Fig 1: ING3 and EPDR1 knockdown increases adipogenic differentiation of hMSCs. a ING3 and EPDR1 siRNA transfected cells were stimulated with adipogenic differentiation medium for 21 days and stained with Oil Red O for visualization of lipid droplets. Representative results of two hMSC donor lines are shown (scale bar = 200 μm). b Relative number of lipid droplets (>25 nm diameter) from 100 different microscopic fields were enumerated from three different experiments (two donor lines) and presented as % adipogenesis compared to control samples. c, d Quantitative gene expression. Gray columns = No differentiation; Black columns = Adipogenic differentiation for 1 week (Adipo). Columns = mean. Error bars = standard deviation. n = 3 unique donor lines. *p < 0.05 comparing no treatment to adipogenic differentiation for each siRNA. #p < 0.05 comparing control siRNA to siRNA for gene of interest (two-way homoscedastic Student’s t-tests). Source data are provided as a Source Data file
Fig 2: RT‐qPCR of clustered regularly interspaced short‐palindromic repeat (CRISPR)‐edited hFOB1.19‐derived RNA reveals EPDR1 mRNA levels increase when normalized to GAPDH expression and permissive control. Three biological replicates are shown for each condition. All samples are normalized to control (empty vector) at permissive (33.5°C) growth. This normalization reveals a large increase (up to 16‐fold) in EPDR1 mRNA levels when hFOB1.19 cells differentiate (39.5°C) but levels return to basal levels when the proxy‐SNPs contain region is deleted by CRISPR‐cas9. p Values (t test): # = empty vector‐33.5°C versus all, * = CRISPR pool‐33.5°C versus CRISPR pool‐39.5°C, ^ = empty vector‐39.5°C versus CRISPR pool‐39.5°C, all marked samples have a p < 0.04.
Fig 3: Western Immunoblotting reveals a decrease in EPDR1 protein levels specific to clustered regularly interspaced short‐palindromic repeat (CRISPR)‐edited hFOB1.19 cells differentiated for 7 days. Three biological replicates are shown for each condition. (A) Western immunoblotting detected bands for all three isotypes of EPDR1 (25 kDa) with a decrease in EPDR1 band intensities in differentiated CRISPR‐edited hFOB1.19 samples. Equal loading was verified with the housekeeping α‐tubulin antibody (55 kDa). (B) Quantification of the upper band (U) and middle band (M) that are present across all samples shows a decrease of approximately 90% on average for both U and M bands when compared with controls (empty vector).
Fig 4: Validation of elevated levels of resident lysosomal proteins in whole‐cell extracts. (a) Immunoblot of ARSA (arylsulfatase A) in total cell homogenates from control or palbociclib‐treated (palbo) SK‐MEL‐103, treated or not with ammonium chloride and leupeptin (N/L) 16 h before cell lysis, as indicated. Quantification of three biological independent replicates (n = 3) (top) and representative immunoblot (bottom). ***p < 0.001, **p < 0.01, *p < 0.05, 2‐way ANOVA test. (b) Immunoblot of NAGLU (N‐acetyl‐alpha‐glucosaminidase), same as in panel (a). (c) Immunoblot of EPDR1 (ependymin related 1), same as in panel (a). (d) Immunoblot of CTSF (cathepsin F), same as in panel (a). (e) Immunoblot of HLA‐DMB (beta subunit of MHC‐II DM), same as in panel (a). (f) Immunoblot of IMR90‐ER:RAS from control and senescent cells. Senescence was induced by irradiation (20 Gy, 2 weeks) or by addition of tamoxifen (1 μM, 3 weeks).
Fig 5: Knockdowns of ING3 and EPDR1 impair osteoblast differentiation. ING3 knockdown at the GWAS implicated CPED1 locus results in a complete disruption of alkaline phosphatase induction and Alizarin red S staining, but there is no effect of CPED1 and WNT16 knockdown. Similarly, EPDR1 disruption at STARD3NL results in a reduction in alkaline phosphatase expression and activity and alizarin red S staining, but there is no effect of SFRP4 knockdown. a–c, g–i Quantitative gene expression. Gray columns = No BMP treatment; Black columns = BMP treatment. Columns = mean. Error bars = Standard deviation. n = 4 (for ING3, CPED1, and WNT16 datasets) and n = 3 (for SFRP4 and EPDR1 datasets) unique donor lines. *p < 0.05 comparing No treatment to BMP treatment for each siRNA. #p < 0.05 comparing control siRNA to siRNA for gene of interest (two-way homoscedastic Student’s t-tests). d, j Representative AlkPhos (purple) and Alizarin (red) stained plates, and e, f, k, and l quantitative image analysis of staining results repeated with four different independent hMSC donor cell lines. Source data are provided as a Source Data file
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