HPV-YAP1 oncogenic alliance drives malignant transformation of fallopian tube epithelial cells

Abstract

High grade serous ovarian carcinoma (HGSOC) is the most common and aggressive ovarian malignancy. Accumulating evidence indicates that HGSOC may originate from human fallopian tube epithelial cells (FTECs), although the exact pathogen(s) and/or molecular mechanism underlying the malignant transformation of FTECs is unclear. Here we show that human papillomavirus (HPV), which could reach FTECs via retrograde menstruation or sperm-carrying, interacts with the yes-associated protein 1 (YAP1) to drive the malignant transformation of FTECs. HPV prevents FTECs from natural replicative and YAP1-induced senescence, thereby promoting YAP1-induced malignant transformation of FTECs. HPV also stimulates proliferation and drives metastasis of YAP1-transformed FTECs. YAP1, in turn, stimulates the expression of the putative HPV receptors and suppresses the innate immune system to facilitate HPV acquisition. These findings provide critical clues for developing new strategies to prevent and treat HGSOC.

Keywords: Fallopian Tube; HGSOC; HPV Infection; Hippo Pathway; Innate Immunity.

Figure 1. HPV inhibits natural replicative and YAP1-promoted senescence.

(A) Representative images showing the morphology and SA-β-galactosidase activity in FTECs (FTEC-MX, control), FTEC-YAP (FTECs with ectopic expression of wild-type YAP1), and FTEC-YAP^S127A cells (FTECs expressing YAP1^S127A) at their 6th passage in the presence and absence of E6/E7 gene expression. Scale bar: 200 µm. (B) Quantitative data showing the percentage of SA-β-gal positive cells in FTEC-MX, FTEC-YAP, and FTEC-YAP^S127A cells at their 6th passage in the presence or absence of E6/E7 gene expression. Each bar represents the mean ± SEM (n = 4 technical replicates). Bars with different letters are significantly different from each other. Data were analyzed for significance using the two-way ANOVA followed by Tukey’s multiple comparisons test. A value of P < 0.05 was considered statistically significant. Exact P values for this analysis are presented in the source data, which is available online. (C) Representative images showing the morphology of control FTEC cells and FTEC cells expressing HPV16 E6/E7 oncoproteins (FTEC-E6/E7 cells) at their 3rd passage. Scale bar: 200 µm. (D) Quantitative data showing the cell number of control FTECs and FTEC-E6/E7 cells at the 3rd passage incubated in the growth media for five days. Each bar represents the mean ± SEM (n = 4 technical replicates). Data were analyzed for significance using one-way ANOVA (with the Tukey’s post hoc test). A value of P < 0.05 was considered statistically significant. *P < 0.05. Exact P value: CTRL vs. E6/E7, P = 0.0112. (E) Representative images showing the morphology and SA-β-gal staining in control FTECs and FTEC-E6/E7 cells at their 6th passage. Scale bar: 200 µm. (F) Growth curves of FTEC-MX (control), FTEC-YAP, FTEC-YAP^S127A, FTEC-E6/E7, FTEC-E6/E7-YAP, and FTEC-E6/E7-YAP^S127A cells at the 6th passage. Data in each time point represented the mean ± SEM (n = 4 rechnical replicates). (G) Growth curves of FNE1-MX (control), FNE1-YAP, FNE1-YAP^S127A, FNE1-E6/E7, FNE1-E6/E7-YAP, and FNE1-E6/E7-YAP^S127A cells. FNE1 cell is a hTERT immortalized FTEC cell line. Data in each time point represented the mean ± SEM (n = 4 technical replicates). (H) Growth curves of FT190-MX (control), FT190-YAP, FT190-YAP^S127A, FT190-E6/E7, FT190-E6/E7-YAP, and FT190-E6/E7-YAP^S127A cells. FT190 is an SV40 immortalized FTEC cell line. Data in each time point represented the mean ± SEM (n = 4 technical replicates). Source data are available online for this figure.

Figure 2. HPV is present in normal and cancerous fallopian tube tissues.

(A) Representative images showing PCR products of PGMY09/PGMY11 primer set in chronic inflammatory fallopian tube tissues and fallopian tube cancer tissues. PCR products were fragmented on a 2% agarose gel, stained with ethidium bromide, and visualized under a UV-transilluminator. The left lane (Lane 1) is the 100 bp nucleic acid molecular marker. Lanes 2 to 8 are chronic inflammatory fallopian tube tissues. Lanes 9 to 15 are fallopian tube cancer tissues. Note that ME180 and Hela cells (HPV-positive cell lines) are used as positive controls. HT3 (HPV negative cell line) and H₂O (to replace the cDNA template in the reaction mixture) are negative controls. The blue arrow points to the expected HPV+ bands. (B, C) Results of HC2 HPV DNA Test in fallopian tube tissues for five low-risk types (6/11/42/43/44) (B) and 13 high-risk types (16/18/31/33/35/39/45/51/52/56/58/59/68) (C). Each bar represents the mean ± SEM (n = 3 technical replicates). Sample preparation, experimental operation, and result interpretation were performed according to a protocol included in QIAGEN’s digene HC2 HPV DNA Test kit (# 5198-1220, QIAGEN Ltd, UK). NC (Negative Calibrator), LRC (Low-Risk HPV Calibrator), HRC (High-Risk HPV Calibrator), HPV16 DNA, and HPV6 DNA were from the QIAGEN Kit. CN: cancer samples with negative PGMY11/09 Result; CP: fallopian tube cancer samples with positive PGMY11/09 result; FP: fallopian tube samples with positive PGMY11/09 result. (D) Representative images showing nested PCR products amplified with the PGMY09/11-GP5 + /6+ system on fallopian tube cancer samples #3 (CP3) and #7 (CP7). These CP3 and CP7 Samples were negative for the GP5 + /6+ test in the primary PCR screen. (E) Representative images showing DNA sequences of PCR products of CP3 and CP7 amplified using PGMY09/11-GP5 + /6 + PCR system. (F, G) Representative images showing that the DNA sequence of nested PCR product of CP3 cDNA was aligned to HPV18 L1 gene (F), while the DNA sequence of nested PCR product of CP7 cDNA was aligned to HPV16 L1 gene (G). Source data are available online for this figure.

Figure 3. HPV differentially infects fallopian tube epithelial and ovarian cancer cells.

(A) Representative images showing HPV16 pseudovirions-derived GFP signal in primary human cervical epithelial cells (hCerEC) and primary fallopian tube epithelial cells (FTEC) incubated with different concentrations (MOI = 0.1, 0.5, 1, and 5) of HPV16 pseudovirions (PsV). The infection efficiencies of HPV16 PsV in hCerEC and FTEC cells are presented as the ratio of GFP-positive cells. Scale bar: 200 µm. (B) Quantitative data showing the ratio of GFP-positive cells in hCerEC and FTEC cells under different concentrations of HPV PsV. Each bar represented the mean ± SEM (n = 4 technical replicates). Data were analyzed for significance using the two-way ANOVA followed by the Tukey’s multiple comparisons post hoc test. A value of P < 0.05 was considered statistically significant. ****P < 0.0001; ***P < 0.001; **P < 0.01; ns: not significant, between indicated two groups. Exact P values for all comparisons are presented with the corresponding source data, which is available online. (C) Representative images showing the presence of HPV16 PsV-derived GFP signal in hCerEC, FTEC, FT194, FT246, COV362, and OVSAHO cells incubated with of HPV16-Psv (MOI = 2.0) for 72 h. Scale bar: 100 µm. (D) Quantitative results showing the ratio of HPV16 PsV positive cells in hCerEC, FTEC, FT194, FT246, COV362, and OVSAHO cells. Each bar represented the mean ± SEM (n = 4 technical replicates). Bars with different letters are significantly different from each other (P < 0.05). Data were analyzed for significance using the one-way ANOVA followed by the Tukey’s post hoc test. A value of P < 0.05 was considered statistically significant. Exact P values when compared to FTECs: P = 0.0059 for hCerEc; P = 0.0526 for FT194; P = 0.9903 for FT246; P < 0.00011 for COV326; P < 0.0001 for OVSAHO. (E) Quantitative data showing mRNA levels of YAP1 and the putative HPV receptors (ITGA6, SDC1, and EGFR) in hCerEC, FTEC, Hela, and OVSAHO cells. Each bar represented the mean ± SEM (n = 3 technical replicates). Bars with different letters are significantly different from each other (P < 0.05). Data were analyzed for significance using the one-way ANOVA followed by the Tukey’s multiple comparisons post hoc test. A value of P < 0.05 was considered statistically significant. Exact P values for all comparisons are presented with corresponding source data, which is available online. (F) HPV16 PsV infected fallopian tube epithelial cells in vivo. HPV16 PsV (1.0 × 10⁶ pfu/µl in 30 µl saline with Evans blue dye) was injected into the right uterus (near the oviduct) of C57BL/6 mice (n = 5 technical replicates). The left uterus was injected with the same amount of saline with Evans blue dye and used as a negative control. The presence of HPV16 PsV in the oviducts and ovarian bursa was monitored by the blue color (left panel). Yellow arrow points to ovary and ovarian bursa. White arrow pints to uterus. Scale bar: 3.0 mm. Representative images showing the presence of HPV16 PsV-derived GFP signal in the epithelium of mouse oviduct. Nuclei were stained with DAPI (blue). Scale bar: 100 µm. Source data are available online for this figure.

Figure 4. YAP regulates expressions of putative HPV receptors.

(A) Representative images showing HPV16 PsV-derived GFP signal in FT246-MX (control), FT246-YAP, and FT246-YAP^S127A cells. The quantitative data is presented in Appendix Fig. S4A. Scale bar: 100 µm. (B) Quantitative data showing mRNA levels of YAP1 and the putative HPV receptors (ITGA6, SDC1, and EGFR) in FT246-MX (control), FT246-YAP, and FT246-YAP^S127A cells. Each bar represented the mean ± SEM (n = 3 technical replicates). Bars with different letters are significantly different from each other. Data were analyzed for significance using the one-way ANOVA followed by the Tukey’s post hoc test. A value of P < 0.05 was considered statistically significant. Exact P values for each gene are presented with the corresponding source data, which is available online. (C) Quantitative data showing mRNA levels of the putative HPV receptors (ITGA6, SDC1, and EGFR) in control (scramble siRNA, CTRL) and YAP1-knockdown (siYAP1) OVSAHO cells. Each bar represented the mean ± SEM (n = 3 technical replicates). Bars with different letters are significantly different from each other. Data were analyzed for significance using the unpaired t test. A value of P < 0.05 was considered statistically significant. Exact P values when compared to their corresponding control (CTRL): P = 0.0048 for YAP1; P = 0.0031 for ITGA6; P = 0.0161 for SDC1; P = 0.0115 for EGFR. (D) RT-PCR analyses showing successful knockdown of ITGA6 in OVSAHO cells using ITGA6 siRNAs (siITGA6). Each bar represented the mean ± SEM (n = 3 technical replicates). Bars with different letters are significantly different from each other (P = 0.0025). Data were analyzed for significance using the unpaired t test. A value of P < 0.05 was considered statistically significant. (E) Representative images showing HPV16 PsV-derived GFP signal in control (scramble siRNA, CTRL) and YAP1-knockdown (siYAP1) OVSAHO cells. GFP signal indicated the infection efficiency of HPV16 PsV in these cells. Scale bar: 100 µm. (F) Quantitative results of (E) to show the ratio of GFP-positive cells in OVSAHO cells with (siYAP1) or without (CTRL) YAP1 knockdown. Each bar represented the mean ± SEM (n = 4 technical replicates). Bars with different letters are significantly different from each other (P = 0.0002). Data were analyzed for significance using the unpaired t test. A value of P < 0.05 was considered statistically significant. (G) Representative images showing HPV16 PsV-derived GFP signal in control (scramble siRNA, CTRL) and ITGA6-knockdown (siITGA6) OVSAHO cells. Scale bar: 100 µm. (H) Quantitative results of (G) to show the ratio of GFP-positive cells in OVSAHO cells with (siITGA6) or without (CTRL) knockdown of ITGA6. Each bar represented the mean ± SEM (n = 4 technical replicates). Bars with different letters are significantly different from each other (P = 0.0002). Data were analyzed for significance using the unpaired t test. A value of P < 0.05 was considered statistically significant. (I) Representative images showing HPV16 PsV-derived GFP signal in FT246-MXIV, FT246-YAP, and FT246-YAP^S127A cells with (siITGA6) or without (siCTRL) knockdown of ITGA6 using RNA interference technique. The quantitative data are presented in Appendix Fig. S5. Source data are available online for this figure.

Figure 5. HPV E6/E7 contributes to the malignant transformation of FTECs and drives metastasis of YAP1-induced HGSOC.

(A) Quantitative data showing colonies formed by modified primary FTECs [FTEC-MX (control), FTEC-YAP, FTEC-YAP^S127A, FTEC-E6/E7, FTEC-E6/E7-YAP and FTEC-E6/E7-YAP^S127A cells], FNE1-derived cells [FNE1-MX (control), FNE1-YAP, FNE1-YAP^S127A, FNE1-E6/E7, FNE1-E6/E7-YAP and FNE1-E6/E7-YAP^S127A cells], and FT190-derived cells [FT190-MX (control), FT190-YAP, FT190-YAP^S127A, FT190-E6/E7, FT190-E6/E7-YAP and FT190-E6/E7-YAP^S127A cells] at their 6th passage in the soft agar assay. Each bar represented the mean ± SEM (n = 5 technical replicates). Data were analyzed for significance using the two-way ANOVA followed by the Tukey’s multiple comparisons post hoc test. A value of P < 0.05 was considered statistically significant. ****P < 0.0001; ***P < 0.001; *P < 0.05; ns: not significant, between indicated two groups. Exact P values for the compared groups in each graph are presented with source data, which is available online. (B) Representative images showing tumorigenesis of FNE1-MX (control) and FNE1-YAP^S127A cells. Please note that no tumor formed in the FNE1-MX control group. (C) Representative images showing H&E staining of FNE1-YAP^S127A cell-derived tumors. Scale bar: 50 µm. (D) Representative IHC images showing YAP1 expression in FNE1-YAP^S127A cell-derived tumors. Scale bar: 50 µm. (E, G) Representative images showing the tumorigenesis of FNE1-E6/E7 and FNE1-E6/E7-YAP^S127A cells. Please note that no tumor formed in E6/E7 alone group (FNE1-E6/E7 cells). Tumors derived from FNE1-E6/E7-YAP^S127A cells metastasized intraperitoneally to multiple organs and tissues. (H, I) Representative images showing the histology (H&E staining) of tumor tissues derived from FNE1-E6/E7-YAP^S127A cells. (J–Q) Representative IHC images showing expressions of known biomarkers (KRT7, PAX8, WT1, and TP53) of high grade serous ovarian carcinoma (HGSOC) in tumor tissues derived from FNE1-E6/E7-YAP^S127A cells. Scale bars in (H), (J), (L), (N), and (P): 200 µm; Scale bars in (I), (K), (M), (O), and (Q): 50 µm. Source data are available online for this figure.

Figure 6. Enrichment of genes and pathways in FNE1 cells with differential expression of YAP1 and HPV E6/E7 oncogenes.

(A) Increased expression of YAP1 and HPV16 E6/E7 downstream genes provides evidence for the successful ectopic expression of YAP1 and HPV16 E6/E7 in FNE1 cells. Each bar represents the mean ± SEM (n ≥ 3 technical replicates). Bars with different letters are significantly different from each other. Data were analyzed for significance using the two-way ANOVA followed by the Tukey’s multiple comparisons post hoc test. A value of P < 0.05 was considered statistically significant. Exact P values for the compared groups in each graph are presented with source data, which is available online. (B–D) Representative GSEA graphs showing the enriched genes and pathways in FNE1-E6/E7 (B), FNE1-YAP ^S127A (C), and FNE1-E6/E7-YAP^S127A (D) cells. GSEA analyses were performed based on RNA-seq data from FNE1-eE6/E7 (B), FNE1-YAP^S127A, and FNE1-E6/E7-YAP^S127A cells. Source data were uploaded to Gene Expression Omnibus (accession: GSE268836). The exact P value for each assay is directly presented on the graph. Source data are available online for this figure.

Figure 7. Constitutive activation of YAP1 suppresses type I interferon production in FTECs.

(A) Quantitative data showing mRNA levels of major components of viral recognition pathway in FNE1-MX (control or CTRL) and FNE1-YAP^S127A cells. Each bar represented the mean + SEM (n = 3 technical replicates). Data were analyzed for significance using the unpaired t test. A value of P < 0.05 was considered statistically significant. ***P < 0.001; ****P < 0.0001, when compared to their control (CTRL). Exact p values for each gene: P < 0.0001 for TLR1, TLR2, TLR3, TLR6, MYD88, TBK1, NFkB1, NFKB2, ReIA, and IRF7; P = 0.0603 for TLR5; P = 0.006 for IRF3. (B) Representative images selected from three biological replicates showing the expressions and locations of IRF3 and NFκB1 in FT246-MXIV, FT246-YAP- and FT246-YAP^S127A cells. IRF3 and NFκB1 proteins were visualized using an Alexa-488 (green) conjugated secondary antibody. Actin filaments were stained with rhodamine-phalloidin (red). Nuclei were stained with DAPI (blue). Scale bar: 50 µm. (C) Quantitative data showing mRNA levels of downstream target genes of IRF3 and NFκB, including type I interferons, pro-inflammatory cytokines, and key antiviral factors in FNE1-MX (control) and FNE1-YAP^S127A cells. Each bar represents the mean + SEM (n = 3 technical replicates). Data were analyzed for significance using the unpaired t test. A value of P < 0.05 was considered statistically significant. ****P < 0.0001, when compared to their control (CTRL). Exact p values for each gene: P < 0.0001 for Il6, IL8, RSAD2, IRF1, and IFNA2; P = 0.0001 for IFNA1; P = 0.0561 for IFNB1; P = 0.0017 for IFNε. (D) Representative western blots selected from three biological replicates showing protein levels of major components of the viral recognition and interferon production pathways in FNE1-MX (control), FNE1-YAP, and FNE1-YAP^S127A cells with or without HPV16 pseudovirions treatment. Source data are available online for this figure.

Figure 8. Constitutive activation of YAP1 suppresses the JAK/STAT/IRF9 pathway in FTECs.

(A) Quantitative data showing mRNA levels of major components of the IFNs/JAK/STAT/IRF9 pathway in control (FNE1-Mx) and FNE1-YAP^S127A cells. Each bar represented the mean + SEM (n = 3 technical replicates). Data were analyzed for significance using the unpaired t test. A value of P < 0.05 was considered statistically significant. **P < 0.01; ***P < 0.001, when compared with control (CTRL). Exact p values for each gene: P = 0.0006 for IFNαR1; P < 0.0001 for IFNαR2; P < 0.0001 for JAK1; P = 0.0001 for JAK2; P < 0.0001 for STAT1; P = 0.0024 for STAT2; P = 0.0004 for STAT4; P = 0.0028 for IRF9. (B) Representative blots selected from three biological replicates showing expression and activation of key proteins and kinases in the JAK/STAT/IRF9 pathway in FNE1-MX (control), FNE1-YAP, and FNE1-YAP^S127A cells. (C) Representative images selected from three biological replicates showing the expressions and locations of STAT1 and IRF9 in FT246-MX (control) and FT246-YAP^S127A cells in the presence or absence of IFNα2b. STAT1 and IRF9 proteins were visualized using an Alexa-488 (green) conjugated secondary antibody. In the left panel, STAT1 and IRF9 proteins were visualized using an Alexa-488 (green) conjugated secondary antibody, while YAP was visualized using an Alexa-594 (red) conjugated secondary antibody. In the right panel, IRF9 proteins were visualized using an Alexa-488 (green) conjugated secondary antibody. Actin was stained with rhodamine-phalloidin (red). Nuclei were stained with DAPI. Scale bar: 30 µm. (D) Representative images selected from three biological replicates showing HPV16 PsV-derived GFP signal in control FT246-MX and FT246-YAP^S127A cells in the presence or absence of Ruxolitinib (JAK inhibitor) or recombined human interferon alpha 2b (IFNα2b). GFP signal indicates the infection efficiency of HPV16 PsV in these cells. Scale bar: 100 µm. Quantitative results of the GFP signal are presented in Appendix Figure S9. Source data are available online for this figure.

Figure EV1. Expression of HPV16 E6/E7 in the Fallopian tube STIC lesion (precursor of HGSOC) detected by RNA scope.

(A) A representative image showing the expression of HPV16 E6/E7 mRNA (in pink) in fallopian tube STIC lesion (arrow) of a human patient (sample-GU980150-E10). E6/E7 were detected and visualized using the RNA scope technique. Scale bar: 50 µm. (B) A representative image showing the expression of YAP1 protein (in brown) in fallopian tube STIC lesion. YAP1 protein was detected and visualized by immunohistochemistry. Arrows point to neoplastic growth of epithelial cells with nuclear YAP1 protein. Scale bar: 50 µm. (C) A representative image showing the expression of HPV16 E6/E7 mRNA (in pink) in SiHa cell xenograft tumor tissues (positive control). Blue arrows point to the HPV16 E6/E7 positive cells (in pink). Scale bar: 50 µm. (D) Representative images showing negative staining (non-targeting probe) of HPV16 E6/E7 mRNA in SiHa cell xenograft tumor tissues and human STIC lesion (insert). Scale bar: 50 µm. Source data are available online for this figure.

Figure EV2. Expression of genes encoding the putative HPV receptor molecules in normal ovarian tissues, primary ovarian tumors, and recurrent ovarian tumor tissues.

The TCGA TARGET GTEx study online tool (https://xenabrowser.net/) was used to compare the expression of genes encoding the putative HPV receptor molecules in normal ovarian tissues (n = 88 normal human samples), primary ovarian tumors (n = 418 patient samples), and recurrent ovarian tumor tissues (n = 8 patient samples). Data were taken from the UCSC RNA-seq Compendium, where TCGA, TARGET, and GTEx samples are re-analyzed using the same RNA-seq pipeline. Extracted data were analyzed for significance using the one-way ANOVA followed by the Tukey’s post hoc test. A value of P < 0.05 was considered statistically significant. ****P < 0.0001, compared to the normal control group (Normal). Exact P values for each gene are presented with the source data of this figure, which is available online. Source data are available online for this figure.

Figure EV3. Constitutive activation of YAP inhibits the type I interferon (JAK/STAT) signaling pathway in fallopian tube secretory epithelial cells (FNE1).

(A) Representative blots showing expression and activation of major components of the JAK/STAT/IRF9 pathway in FNE1-MX (control), FNE1-YAP, and FNE1-YAP^S127 cells with or without IFNα2b treatment for 30 min. Ectopic expression of YAP or YAP^S127A in FNE1 cells suppressed IFNα2-induced phosphorylation of STAT1/2. (B) Quantitative data showing that transcription of STATs are suppressed by YAP^S127A in FNE1-YAP^S127A cells. Each bar represents the mean ± SEM (n = 3 technical replicates). Data were analyzed for significance using unpaired t test. A value of P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01; ***P < 0.001, when compared with MX control (CTRL). Exact P values for each gene: P < 0.0001 for STAT3; P = 0.0004 for STAT4; P = 0.0448 for STAT5; P = 0.0002 for STAT6; P = 0.0027 for IRF9. Source data are available online for this figure.

Figure EV4. Expression of genes encoding the key molecules of the innate immune signaling pathway in normal ovarian tissues, primary ovarian tumors, and recurrent ovarian tumors.

The TCGA TARGET GTEx study online tool (https://xenabrowser.net/) was used to compare the expression of genes encoding the putative HPV receptor molecules in normal ovarian tissues (n = 88 normal ovarian samples), primary ovarian tumor (n = 418 patient samples), and recurrent ovarian tumor tissues (n = 8 patient samples). Data were from the UCSC RNA-seq Compendium, where TCGA, TARGET, and GTEx samples are re-analyzed using the same RNA-seq pipeline. Extracted data were analyzed for significance using the one-way ANOVA followed by the Tukey’s post hoc test. A value of P < 0.05 was considered statistically significant. ***P < 0.001; ****P < 0.0001, when compared to the normal control group (Norm-OV). Exact P values for each gene are presented with the source data of this figure, which is available online. Source data are available online for this figure.

Figure EV5. Constitutive activation of YAP1 blocks basal, pathogen-induced, or IFNα2b-induced production of antiviral molecules in FTECs.

(A) Quantitative data showing mRNA levels of several major antiviral interferon-stimulated genes (ISGs) in control (FNE1-MX) and YAP^S127A-expressing FNE1 (FNE1-YAP^S127A) cells with or without HPV16 pseudovirions treatment. (B) Quantitative data showing mRNA levels of major components of the JAK/STAT/IRF9 pathway and some antiviral ISGs in FNE1-MX and FNE1-YAP^S127A cells with or without IFNα2b treatment. Each bar represents the mean + SEM (n = 4 technical replicates). Bars with different letters are significantly different from each other. Data were analyzed for significance using the two-way ANOVA followed by the Tukey’s multiple comparisons post hoc test. A value of P < 0.05 was considered statistically significant. Exact P values between the compared groups for each gene are presented with the source data of Fig. EV5A and Fig. EV5B, which are available online. Source data are available online for this figure.