Fig. 1.

DGCR8 silencing in hMSCs induces severe proliferation defects and cellular senescence. (A) Growth curves of hMSCs transfected with either siGFP (white dots) or siDGCR8 (black dots). Error bars indicate the standard error of the mean of at least three independent experiments (**P < 0.01, ***P < 0.001). (B) BrdU incorporation was quantified 5 days after siRNA transfection. Error bars indicate the standard error of the mean of three independent experiments (**P < 0.01). (C) Senescence-associated-β-galactosidase (SA-β-gal) staining. Compared with the siGFP-transfected group, siDGCR8-transfected hMSCs showed increased numbers of SA-β-gal-positive cells and enlarged size (left). The percentage of SA-β-gal-positive cells was significantly higher in siDGCR8-transfected hMSCs (right; ***P < 0.001). Magnification, 40 × ; scale bar, 150 μm. (D) Western blot showing the protein levels of tumour suppressors and senescence markers, including p21, p53, and p-p53 (ser15). (E) A heatmap of the canonical pathways enriched in replicatively senescent or DGCR8-depleted cells. Pathways were sorted by the activation z-score, which was calculated by total −log (P-value) from Fisher's Exact test across compared observations (observation 1, early-vs. late-passage; observation 2, siGFP- vs. siDGCR8-transfected cells). Orange (positive z-score) or blue (negative z-score) colour codes represent the activation or inhibition of the given pathway. (F) The levels of IL-8 secretion upon DGCR8 knockdown were quantified by enzyme-linked immunosorbent assay (ELISA). Error bars indicate the standard error of the mean of three independent experiments (***P < 0.001).