Fig. 2: SPT6 is necessary for human epidermal differentiation. | Nature Communications

Fig. 2: SPT6 is necessary for human epidermal differentiation.

From: SPT6 promotes epidermal differentiation and blockade of an intestinal-like phenotype through control of transcriptional elongation

Fig. 2

a Eight elongation factors were knocked down greater than 75% by siRNAs. Knockdown cells were placed in high confluence and calcium for 3 days to induce differentiation. RT-qPCR was used to quantify the knockdown levels of each elongation factor shown on the X-axis. Two separate siRNAs (SPT6-1i and SPT6-2i) targeting different regions of SPT6 were used. qPCR values were normalized to L32. n = 3 independent experiments. b Western blot for knockdown of SPT5 and ELL. N = 3 independent experiments which all showed similar results. c, d RT-qPCR quantifying the relative mRNA expression levels of epidermal differentiation genes TGM1 and GRHL1 in differentiated cells as described in a. n = 3 independent experiments. e Immunofluorescent staining of late differentiation markers FLG (red) and LOR (green) in day 6 regenerated human epidermis treated with control (CTLi) or two distinct siRNAs targeting SPT6 (SPT6-1i and SPT6-2i). Merged image includes Hoechst staining of nuclei. n = 3 independent experiments, scale bar = 50 μm. f Immunofluorescent staining of early differentiation marker K10 (red) and proliferation marker ki67 (green) in day 6 regenerated human epidermis treated with control (CTLi) or two distinct siRNAs targeting SPT6 (SPT6-1i and SPT6-2i). Merged image includes Hoechst staining of nuclei. n = 3 independent experiments. Scale bar = 50 μm. g Quantification of the %ki67 positive cells in the basal layer from images taken in f. n = 3 independent experiments. At least 100 basal layer cells were quantitated per experiment. h Hematoxylin and eosin staining of CTLi or SPT6i knockdown regenerated human epidermis (day 6). N = 3 independent experiments which all showed similar results. Scale bar = 50 μm. i Quantification of the tissue thickness from images taken from e, f. n = 3 independent experiments. j RT-qPCR quantifying the relative mRNA expression levels of epidermal differentiation genes in CTL and SPT6 knockdown regenerated human epidermis (day 6). N = 3 independent experiments. k RNA-Seq analysis of CTL and SPT6 knockdown regenerated human epidermis (day 6). N = 3 independent experiments. In total, 4275 genes were upregulated (red) and 1465 genes were downregulated (blue) upon SPT6 depletion. Heatmaps are shown in Log2 scale. l Gene ontology (GO) terms for the 4275 genes upregulated in SPT6i tissue using hypergeometric distribution. m GO terms for the 1465 genes reduced in expression upon SPT6 loss using hypergeometric distribution. n Overlap of the SPT6 knockdown gene expression signature with the differentiation signature. p value was calculated using hypergeometric distribution. Mean values are shown with error bars = SD. ****p < 0.0001 (two-sided t-test for c, d, g, i, j).

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