Fig 1: Sleeping Beauty transposon insertion profile predicts Srgap2 is a tumor suppressor.Arrows denote transposon insertion sites, which point in the direction of the promoter. Three animals had multiple insertions in Srgap2, represented by the red open arrows, the blue shaded arrows, and the solid orange arrows. All remaining insertion sites, represented by solid purple arrows belong to different animals. The insertion profile predicts Srgap2 is a tumor suppressor due to the unbiased transposon insertion throughout the gene without a promoter direction preference.
Fig 2: The SRGAP2a expressions and actin cytoskeleton of podocytes following HG and HG plus klotho treatment. A The immunofluorescence staining of SRGAP2a with HG and HG plus klotho treatment. B The qRT-PCR results showing SRGAP2a mRNA expressions with HG and HG plus klotho treatment. C The western blotting results showing SRGAP2a protein expressions with HG and HG plus klotho treatment. D The quantitative analysis of the SRGAP2a protein levels in C. E The fluorescein-conjugated phalloidin staining of podocytes with HG and HG plus klotho treatment. F The quantitative analysis of the mean actin content per cell in E. All data were mean ± SD, (n = 6); *p < 0.05, **p < 0.01. Bars = 50 μm in A, 5 μm in E. NC, negative controls without additional treatment
Fig 3: SRGAP2 expression affects in vitro proliferation and migration but not anchorage independent growth in murine cell lines.(a) MTS assay was used to evaluate cellular proliferation. Readings were taken at 24, 48, 72, and 96 hours of incubation. A slowed cellular proliferation was observed in K12 and K7M2 KO cell lines. Each point shows mean ± SEM (n = 18). (b) Wound-healing assay was used to evaluate cellular migration. Overexpression of SRGAP2 suppressed cellular migration in both K12 and K7M2 cell lines. Knockout of Srgap2 enhanced cellular migration in K12 but not K7M2 cell lines. Bar graphs show mean ± SEM (n ≥ 16). (c) Soft agar colony formation assay was used to evaluate anchorage independent growth. SRGAP2 expression did not influence anchorage independent growth of K12 cell lines, whereas SRGAP2 overexpression decreased the K7M2 cell line’s ability for anchorage independent growth. Bar graphs show mean ± SEM (n = 36). *p-value < 0.05, **p-value < 0.01 ****p-value < 0.0001 (two-tailed, unpaired t-test).
Fig 4: The HE staining A and Nephrin, Synaptopodin and SRGAP2a immunochemistry B of the kidneys from the modelled DN and DN plus klotho rats. Quantitative analysis of the average optical density of Nephrin C, Synaptopodin D and SRGAP2a E by immunohistochemistry. Bars = 200 μm. All data were mean ± SD, (n = 6); *p < 0.05, **p < 0.01
Fig 5: SRGAP2 mRNA xpression levels in K12 and K7M2 cell lines by quantitative RT-PCR and protein quantification by Western blotting.(a,b) SRGAP2 mRNA levels were quantified by RT-PCR. The expression of SRGAP2 mRNA is >2 fold overexpressed in cell lines with the addition of doxycycline. Expression of Srgap2 mRNA is (a) halved in the K12 KO and (b) not detectable in the K7M2 KO. Bar graph shows mean ± SEM (n = 3). (c,d) Western blotting showing the addition of doxycycline to culture media induces expression of human SRGAP2 protein in the (c) K12 and (d) K7M2 OE cell lines. The antibody does not bind endogenous murine SRGAP2 protein. Four bands are observed: SRGAP2 (140 KDa), beta-actin control (42 KDa), and a two unknown bands (120 KDa and 100 KDa). ****p-value < 0.0001 (two-tailed, unpaired t-test).
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