Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Critical roles of SMYD2 lysine methyltransferase in mediating renal fibroblast activation and kidney fibrosis

doi: 10.1096/fj.202000554RRR

Figure Lengend Snippet: Blocking SMYD2 with AZ505 inhibits TGF-β1-induced activation of renal interstitial fibroblasts. Starved NRK-49F cells were cultured in the DMEM with TGFβ1 (2 ng/mL) in the presence or absence of AZ505 at the doses as indicated for 36 hours (A-D) or Serum–starved NRK-49F cells were transfected with siRNA targeting SMYD2 or scrambled siRNA and then, incubated in 2 ng/mL TGFβ1 for an additional 24 hours (E-H). Cell lysates were prepared for immunoblot analysis with antibodies against fibronectin, collagen 1, α-SMA, SMYD2, H3K36me3, Histone H3, or GAPDH (A, B, E, F). The levels of fibronectin, collagen I and α-SMA (C, G), as well as SMYD2 (D, H) were quantified by densitometry and normalized with GAPDH. H3K36me3 levels were normalized with Histone H3 (D, H). Values are the means ± SDs of at least three independent experiments. Bars with different letters (a-c) for each molecule are significantly different from one another (P < .05)

Article Snippet: Cell culture and treatment Rat renal interstitial fibroblasts (NRK-49F) were purchased from the ATCC (Manassas, VA) and cultured in DMEM with F12 containing 5% FBS and 0.5% penicillin and streptomycin in an atmosphere of 5% CO 2 and 95% ambient air at 37°C.

Techniques: Blocking Assay, Activation Assay, Cell Culture, Transfection, Incubation, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Critical roles of SMYD2 lysine methyltransferase in mediating renal fibroblast activation and kidney fibrosis

doi: 10.1096/fj.202000554RRR

Figure Lengend Snippet: AZ505 inhibits cell proliferation in the kidney after UUO injury and in the culture of renal interstitial fibroblasts. A, Photomicrographs illustrating the kidney tissue stained with DAPI and antibodies against α-SMA and proliferating cell nuclear antigen (PCNA) (magnification ×200). B, The α-SMA (+)/PCNA (+) cells in the interstitium were calculated in ten high-power fields and expressed as means ± SDs. White arrows indicate PCNA-positive myofibroblasts. Kidney tissue lysates were subjected to immunoblot analysis with antibodies against PCNA or GAPDH (D). Expression levels of PCNA were quantified by densitometry and normalized with GAPDH (C). NRK-49F cells were cultured in the DMEM with 5% FBS (E, G) or 2 ng/mL TGFβ1 (F, H) in the presence or absence of AZ505 at the doses as indicated for 36 hours. Cell lysates were subjected to immunoblot analysis with antibodies against PCNA, cyclin D1, p21, or GAPDH (E, F). Expression levels of PCNA, cyclin D1, p21, or GAPDH were quantified by densitometry, and the levels of PCNA, cyclin D1, p21 were normalized with GAPDH (G, H). Values are the means ± SDs of at least three independent experiments. Means with different letters (a-d) are significantly different from one another (P < .05)

Article Snippet: Cell culture and treatment Rat renal interstitial fibroblasts (NRK-49F) were purchased from the ATCC (Manassas, VA) and cultured in DMEM with F12 containing 5% FBS and 0.5% penicillin and streptomycin in an atmosphere of 5% CO 2 and 95% ambient air at 37°C.

Techniques: Staining, Western Blot, Expressing, Cell Culture

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Critical roles of SMYD2 lysine methyltransferase in mediating renal fibroblast activation and kidney fibrosis

doi: 10.1096/fj.202000554RRR

Figure Lengend Snippet: AZ505 inhibits Smad3 phosphorylation and restores Smad-7 expression in the kidney after UUO injury and in the culture of renal interstitial fibroblasts exposed to TGFβ1. Kidney tissue (A-D) or cell lysates (E-G) were subjected to immunoblot analysis with antibodies against Phospho-Smad3, Smad3, Smad7, or GAPDH (A, E). Expression levels of Phospho-Smad3 (B, F), Smad3 (C, G), Smad7 (D, H), or GAPDH were quantified by densitometry, and normalized with GAPDH. Values are the means ± SDs of at least three independent experiments. Means with different letters (a-e) are significantly different from one another (P < .05)

Article Snippet: Cell culture and treatment Rat renal interstitial fibroblasts (NRK-49F) were purchased from the ATCC (Manassas, VA) and cultured in DMEM with F12 containing 5% FBS and 0.5% penicillin and streptomycin in an atmosphere of 5% CO 2 and 95% ambient air at 37°C.

Techniques: Phospho-proteomics, Expressing, Western Blot