Fig 2: Expression and localization of TP63 isoforms and other selected factors associated with CE homeostasis and stratification in absence of Myc. (A, left panel) CE immunostaining of Krt12 (red) and DAPI (blue) on cross-sections of the paraffin-embedded cornea. The Abs utilized were directed to either the N- or the C-terminus of the Krt12 protein. Krt12 accumulation at the CE surface observed in the mutant using either Ab was severely reduced in the CE of the CKO. Bar graphs represent the percentage of Krt12 apical staining along the CE length of cornea cross-sections (mean ± SD, n = 3). (A, right panel) Line-intensity profile plot of Krt12 fluorescence signal across a CE cross-section. The y-axis represents the average intensity of 15 line-intensity scans (31 µm wide) from the basal membrane (BM) to the apical side of the CE taken every 100 µm along the length of each cornea section. The x-axis represents the length of the line scan from the BM to the apical side of the CE (n = 3). (B) CE immunostaining with Ab directed to the epithelial-specific ?Np63 (n = 4/group). (C) CE immunostaining with the pan (TP) p63 Abs (n = 2 control and 4 CKO). The bar graphs in B and C describe the mean ± SD values of p63 intensities (green fluorescence) over the whole epithelium, normalized by the corresponding nuclear stain (blue fluorescence). (D, left panel) GAPDH-normalized CKO/control chemiluminescence signal ratios (mean ± SD). Number of independent replicates for each protein are indicated on top of the bars. Myc ablation led to a fourfold increase of ?Np63 and about twofold increases in ETS1 and Notch 1. Krt14 and Krt 12 displayed no substantial differences in two duplicates and were not quantitated. (E) Level of expression of p63 isoforms relative to Gapdh in the wild-type mouse (mean ± SD, n = 3/group). (F) Expression of p63 isoforms and genes involved in the CE differentiation. Data are represented as the fold change in the CKO relative to the control (n = 3/group). ns, not significant (P = 0.05, Student's t-test). *P < 0.05, **P < 0.001. Scale bars: 25 µm.

Fig 3: Fibroblasts expressing BMP-2 increase epithelial thickness, restore cell polarity, increase planar cell divisions, and ordered differentiation to human keratinocytes in OCs.(A, C, E, G, H) OCs populated with control (F_GFP) or BMP-2–transduced (F_BMP2) fibroblasts were analyzed after hematoxylin and eosin staining (A) or immunostaining with Ki67 (C), ?N-p63 (E), LAMA5 (G), and K10 (H) to reveal improved basal cell polarity, increased expression of Ki67, ?N-p63, and LAMA5 and better suprabasal K10 expression in the presence of F_BMP-2 compared with F_GFP controls. Images are representative of three independent experiments. (B, D, F) Quantitation of epithelial thickness (B), number of Ki67+ (D), and ?N-p63+ (F) cells in OCs populated with control (F_GFP) or BMP-2–transduced fibroblasts (F_BMP2). Error bars represent mean ± SD; statistical analysis performed using unpaired t test; n.s. not significant; *P < 0.05; ***P < 0.00. All quantitative data are derived from three independent experiments. (I, J, L, M) Immunostaining of OCs for pH 3 (green) and ?-tubulin (red) to determine keratinocyte cell division orientation in the presence of BMP-2–transduced (I, J: F_GFP, n = 28 mitoses) and control fibroblasts (L, M: F_BMP2, n = 27 mitoses). (K, N) Radial bar charts illustrating angle of keratinocyte mitoses obtained with F_BMP2 (K) and F_GFP control (N) demonstrating increased planar divisions in the latter.

Fig 4: TP63 regulates NRG1 in SCC.(A) Correlation of TP63 and NRG1 in LUSC (n = 223) and Esophageal (n = 263) patient samples in TCGA data set. Statistical significance was determined by two-tailed test and Pearson correlation (r) was determined. (B) Relative expression of TP63, NRG1a and NRG1ß upon TP63 knockdown using siRNA in OE21 and KYSE-140 SCCs. (C) Relative expression of TP63 (all, TA and deltaN isoforms), NRG1a, NRG1ß upon TP63 knockdown using siRNA in KYSE-180 SCC. Expression of TA and deltaN TP63 and upon TP63 knockdown by siRNA. siRNAs for TA-TP63 (#13), all isoform-TP63 (#14), deltaN-TP63 [#1, #2, #3) and non-target control (NTC#3, NTC#4). Relative quantitation by qPCR in B and C is mean with standard deviation relative to dharmafect transfection reagent control. (D) ChIP analysis of p63alpha and deltaNp63 binding -30 kB from the NRG1 transcriptional start site (TSS) in KYSE-180 SCC. IgG and primers amplifying the -21 kB region were used as controls. Data is represented as average with standard error of mean from three independent experiments. One-way ANOVA test was used to determine the statistical significance in comparison to IgG. *p<0.05.

Fig 5: The C-terminus of p63 protein is required for the ability to inhibit R-mediated lytic reactivation.NOKs-Akata cells were transfected with or without an R expression vector in the presence or absence of various different co-transfected p63 isoform vectors, including the human ?Np63? protein, the mouse ?Np63? protein, the mouse ?Np63ß isoform (homologous to ?Np63? but lacking 121 amino acids at the C-terminal domain), or the mouse TAp63a isoform (which is identical to ?Np63? except for the addition of an N terminal transactivating domain). Two days after transfection, immunoblots were performed to assess expression levels of the Z, R, BMRF1, p63 protein isoforms, and tubulin as indicated. The quantification of Z protein expression was determined through ImageJ, normalized to tubulin, with the R alone condition set as 1. The black lines show where irrelevant lanes were removed; the original western blots used to construct this figure are shown in supplemental Fig 7. This experiment is representative of two independent biological replicates.