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. 2021 Feb 4;12:784. doi: 10.1038/s41467-021-21067-w

Fig. 4. PAF1 is necessary for differentiation by promoting transcription elongation.

Fig. 4

a Immunofluorescent staining of early differentiation markers Keratin 10 (K10: red) and Keratin 1 (K1: green) in day 6 regenerated human epidermis treated with control (CTLi) or PAF1 siRNAs (PAF1i). Merged image includes Hoechst staining of nuclei. n = 3 independent experiments. Scale bar = 50 μm. b Immunofluorescent staining of late differentiation markers FLG (red) and LOR (green) in day 6 regenerated human epidermis treated with control (CTLi) or PAF1 siRNAs (PAF1i). Merged image includes Hoechst staining of nuclei. n = 3 independent experiments, scale bar = 50 μm. c RT-qPCR quantifying the relative mRNA expression levels of epidermal differentiation genes in CTL and PAF1 knockdown regenerated human epidermis (day 6). N = 3 independent experiments, two-sided t-test. *** = 0.003 for FLG, *** = 0.0001 for HOPX, *** = 0.0001 for DSG3. ****p < 0.0001. d ChIP-QPCR of differentiation genes in differentiated primary human keratinocytes with PAF1 pulldown. IGG pulldowns were used as a negative control. All primers were targeted toward the transcription start site (TSS) of each differentiation gene. Results are shown as a percent of input. N = 3 independent experiments. e ChIP-QPCR of RNA Pol II pulldown in both CTLi and PAF1i differentiated cells. Each pulldown was normalized to its respective input and enrichment shown as a percent of input. All primers were targeted toward the TSS of each differentiation gene. N = 2 independent experiments. Mean values are shown with error bars = SD.