Fig. 1: SGF29 forms nuclear condensate in senescent cells and its overexpression promotes cellular aging in hMPCs.

a SA-β-Gal staining of WT hMPCs at early passage (EP, P5) and late passage (LP, P16). Left, representative images of SA-β-Gal staining. Scale bars, 50 μm. Right, quantitation of the relative percentages of SA-β-Gal-positive cells. Data are presented as the mean ± SEM. n = 3 biological replicates. Over 100 cells were quantified in each replicate. ***P < 0.001 (t-test). b Clonal expansion assay in WT hMPCs at EP (P5) and LP (P16). Left, representative images of crystal violet staining. Right, quantification of the relative clonal expansion ability of EP and LP hMPCs. Data are presented as the mean ± SEM. n = 3 biological replicates. ***P < 0.001 (t-test). c Immunofluorescence staining of p21^Cip1 and SGF29 in hMPCs at EP (P5) and LP (P16). Scale bars, 10 μm and 2.5 μm (zoomed-in image). d Quantification of the fluorescence intensity along the inset arrows in (c) following the arrow direction. e Correlation between the fluorescence intensity of p21^Cip1 and the number of nuclear SGF29 puncta. n = 100 hMPCs. Each dot represents one cell. The SGF29 condensate with a size greater than 0.196 μm² (area) was quantified as a SGF29 punctum. f Immunofluorescence staining of HP1α and SGF29 in hMPCs at EP (P5) and LP (P16). Scale bars, 10 μm and 2.5 μm (zoomed-in image). g Correlation between the fluorescence intensity of HP1α and the number of nuclear SGF29 puncta. n = 100 hMPCs. Each dot represents one cell. The SGF29 condensate with a size greater than 0.196 μm² (area) was quantified as a SGF29 punctum. h Western blot analysis of the abundance of SGF29 in the cytoplasmic (Cyt), nuclear soluble (Nuc) and chromatin-associated (Chr) fractions in hMPCs at EP (P5) and LP (P16). β-Tubulin, TAP1, and H4 were used as the loading control for indicated fraction, respectively. Left, representative images of western blotting. Right, quantification of the relative protein levels of SGF29 in indicated fractions. n =3 independent experiments. Data are presented as the means ± SEM. ns, not significant; *P < 0.05 (t-test). i Immunofluorescence staining of SGF29 in EP WT hMPCs (P5, young hMPCs) transduced with lentiviruses expressing either EGFP or EGFP-SGF29. The white arrowheads denote the SGF29 puncta. Scale bars, 10 μm and 2.5 μm (zoomed-in image). j Western blot analysis of p21^Cip1 in EP WT hMPCs (P5, young hMPCs) transduced with lentiviruses expressing either EGFP or EGFP-SGF29. Left, representative images of western blotting. The band of exogenous EGFP-SGF29 protein is marked with *. β-Tubulin was used as the loading control. Right, quantitation of the relative protein levels of p21^Cip1. Data are presented as the means ± SEM. n = 3 biological replicates. *P < 0.05 (t-test). k SA-β-Gal staining of EP WT hMPCs (P5, young hMPCs) transduced with lentiviruses expressing either EGFP or EGFP-SGF29. Left, representative images of SA-β-Gal staining. Scale bars, 20 μm. Right, quantitation of the relative percentages of SA-β-Gal-positive cells. Data are presented as the mean ± SEM. n = 3 biological replicates. Over 100 cells were quantified in each replicate. ***P < 0.001 (t-test). l Immunofluorescence staining of Ki67 in EP WT hMPCs (P5, young hMPCs) transduced with lentiviruses expressing either EGFP or EGFP-SGF29. Left, representative images of Ki67 immunofluorescence. The white arrowheads denote the Ki67-positive cells. Scale bars, 20 μm. Right, quantification of the relative percentages of Ki67-positive cells. Data are presented as the mean ± SEM. n = 3 biological replicates. Over 100 cells were quantified in each replicate. ***P < 0.001 (t-test). m Immunofluorescence staining of γH2A.X and 53BP1 in EP WT hMPCs (P5, young hMPCs) transduced with lentiviruses expressing either EGFP or EGFP-SGF29. Left, representative images of γH2A.X and 53BP1 immunofluorescence. The white arrowheads denote the γH2A.X and 53BP1-positive cells. Scale bars, 20 μm. Right, quantification of the relative percentages of γH2A.X and 53BP1-positive cells. Data are presented as the mean ± SEM. n = 3 biological replicates. Over 100 cells were quantified in each replicate. **P < 0.01 (t-test). n Western blot analysis of SGF29 in LP WT hMPCs (P16, aged hMPCs) after treatment with si-Control or si-SGF29. β-Tubulin was used as the loading control. o SA-β-Gal staining of LP WT hMPCs (P16, aged hMPCs) after treatment with si-Control or si-SGF29. Left, representative images of SA-β-Gal staining. Scale bars, 50 μm. Right, quantitation of the relative percentages of SA-β-Gal-positive cells. Data are presented as the mean ± SEM. n = 3 biological replicates. Over 100 cells were quantified in each replicate; ***P < 0.001 (t-test). p Immunofluorescence staining of SPIDER-βGal and SGF29 in human fibroblasts (hFib) at EP (P13) and LP (P23). Left, representative images of SPIDER-βGal and SGF29 immunofluorescence. Right, quantification of the fluorescence intensity along the line embedded in the zoomed-in images following the arrow direction. Scale bars, 10 μm and 2.5 μm (zoomed-in image). q Quantification of the number of SGF29 puncta in human fibroblasts at EP (P13) and LP (P23) in Fig. 1p. n = 80 hFib cells. Data are shown as means ± SEM. ***P < 0.001 (t-test).