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. 2021 Nov 4;17(11):e1010032. doi: 10.1371/journal.ppat.1010032

Fig 7. Abrogation of CTCF binding to the HPV18 genome causes a significant reduction in differentiation-dependent late protein abundance.

Fig 7

(A) HPV18 genome containing keratinocytes (HPV18 or ΔCTCF-HPV18) grown in monolayer (undifferentiated, 0h) or differentiated in methylcellulose (48h) and E1^E4, involucrin (IVL) and GAPDH protein expression analysed by Western blotting. (B) Relative E1^E4 protein expression in comparison to GAPDH was quantified in three independent experiments by densitometry. Data are the mean +/- standard deviation. * denotes p<0.05. (C) E1^E4 (red) and L1 (green) protein abundance was analysed by indirect immunofluorescence in epithelia derived from HPV18 and ΔCTCF-HPV18 genome-containing keratinocytes grown in organotypic raft culture. Cellular nuclei are shown in blue, and the basal layer indicated with white arrows. Scale bar indicates 10 μm. (D) The total number of L1 positive cells per section of three independent raft cultures grown from two independent keratinocyte donors was counted. Data show the mean +/- standard deviation. *** p<0.001, **** p<0.0001. (E) E1^E4, E6 and E7, and (F) E2 protein expression in organotypic raft cultures was assessed by Western blotting lysates harvested from three independent raft cultures alongside GADPH loading control. Molecular weight markers are indicated on the left of Western blots (kDa).

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