polyjet transfection reagent sl100688  (SignaGen)

Journal: Advanced Science

Article Title: HUWE1‐Mediated Degradation of MUTYH Facilitates DNA Damage and Mitochondrial Dysfunction to Promote Acute Kidney Injury

doi: 10.1002/advs.202412250

Figure Lengend Snippet: Overexpression of type 2 MUTYH ameliorates cisplatin‐induced apoptosis, DNA damage, and mitochondrial dysfunction in vitro. a) After transfection of mPTCs with type 1 or 2 human MUTYH, subcellular localization of human MUTYH were determined by immunofluorescence staining for anti‐FLAG. FLAG tag was added at the C‐terminus of the MUTYH protein. (Green: FLAG, red: Mitotracker, blue: DAPI; magnification: 630×, scale bar: 10 µm). b) Flow cytometry analysis of annexin V and PI staining and quantification of apoptosis in cisplatin‐treated (24 h) mPTCs after overexpression of type 1 or 2 human MUTYH. c) The protein levels of MUTYH and cleaved caspase3 in mPTCs overexpressing type 1 or 2 MUTYH induced by cisplatin were analyzed by Western blot. The graph on the right displays the results of the densitometry analysis. d) Representative images of the comet tail moment induced by cisplatin after type 1 or 2 human MUTYH overexpression in mPTCs. e) Fluorescent images of TMRM in MUTYH overexpressing mPTCs treated with cisplatin for 24 h. f) OCR in type 1 or type 2 MUTYH overexpression mPTCs measured using a Seahorse 96 XF‐analyzer. Cell experiments were performed three times, and data are expressed as mean ± S.D. ( n = 3). ctrl: control, Cis: cisplatin; ***P < 0.001, **P < 0.01, *P < 0.05 (one‐ or two‐way ANOVA).

Article Snippet: The sequenced CRISPR/Cas9 plasmids targeting MUTYH or HUWE1 were transfected into mPTCs with PolyJet DNA transfection reagent (SignaGen, SL100688), and positive cells were selected using puromycin (2 μg mL −1 ) for 3 days prior to clonal expansion.

Techniques: Over Expression, In Vitro, Transfection, Immunofluorescence, Staining, FLAG-tag, Flow Cytometry, Western Blot, Control