Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: eQTL analyses were performed for rs10931936 combining genotype and expression level data derived from (A) 106 human primary melanocyte cultures (TT, n =7; TC, n =39; CC, n =60), (B) 349 melanoma tumors from TCGA-SKCM (TT, n =42; TC, n =126; CC, n =181), and (C) a panel of 59 early-passage melanoma cell lines (TT, n =8; TC, n =23; CC, n =25), where the risk-T allele is labeled in red. A significant eQTL effect with higher expression driven from the C-protective allele was observed for CASP8 in both melanocytes and melanoma tumors ( P = 1.2 x 10 -9 and P = 6.8 x 10 -3 , respectively), while the result was marginal but in the same direction in melanoma cell lines ( P = 0.07). Significance determined by linear regression; mean with SEM are plotted along with individual data values.

Article Snippet: Taqman real-time PCR assays targeting unique junctions of CASP8 transcript isoforms were obtained from Thermo Fisher Scientific (Waltham, MA; all isoform transcripts: Hs01018151_m1; exon 8 to 9 junction: Hs01018160_m1; exon 8L (alternative spliced) to 9 junction: Hs04405665_m1).

Techniques: Expressing, Derivative Assay, Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) LocusZoom plots present –log10 P -values for melanoma GWAS (upper) and melanocyte CASP8 eQTL (lower) for a 1 Mb region encompassing rs10931936. The melanoma risk lead SNP rs10931936 is labeled and highlighted in purple in both panels, and LD (r 2 based on 1000G EUR) of all other SNPs to the melanoma GWAS lead SNP is color-coded. (B) A LocusCompare plot compares P -values between melanoma GWAS ( x -axis) and melanocyte CASP8 eQTL ( y axis) for the same region. Genomic coordinates are based on hg19.

Article Snippet: Taqman real-time PCR assays targeting unique junctions of CASP8 transcript isoforms were obtained from Thermo Fisher Scientific (Waltham, MA; all isoform transcripts: Hs01018151_m1; exon 8 to 9 junction: Hs01018160_m1; exon 8L (alternative spliced) to 9 junction: Hs04405665_m1).

Techniques: Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) E4F1 or IRF2 were knocked down using respective pools of four different siRNAs in UACC903 melanoma cells, and E4F1 , IRF2 and CASP8 levels were measured. GAPDH - normalized E4F1 , IRF2 or CASP8 mRNA levels are shown as fold change over those from non-targeting siRNA. A representative experiment from three biological replicates is shown (individual datapoints, mean, and SEM are plotted). P -values are shown from one representative set. (B) Protein levels were examined using anti-E4F1, anti-IRF2, or anti-GAPDH antibody with UACC903 cell lysates of siRNAs transfected with either non-targeting control, E4F1 , or IRF2 siRNAs. GAPDH was used as a loading control. One representative set of three replicate experiments is shown. (C) Individual luciferase assays were performed by co-transfecting rs3769823 luciferase constructs with E4F1 , IRF2, or non-targeting control siRNAs into UACC903 melanoma cells. Renilla -normalized relative luciferase activities are shown relative to an empty construct containing only a minimal promoter (TATA). One representative set is shown from three biological replicates. Mean with SEM is plotted, n = 6 technical replicates. A two-tailed t-test assuming unequal variances was used to calculate all P values shown against control siRNA.

Article Snippet: Taqman real-time PCR assays targeting unique junctions of CASP8 transcript isoforms were obtained from Thermo Fisher Scientific (Waltham, MA; all isoform transcripts: Hs01018151_m1; exon 8 to 9 junction: Hs01018160_m1; exon 8L (alternative spliced) to 9 junction: Hs04405665_m1).

Techniques: Transfection, Control, Luciferase, Construct, Two Tailed Test

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

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: eQTL analyses were performed for rs10931936 combining genotype and expression level data derived from (A) 106 human primary melanocyte cultures (TT, n =7; TC, n =39; CC, n =60), (B) 349 melanoma tumors from TCGA-SKCM (TT, n =42; TC, n =126; CC, n =181), and (C) a panel of 59 early-passage melanoma cell lines (TT, n =8; TC, n =23; CC, n =25), where the risk-T allele is labeled in red. A significant eQTL effect with higher expression driven from the C-protective allele was observed for CASP8 in both melanocytes and melanoma tumors ( P = 1.2 x 10 -9 and P = 6.8 x 10 -3 , respectively), while the result was marginal but in the same direction in melanoma cell lines ( P = 0.07). Significance determined by linear regression; mean with SEM are plotted along with individual data values.

Article Snippet: Gene expression levels were quantified by qPCR using Taqman assays for E4F1 (Hs00231773_m1), IRF2 (Hs01082884_m1), CASP8 (Hs01018151_m1, Hs01018149, Hs01018160_m1, Hs04405665_m1 and Hs01022432_m1), and GAPDH (Hs99999905_m1) from Thermo Fisher Scientific.

Techniques: Expressing, Derivative Assay, Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) LocusZoom plots present –log10 P -values for melanoma GWAS (upper) and melanocyte CASP8 eQTL (lower) for a 1 Mb region encompassing rs10931936. The melanoma risk lead SNP rs10931936 is labeled and highlighted in purple in both panels, and LD (r 2 based on 1000G EUR) of all other SNPs to the melanoma GWAS lead SNP is color-coded. (B) A LocusCompare plot compares P -values between melanoma GWAS ( x -axis) and melanocyte CASP8 eQTL ( y axis) for the same region. Genomic coordinates are based on hg19.

Article Snippet: Gene expression levels were quantified by qPCR using Taqman assays for E4F1 (Hs00231773_m1), IRF2 (Hs01082884_m1), CASP8 (Hs01018151_m1, Hs01018149, Hs01018160_m1, Hs04405665_m1 and Hs01022432_m1), and GAPDH (Hs99999905_m1) from Thermo Fisher Scientific.

Techniques: Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) E4F1 or IRF2 were knocked down using respective pools of four different siRNAs in UACC903 melanoma cells, and E4F1 , IRF2 and CASP8 levels were measured. GAPDH - normalized E4F1 , IRF2 or CASP8 mRNA levels are shown as fold change over those from non-targeting siRNA. A representative experiment from three biological replicates is shown (individual datapoints, mean, and SEM are plotted). P -values are shown from one representative set. (B) Protein levels were examined using anti-E4F1, anti-IRF2, or anti-GAPDH antibody with UACC903 cell lysates of siRNAs transfected with either non-targeting control, E4F1 , or IRF2 siRNAs. GAPDH was used as a loading control. One representative set of three replicate experiments is shown. (C) Individual luciferase assays were performed by co-transfecting rs3769823 luciferase constructs with E4F1 , IRF2, or non-targeting control siRNAs into UACC903 melanoma cells. Renilla -normalized relative luciferase activities are shown relative to an empty construct containing only a minimal promoter (TATA). One representative set is shown from three biological replicates. Mean with SEM is plotted, n = 6 technical replicates. A two-tailed t-test assuming unequal variances was used to calculate all P values shown against control siRNA.

Article Snippet: Gene expression levels were quantified by qPCR using Taqman assays for E4F1 (Hs00231773_m1), IRF2 (Hs01082884_m1), CASP8 (Hs01018151_m1, Hs01018149, Hs01018160_m1, Hs04405665_m1 and Hs01022432_m1), and GAPDH (Hs99999905_m1) from Thermo Fisher Scientific.

Techniques: Transfection, Control, Luciferase, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: eQTL analyses were performed for rs10931936 combining genotype and expression level data derived from (A) 106 human primary melanocyte cultures (TT, n =7; TC, n =39; CC, n =60), (B) 349 melanoma tumors from TCGA-SKCM (TT, n =42; TC, n =126; CC, n =181), and (C) a panel of 59 early-passage melanoma cell lines (TT, n =8; TC, n =23; CC, n =25), where the risk-T allele is labeled in red. A significant eQTL effect with higher expression driven from the C-protective allele was observed for CASP8 in both melanocytes and melanoma tumors ( P = 1.2 x 10 -9 and P = 6.8 x 10 -3 , respectively), while the result was marginal but in the same direction in melanoma cell lines ( P = 0.07). Significance determined by linear regression; mean with SEM are plotted along with individual data values.

Article Snippet: Taqman real-time PCR assays targeting unique junctions of CASP8 transcript isoforms were obtained from Thermo Fisher Scientific (Waltham, MA; all isoform transcripts: Hs01018151_m1; exon 8 to 9 junction: Hs01018160_m1; exon 8L (alternative spliced) to 9 junction: Hs04405665_m1).

Techniques: Expressing, Derivative Assay, Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) LocusZoom plots present –log10 P -values for melanoma GWAS (upper) and melanocyte CASP8 eQTL (lower) for a 1 Mb region encompassing rs10931936. The melanoma risk lead SNP rs10931936 is labeled and highlighted in purple in both panels, and LD (r 2 based on 1000G EUR) of all other SNPs to the melanoma GWAS lead SNP is color-coded. (B) A LocusCompare plot compares P -values between melanoma GWAS ( x -axis) and melanocyte CASP8 eQTL ( y axis) for the same region. Genomic coordinates are based on hg19.

Article Snippet: Taqman real-time PCR assays targeting unique junctions of CASP8 transcript isoforms were obtained from Thermo Fisher Scientific (Waltham, MA; all isoform transcripts: Hs01018151_m1; exon 8 to 9 junction: Hs01018160_m1; exon 8L (alternative spliced) to 9 junction: Hs04405665_m1).

Techniques: Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) E4F1 or IRF2 were knocked down using respective pools of four different siRNAs in UACC903 melanoma cells, and E4F1 , IRF2 and CASP8 levels were measured. GAPDH - normalized E4F1 , IRF2 or CASP8 mRNA levels are shown as fold change over those from non-targeting siRNA. A representative experiment from three biological replicates is shown (individual datapoints, mean, and SEM are plotted). P -values are shown from one representative set. (B) Protein levels were examined using anti-E4F1, anti-IRF2, or anti-GAPDH antibody with UACC903 cell lysates of siRNAs transfected with either non-targeting control, E4F1 , or IRF2 siRNAs. GAPDH was used as a loading control. One representative set of three replicate experiments is shown. (C) Individual luciferase assays were performed by co-transfecting rs3769823 luciferase constructs with E4F1 , IRF2, or non-targeting control siRNAs into UACC903 melanoma cells. Renilla -normalized relative luciferase activities are shown relative to an empty construct containing only a minimal promoter (TATA). One representative set is shown from three biological replicates. Mean with SEM is plotted, n = 6 technical replicates. A two-tailed t-test assuming unequal variances was used to calculate all P values shown against control siRNA.

Article Snippet: Taqman real-time PCR assays targeting unique junctions of CASP8 transcript isoforms were obtained from Thermo Fisher Scientific (Waltham, MA; all isoform transcripts: Hs01018151_m1; exon 8 to 9 junction: Hs01018160_m1; exon 8L (alternative spliced) to 9 junction: Hs04405665_m1).

Techniques: Transfection, Control, Luciferase, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: eQTL analyses were performed for rs10931936 combining genotype and expression level data derived from (A) 106 human primary melanocyte cultures (TT, n =7; TC, n =39; CC, n =60), (B) 349 melanoma tumors from TCGA-SKCM (TT, n =42; TC, n =126; CC, n =181), and (C) a panel of 59 early-passage melanoma cell lines (TT, n =8; TC, n =23; CC, n =25), where the risk-T allele is labeled in red. A significant eQTL effect with higher expression driven from the C-protective allele was observed for CASP8 in both melanocytes and melanoma tumors ( P = 1.2 x 10 -9 and P = 6.8 x 10 -3 , respectively), while the result was marginal but in the same direction in melanoma cell lines ( P = 0.07). Significance determined by linear regression; mean with SEM are plotted along with individual data values.

Article Snippet: Gene expression levels were quantified by qPCR using Taqman assays for E4F1 (Hs00231773_m1), IRF2 (Hs01082884_m1), CASP8 (Hs01018151_m1, Hs01018149, Hs01018160_m1, Hs04405665_m1 and Hs01022432_m1), and GAPDH (Hs99999905_m1) from Thermo Fisher Scientific.

Techniques: Expressing, Derivative Assay, Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) LocusZoom plots present –log10 P -values for melanoma GWAS (upper) and melanocyte CASP8 eQTL (lower) for a 1 Mb region encompassing rs10931936. The melanoma risk lead SNP rs10931936 is labeled and highlighted in purple in both panels, and LD (r 2 based on 1000G EUR) of all other SNPs to the melanoma GWAS lead SNP is color-coded. (B) A LocusCompare plot compares P -values between melanoma GWAS ( x -axis) and melanocyte CASP8 eQTL ( y axis) for the same region. Genomic coordinates are based on hg19.

Article Snippet: Gene expression levels were quantified by qPCR using Taqman assays for E4F1 (Hs00231773_m1), IRF2 (Hs01082884_m1), CASP8 (Hs01018151_m1, Hs01018149, Hs01018160_m1, Hs04405665_m1 and Hs01022432_m1), and GAPDH (Hs99999905_m1) from Thermo Fisher Scientific.

Techniques: Labeling

Journal: bioRxiv

Article Title: Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

doi: 10.64898/2026.01.06.697866

Figure Lengend Snippet: (A) E4F1 or IRF2 were knocked down using respective pools of four different siRNAs in UACC903 melanoma cells, and E4F1 , IRF2 and CASP8 levels were measured. GAPDH - normalized E4F1 , IRF2 or CASP8 mRNA levels are shown as fold change over those from non-targeting siRNA. A representative experiment from three biological replicates is shown (individual datapoints, mean, and SEM are plotted). P -values are shown from one representative set. (B) Protein levels were examined using anti-E4F1, anti-IRF2, or anti-GAPDH antibody with UACC903 cell lysates of siRNAs transfected with either non-targeting control, E4F1 , or IRF2 siRNAs. GAPDH was used as a loading control. One representative set of three replicate experiments is shown. (C) Individual luciferase assays were performed by co-transfecting rs3769823 luciferase constructs with E4F1 , IRF2, or non-targeting control siRNAs into UACC903 melanoma cells. Renilla -normalized relative luciferase activities are shown relative to an empty construct containing only a minimal promoter (TATA). One representative set is shown from three biological replicates. Mean with SEM is plotted, n = 6 technical replicates. A two-tailed t-test assuming unequal variances was used to calculate all P values shown against control siRNA.

Article Snippet: Gene expression levels were quantified by qPCR using Taqman assays for E4F1 (Hs00231773_m1), IRF2 (Hs01082884_m1), CASP8 (Hs01018151_m1, Hs01018149, Hs01018160_m1, Hs04405665_m1 and Hs01022432_m1), and GAPDH (Hs99999905_m1) from Thermo Fisher Scientific.

Techniques: Transfection, Control, Luciferase, Construct, Two Tailed Test

Journal: Science Advances

Article Title: Caspase-8 expression in CD8 + T cells promotes pathogen restriction in the brain during Toxoplasma gondii infection

doi: 10.1126/sciadv.adz4468

Figure Lengend Snippet: Casp8 −/− Ripk3 −/− , Casp8 +/− Ripk3 −/− , and WT C57BL/6 mice were infected with 10 cysts of the Me49 strain of T. gondii intraperitoneally and analyzed at 4 wpi. ( A ) Cyst burden was quantified by light microscopy. ( B ) Survival curve of infected WT, Casp8 +/− Ripk3 −/− , and Casp8 −/− Ripk3 −/− mice. Representative H&E image of ( C ) WT and ( D ) Casp8 −/− Ripk3 −/− brain at 4 wpi. Arrows indicate inflamed blood vessels, and arrowheads indicate T. gondii cysts. Scale bars, 50 μm. Brain immune cell populations were quantified by spectral flow cytometry at 4 wpi: ( E ) CD8 + T cells, ( F ) CD8 + IFN-γ + T cells, ( G ) CD8 + TNFα + T cells, ( H ) CD8 + FasL + T cells, ( I ) CD8 + GranzymeB + T cells, ( J ) CD4 + T cells, ( K ) CD4 + IFN-γ + T cells, ( L ) CD4 + TNFα + T cells, and ( M ) infiltrating iNOS + monocytes (CD45 hi CD11b + Ly6G − Ly6C hi NOS + ). ( N ) Gene expression was measured by RT-qPCR for Ifng , Tnf , and Nos2 . Statistical significance determined by randomized block analysis of variance (ANOVA) and least-squares means: (A) WT C57BL/6 ( n = 16), Casp8 +/− Ripk3 −/− ( n = 14), and Casp8 −/− Ripk3 −/− ( n = 22) (four experiments); (E, J, and M) WT C57BL/6 ( n = 15), Casp8 +/− Ripk3 −/− ( n = 9), and Casp8 −/− Ripk3 −/− ( n = 14) (three experiments); (F and K) WT C57BL/6 ( n = 15), Casp8 +/− Ripk3 −/− ( n = 8), and Casp8 −/− Ripk3 −/− ( n = 18) (three experiments); (N) WT C57BL/6 ( n = 10), Casp8 +/− Ripk3 −/− ( n = 10), and Casp8 −/− Ripk3 −/− ( n = 14) (two experiments). Statistical significance determined by log-rank (Mantel-Cox) test: (B) WT C57BL/6 ( n = 10), Casp8 +/− Ripk3 −/− ( n = 8), and Casp8 −/− Ripk3 −/− ( n = 9) (two experiments). Statistical significance determined by ordinary one-way ANOVA: (G, I, and L) WT C57BL/6 ( n = 5), Casp8 +/− Ripk3 −/− ( n = 5), and Casp8 −/− Ripk3 −/− ( n = 9) (one experiment). (H) WT C57BL/6 ( n = 5), Casp8 +/− Ripk3 −/− ( n = 6), and Casp8 −/− Ripk3 −/− ( n = 5) (one experiment). Data are presented as mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following Thermo Fisher Scientific mouse gene probes were used: Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), and Casp8 (Mm01255716_m1).

Techniques: Infection, Light Microscopy, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Blocking Assay

Journal: Science Advances

Article Title: Caspase-8 expression in CD8 + T cells promotes pathogen restriction in the brain during Toxoplasma gondii infection

doi: 10.1126/sciadv.adz4468

Figure Lengend Snippet: MERFISH was used to measure spatially resolved transcriptomics at a single nuclei level in whole-brain slices of WT C57BL/6 mice. A 342-gene panel was used to call cell types and measure the expression of numerous inflammation and cell death–related genes. ( A ) Nuclei from uninfected brains were annotated through Leiden clustering and differential expression testing of marker genes by Wilcoxon rank sum test ( n = 55,399 cells, 32,246.9 transcripts per FOV), and cell populations were identified. OPC, oligodendrocyte precursor cell. ( B ) Casp8 expression was mapped to the cell populations. ( C ) Expression of genes associated with cell death was analyzed by cell population. ( D to F ) Cell clustering, Casp8 expression mapping, and gene expression were similarly examined in the infected brain at 4 wpi ( n = 58,479 cells, 29,757.7 transcripts per FOV). ( G ) Outline of experiments in WT mice. ( H ) Confocal microscopy was used to image cells with cleaved caspase-8 (magenta) with 4′,6-diamidino-2-phenylindole (DAPI) (blue). ( I ) Magnified image of cleaved caspase-8 (magenta) with DAPI (blue) and ( J ) cleaved caspase-8 (magenta) signal alone in WT mice at 4 wpi. ( K ) Outline of experiments using caspase-3 inhibition in WT mice. ( L to Q ) Confocal microscopy of cleaved caspase-8 (magenta) with DAPI (blue) and magnified images of WT mice treated with caspase-3 inhibitor. Scale bars, 50 μm.

Article Snippet: The following Thermo Fisher Scientific mouse gene probes were used: Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), and Casp8 (Mm01255716_m1).

Techniques: Expressing, Quantitative Proteomics, Marker, Gene Expression, Infection, Confocal Microscopy, Inhibition

Journal: Science Advances

Article Title: Caspase-8 expression in CD8 + T cells promotes pathogen restriction in the brain during Toxoplasma gondii infection

doi: 10.1126/sciadv.adz4468

Figure Lengend Snippet: Mice were intraperitoneally infected with TF-Pru to identify cell populations with parasite-driven cre activity at 4 wpi. Confocal microscopy was used to image cre activity (green) and T. gondii parasites (magenta) in ( A and B ) WT Ai6 and ( C to F ) Casp8 −/− Ripk3 −/−Ai6 mice. (C) inset represents neurons, (D) inset represents glia, and (E) inset represents immune cells. Images are representative of three independent experiments. Scale bars, 100 μm. ZsGreen + cell populations in the brain of ( G ) WT Ai6 and ( H ) Casp8 −/− Ripk3 −/−Ai6 mice were ( I ) quantified by spectral flow cytometry. SS-A, side scatter-area. ZsGreen + cell populations in the brains of Casp8 −/− Ripk3 −/−Ai6 mice were analyzed for ( J ) CD8, ( K ) CD4, and ( L ) CD11b expression and ( M ) quantified. Statistical significance determined by randomized block ANOVA and least-squares means: (I) WT Ai6 ( n = 11), Casp8 +/− Ripk3 −/−Ai6 ( n = 16), and Casp8 −/− Ripk3 −/−Ai6 ( n = 19) (five experiments). Data are presented as mean ± SEM; * P < 0.05.

Article Snippet: The following Thermo Fisher Scientific mouse gene probes were used: Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), and Casp8 (Mm01255716_m1).

Techniques: Infection, Activity Assay, Confocal Microscopy, Flow Cytometry, Expressing, Blocking Assay

Journal: Science Advances

Article Title: Caspase-8 expression in CD8 + T cells promotes pathogen restriction in the brain during Toxoplasma gondii infection

doi: 10.1126/sciadv.adz4468

Figure Lengend Snippet: ( A ) Levels of Casp8 were measured by RT-qPCR from neurons purified from naïve Syn Cre Casp8 fl/fl Ripk3 −/−Ai6 mice (Syn Cre ) and Casp8 fl/fl Ripk3 −/−Ai6 mice (control). ( B to D ) Cre-reporter activity (ZsGreen) in neurons (NeuN; magenta) was assessed by IHC in naïve Syn Cre Casp8 fl/fl Ripk3 −/−Ai6 mice. Scale bars, 50 μm. ( E ) Syn Cre Casp8 fl/fl Ripk3 −/−Ai6 mice and control Casp8 fl/fl Ripk3 −/−Ai6 mice were infected for 6 weeks, and cyst burden was measured. Immune responses in the brain were measured by flow cytometry: ( F ) CD8 + T cells, ( G ) CD4 + T cells, and ( H ) monocytes producing iNOS (CD45 hi CD11b + Ly6G − Ly6C hi NOS). ( I ) Levels of Casp8 were measured by RT-qPCR from ACSA2 + astrocytes purified from naïve Aldh1l1 CreERT2 Casp8 fl/fl Ripk3 −/−Ai6 ( Casp8 fl/fl ) and Aldh1l1 CreERT2 Casp8 fl/wt Ripk3 −/−Ai6 ( Casp8 fl/wt ) control mice. ( J to L ) Cre-reporter activity (ZsGreen) in astrocytes (GFAP/ALDH1L1/S100β; magenta) was assessed in naïve Aldh1l1 CreERT2 Casp8 fl/fl Ripk3 −/−Ai6 mice. Scale bars, 50 μm. ( M ) Aldh1l1 CreERT2 Casp8 fl/fl Ripk3 −/−Ai6 and control Aldh1l1 CreERT2 Casp8 fl/wt Ripk3 −/−Ai6 mice were infected for 6 weeks, and cyst burden was measured. Immune responses in the brain were measured by flow cytometry: ( N ) CD8 + T cells, ( O ) CD4 + T cells, and ( P ) brain-infiltrating inflammatory monocytes producing iNOS (CD45 hi CD11b + Ly6G − Ly6C hi NOS + ). Statistical significance determined by unpaired t test: (A) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 4) and Syn Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 4) (one experiment). Statistical significance was determined by randomized block ANOVA and least-squares means: (E to H) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 14) and Syn Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 11) (three experiments); (I) Aldh1l1 CreERT2 Casp8 fl/wt Ripk3 −/−Ai6 ( n = 6) and Aldh1l1 CreERT2 Casp8 fl/fl Ripk3 −/−Ai6 ( n = 5) (two experiments); (M) Aldh1l1 CreERT2 Casp8 fl/wt Ripk3 −/−Ai6 ( n = 9) Aldh1l1 CreERT2 Casp8 fl/fl Ripk3 −/−Ai6 ( n = 11) (three experiments); (N to P) Aldh1l1 CreERT2 Casp8 fl/wt Ripk3 −/−Ai6 ( n = 6) and Aldh1l1 CreERT2 Casp8 fl/fl Ripk3 −/−Ai6 ( n = 6) (two experiments). Data are presented as mean ± SEM; * P < 0.05.

Article Snippet: The following Thermo Fisher Scientific mouse gene probes were used: Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), and Casp8 (Mm01255716_m1).

Techniques: Quantitative RT-PCR, Purification, Control, Activity Assay, Infection, Flow Cytometry, Blocking Assay

Journal: Science Advances

Article Title: Caspase-8 expression in CD8 + T cells promotes pathogen restriction in the brain during Toxoplasma gondii infection

doi: 10.1126/sciadv.adz4468

Figure Lengend Snippet: Cd8 α CreAi6 mice were infected with 10 cysts of the Me49 strain of T. gondii intraperitoneally. At the chronic phase of infection, confocal microscopy was used to visualize ( A ) cleaved caspase-8 (magenta) with DAPI (blue), ( B ) cleaved caspase-8 (magenta) with ZsGreen-expressing CD8 + T cells (green), and ( C ) ZsGreen-expressing CD8 + T cells (green) alone. Scale bars, 20 μm. Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 mice ( Cd8 α Cre ) and Casp8 fl/fl Ripk3 −/−Ai6 mice (control) were infected with 10 cysts of the Me49 strain of T. gondii intraperitoneally, and cyst burden was analyzed at ( D to F ) 4, 6, and 8 wpi. ( G ) Survival curve of infected Casp8 fl/fl Ripk3 −/−Ai6 control and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 mice. Immune cells in the brain were analyzed by flow cytometry at 6 wpi: ( H ) CD8 + T cells, ( I ) CD8 + IFN-γ + T cells, and ( J ) CD8 + SIINFEKL + T cells, ( K ) CD4 + T cells, ( L ) CD4 + IFN-γ + T cells, and ( M ) inflammatory monocytes (CD45 hi CD11b + Ly6G − Ly6C hi NOS + ). Gene expression was measured from whole-brain homogenate by RT-qPCR : ( N ) Ifng and ( O ) Nos2 . ( P to R ) Infection of CD8 + T cells in the brain was observed by IHC in Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 mice. Parasite staining is shown in magenta, and ZsGreen-expressing CD8 + T cells in green. Scale bars, 20 μm. Statistical significance determined by randomized block ANOVA and least-squares means: (D) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 16) and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 19) (three experiments); (E, H, K, and M) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 12) and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 18) (three experiments); (F) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 16) and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 11) (two experiments); (I and L) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 6) and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 8) (two experiments); (N and O) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 5) and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 14) (two experiments). Statistical significance determined by log-rank (Mantel-Cox) test: (G) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 13) and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 11) (two experiments). Statistical significance determined by unpaired t test: (J) Casp8 fl/fl Ripk3 −/−Ai6 ( n = 4) and Cd8 α Cre Casp8 fl/fl Ripk3 −/−Ai6 ( n = 4) (one experiment). Data are presented as mean ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following Thermo Fisher Scientific mouse gene probes were used: Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), and Casp8 (Mm01255716_m1).

Techniques: Infection, Confocal Microscopy, Expressing, Control, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Staining, Blocking Assay