Fig 2: CLDN1 participates in cell cycle regulation. Overexpression of CLDN1 decreased the proportion of cells in S phase and increased the proportion of cells in G1 phase in (A and B) SiHa (P=0.0085) and (C and D) ME-180/shp63 (P=0.0015) cells, as detected by flow cytometry with PI staining. (E and F) CLDN1 knockdown in SiHa/p63 cells increased the proportion of cells in S phase and decreased the proportion of cells in G1 phase (P=0.0005). Data are expressed as the mean ± SD in triplicates and analyzed using a two-tailed unpaired Student's t-test from three independent experiments. CLDN1, claudin 1; Con, control; sh, short hairpin.

Fig 3: Loss of Foxa1 drives prostatic cell-plasticity. Paraffin sections from PtenΔP and Pten; Foxa1; Foxp1ΔP prostates were stained with antibodies. (A,B) Immunofluorescence staining for p-Akt (green) and Foxa1 (red). White arrowheads mark Foxa1 and pAkt double positive cells (n = 3). (C) IHC staining for basal cell marker p63. Red dotted line marks abnormal areas positive for basal cells in the prostatic lumen (n = 5). (D) Immunofluorescence staining for Ck5 (basal marker; green) and Foxa1 (red). White arrows mark basal cells towards the stroma in Pten-deficient samples. White arrowheads marks Ck5-positive and Foxa1-negative cells in the lumen of Pten; Foxa1; Foxp1-deficient anterior prostate lobes (n = 3). (E,F) Immunofluorescence staining for Ck5 (basal marker; red) and Ck8 (luminal marker; green). White arrowheads mark Ck5-positive and Ck8-negative cells in the prostatic lumen (n = 3). Panels on the left are taken with 10× magnification and panels on the right are taken with 40× magnification.

Fig 4: (A) Hematoxylin and eosin (H&E) staining of the nasopharyngeal tumor specimen under ×100 magnification. (B) Hematoxylin and eosin (H&E) staining of the nasopharyngeal tumor specimen under ×200 magnification. The tumor mass is immunopositive for (C) CD56, (D) creatine kinase (CK), (E) Ki67, and (F) synaptophysin (SYN; ×200 magnification); it is immunonegative for (F) chromogranin A (CgA), (G) EBER, (H) p40, and (I) p63.

Fig 5: CRISPR generated loss of Foxp1 and Pten in the murine prostate. (A) AAV constructs for expression of sgRNAs to Pten or Pten and Foxp1 together with expression of Cre protein were generated. (B) Validation of the AAV particles were conducted in MEF isolated from Cas9-EGFPflox/flox mice. The expression of EGFP revealed viral transduction and Cre expression from the viral construct. Images were taken with 10× magnification (C) The efficiency of the sgRNA was determined with Sanger Sequencing and analysed with Synthego ICE software. (D) The AAV particles were delivered to the murine prostate with surgical injections and prostate tissues were examined 4 months post-treatment. White arrowheads mark transformed areas in the prostatic tissues (representative image, n = 5). (E) Paraffin sections from the prostates were stained for H & E (arrows mark PIN and arrowheads mark normal prostatic epithelial), p-Akt, Foxp1 and p63 (brown stain) to confirm PIN formation and disruption to the Pten pathway and loss of Foxp1 in the Cas9-EGFP transgene mice (n = 5). Arrows mark cells depleted for Foxp1 and arrowheads mark cells positive for Foxp1 or p63. (F) Indel frequency was measured on EGFP-positive biopics from the anterior prostate lobe. Sanger Sequencing data were analysed with Synthego ICE software (n = 4–5).