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. 2021 Mar 17;40(8):e106276. doi: 10.15252/embj.2020106276

Figure 3. Pvt1 regulates epidermal stemness and tissue homeostasis.

Figure 3

  1. Profiling of RNA methylome by m6A‐seq shows significant changes of lncRNA modifications upon epidermal differentiation in vitro.
  2. Mutations in the predicted m6A modification sites can significantly reduce Pvt1 methylation. n = 3. Error bar represents s.d.
  3. Deletion of endogenous Pvt1 by an inducible Cas9 (iCas9) system can significantly reduce Pvt1 RNA level (quantification from RT–PCR, left panel) and MYC protein level (quantification from immunoblot, right panel) expression. n = 3, P < 0.01 (Student’s t‐test). Error bar represents s.d.
  4. CFE of WT, Pvt1‐inducible KO, and Pvt1‐inducible KO cells rescued with WT or mutant Pvt1 was quantified and presented as bar graph. n = 3, **P < 0.01 (Student’s t‐test). Error bar represents s.d.
  5. Skin organoids derived from WT or Pvt1‐inducible KO cells were grafted to nude mice. The regenerated skin was analyzed by H/E staining.
  6. Epidermal thickness of WT and Pvt1 KO skin grafts was quantified and presented as box and whisker plots. The plot indicates the mean (open circle within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles), and minimum and maximum measurements (solid squares). n = 6 (biological repeats), P < 0.05 (Student’s t‐test). n = 18, P < 0.01 (Student’s t‐test).
  7. Number of p63‐positive cells in WT and cKO skin was quantified and shown as bar graph. n = 5, P < 0.01 (Student’s t‐test). Error bar represents s.d.
  8. Fluorescence microscopy demonstrates different survival capability of WT and Pvt1‐inducible KO cells with or without Doxycycline (Dox) treatment.
  9. Ratio of WT and Pvt1‐inducible KO cells in the co‐culture model was quantified and shown as dot plots. n = 8, P < 0.01 (Student’s t‐test) for KO cells with Dox treatment compared with WT cells or KO cells without Dox stimulation at both Days 7 and 14. Error bar represents s.d.