Fig. 2

MYPOP protein level negatively correlates with HPV16 PsV infection and early gene expression. a–c MYPOP in HPV16 infection assay. a HaCaTs were transfected with control siRNA or MYPOP-specific siRNA #9 for 48 h and then re-transfected for additional 48 h. Seventy-two hours after initial siRNA transfection, knockdown efficiency of MYPOP was analyzed by western blot (upper panel) or infected with HPV16 LCR PsV (lower panel). Relative luciferase activity as measure for infection was assessed 24 h later and normalized to lactate dehydrogenase (LDH) measurements. Control siRNA-treated cells were set to 100% and data (n = 16) were analyzed using Wilcoxon rank sum test: p = 0.0004666, W = 39 (#9). b HaCaT cells were transduced with lentiviruses containing MYPOP-specific shRNAs. Knockdown efficiency (upper panel) and relative luciferase activity (lower panel) were measured as in a. Control shRNA-treated cells were set to 100% and data (n = 8) were analyzed using Welch two-tailed t-test: p = 0.0003732, t = −6.2553, dF = 7.216 (#1), p = 1.792 × 10⁻⁸, t = −19.682, dF = 8.6327 (#2), p = 1.893 × 10⁻⁵, t = −9.4527, dF = 7.5528 (#4). c Western blot analysis of MYPOP expression levels in HaCaT and NHEK cells (upper panel). HaCaT and NHEK cells were infected with HPV16 LCR PsV. Luciferase activity as a measure of infectivity was assessed 24 h later and normalized by LDH measurement. HaCaT cells were set to 100% and data (n = 11) were analyzed using Wilcoxon rank sum test p = 2.835 × 10⁻⁶, W = 121. d, e MYPOP in HPV16 early gene expression. d SCC-13 cells were co-transfected with re-circularized HPV16 wt (114/B) and FLAG-MYPOP or empty FLAG vector. After 48 h of transfection, total cellular RNA was isolated and analyzed for HPV16 E6*I spliced early transcripts. Control-transfected cells were set to 100% and data (n = 6) were analyzed using two-tailed unpaired t-test: p = 1.067 × 10⁻⁷, t = 13.348, dF = 10 (E6*I). e Experiments were performed as described for panel d, but analyzed for HPV16 E1^E4 spliced early transcripts. Control-transfected cells were set to 100% and data (n = 6) were analyzed using two-tailed unpaired t-test: p = 7.417 × 10⁻⁶, t = 8.4307, dF = 10 (E1^E4). The values obtained from three (or two for b) independent experiments are given as boxplots (a–c lower panel, d, e). Detection of endogenous MYPOP was performed using polyclonal MYPOP antibody (Abcam) (a–c). Due to clarity and conciseness the western blot images are cropped (a–c upper panel). The lower panel shows β-actin as a loading control (a, c). ***p ≤ 0.001 (a–e)