<0.05; ** P<0.01; *** P<0.001 vs. vector. (H) Schematic illustration of the regulatory mechanism of STAT1 on the reversal of PTX resistance in OC cells. In PTX-resistant cells, the expression levels of STAT1 and apoptotic factors FAS and CASP3 were low. Administration of PTX increased STAT1 expression and the overexpression of STAT1 upregulated FAS and CASP8 at the transcriptional level, thus promoting PTX-resistant cell apoptosis. CASP8, caspase-8; FAS, Fas cell surface death receptor; ns, not significant; oe, overexpression vector; OC, ovarian cancer; PTX, paclitaxel; PWM, position weight matrix; r, R-value; TSS, transcription start site.

Article Snippet: Cells were incubated with either rabbit anti-FAS antibody (cat. no. 13098-1-AP; dilution, 1:400; Proteintech Group, Inc.) or mouse anti-CASP8 antibody (cat. no. 66093-1-Ig; dilution, 1:400; Proteintech Group, Inc.) at 4°C for 12 h, followed by incubation with rabbit anti-STAT1 antibody (cat. no. 42H3; dilution, 1:4,000; Cell Signaling Technology, Inc.) at 4°C for another 12 h. Subsequently, the cells were treated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (cat. no. 8760S; dilution, 1:1,000) or Alexa Fluor 647-conjugated goat anti-mouse IgG (cat. no. 4410S; dilution, 1:1,000) secondary antibody (Cell Signaling Technology, Inc.) for 2 h at room temperature in the dark.

Techniques: Binding Assay, Sequencing, Luciferase, Transfection, Plasmid Preparation, Expressing, Over Expression