Journal: The American Journal of Pathology
Article Title: Matrix Gla Protein Expression in Pericytes and Myofibroblasts Contributes to Renal Fibrosis
doi: 10.1016/j.ajpath.2025.12.003
Figure Lengend Snippet: Pericytes express Mgp in the kidney. A: Scheme represents Rosa Tomato/+ ;Mgp-Cre mice generation. Cre recombinase expression following the Mgp gene expression deleted the stop cassette and expressed red fluorescent protein (RFP) fluorescence. B: Fluorescence microscopy showing the endogenous RFP expression in tubular interstitial cells of control ( Rosa Tomato/+ ) and Rosa Tomato/+ ;Mgp-Cre kidneys. Nuclei were stained with Hoechst (blue). Bottom panels: Magnified images of yellow dotted boxed areas are presented. C: Mgp target sequence, the sequence of the guide RNA (blue), and single-stranded oligodeoxynucleotide (ssODN) to introduce the desired insertion. The ssODN carries homology arms flanking each side of the target nucleotide with hemagglutinin (HA) sequences ( yellow boxed area ). As shown, the guide RNA anneals on the stop codon (TAG) of Mgp ( red boxed area ), complementary to the protospacer adjacent motif ( green circle ), where co-injected clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is expected to generate a double-stranded break. D: Left: A representative agarose gel image of genotyping PCR using HA/F3 and HA/R3 primers. Wild-type (WT) Mgp allele produces a 297-bp fragment, and HA- Mgp allele produces a 324-bp fragment. Right: Sanger sequencing analyses confirm the insertion of the HA sequence (yellow) before the stop codon (red). E: Confocal microscopy showing matrix Gla protein (MGP)–expressing cells in a healthy kidney. Tissues were stained using rabbit anti-CD31 or rabbit anti–platelet-derived growth factor receptor (PDGFR)-β with mouse anti-HA antibody, followed by anti-rabbit Alexa 555 (green or white) and anti-mouse Alexa 647 (red) antibodies. Nuclei were stained with Hoechst (blue). Right: Magnified images of yellow dotted boxed areas are presented. Scale bars: 10 μm ( B , bottom panels, and E , left ); 20 μm ( B , top panels ); 2 μm ( E , right ). IRES, internal ribosome entry site.
Article Snippet: The following primary antibodies were used: anti-HA tag antibody (1:50; 2367; Cell Signaling Technology, Danvers, MA), anti-CD31 (1:200; 77699; Cell Signaling Technology), anti–platelet-derived growth factor receptor β (1:50; 3169; Cell Signaling Technology), and anti–α smooth muscle actin (SMA) (1:200; 19245; Cell Signaling Technology).
Techniques: Expressing, Gene Expression, Fluorescence, Microscopy, Control, Staining, Sequencing, Introduce, Injection, CRISPR, Agarose Gel Electrophoresis, Confocal Microscopy, Derivative Assay