Journal: bioRxiv

Article Title: GM-CSF drives immune-mediated glomerular disease by licensing monocyte-derived cells to produce MMP12

doi: 10.1101/2022.06.13.495915

Figure Lengend Snippet: (A) Unsupervised clustering and UMAP of FACS-analysis of renal leukocytes from nephritic wild-type and GM-CSF/ Cf2 −/− mice. Cell counts in the respective clusters from wildtype and GM-CSF/ Csf2 -/- animals. (B) Unsupervised clustering and UMAP dimensional reduction of scRNA-seq of renal leukocytes from wildtype and GM-CSF/ Csf2 −/− mice ten days after cGN induction. Clusters were annotated according to their gene expression profiles. Fraction of cells in the respective clusters from wildtype and GM-CSF/ Cf2 −/− animals. (C) Heatmap of key marker gene expression of the indicated clusters form scRNA-seq. (D) RT-PCR analysis of renal Mmp12 expression in wildtype and GM-CSF −/− mice. (E) Combined immunofluorescence staining of CD3 + (green), MAC2 + (turquoise), and FISH ( Mmp12 , red) of renal leukocytes in wildtype and GM-CSF/ Csf2 −/− mice. (F) Pathway analysis of differentially expressed genes of renal MdCs from nephritic wildtype and GM-CSF/ Csf2 −/− mice (color code indicating the log2-fold change in wildtype versus GM-CSF/ Csf2 −/− animals). (G) FACS sorted MdC like cells from the spleen of control mice were in vitro cultured for 24 hours with/without addition of recombinant mouse GM-CSF. Mmp12 gene expression and protein expression in the supernatant were quantified. (H) FACS sorted MdCs from kidneys of cGN mice were in vitro cultured for 24 hours with/without addition of recombinant mouse GM-CSF. Mmp12 gene expression and protein expression in the supernatant were quantified. (I) FACS sorted MdC like cells from spleens of control mice were in vitro cultured for 1 hour with/without addition of recombinant mouse GM-CSF and with addition of indicated pharmacological inhibitors. Mmp12 gene expression was quantified by RT-PCR. (J-M) Crescentic GN induction and treatment with pharmacological MMP12 inhibitor or vehicle. (J) cGN was induced in wildtype mice and MMP12 inhibitor was given by oral gavage at indicated time points (bis in die, B.I.D.). (K) Quantification of glomerular crescents, tubulointerstitial damage, and BUN levels. (L) Representative confocal micrographs depicting extent of glomerular injury in kidneys of mice with/without MMP12 inhibitor treatment. Note the strong fibrin/fibrinogen signal within the capillaries of the cGN + vehicle glomerulus, whereas msIgG and shIgG/CollagenIV signals at the glomerular filtration barrier are comparable between both groups. (M) Quantification of fibrin positive glomeruli detected by immunohistochemistry. (N) cGN was induced in wildtype mice and GM-CSF-deficient mice as well as MMP12 inhibitor was given by oral gavage at indicated time points (bis in die, B.I.D.). (O) Quantification of glomerular crescents, tubulointerstitial damage, and BUN levels. Symbols represent individual data points with the mean as a bar (* P<0.05, ** P<0.01, *** P<0.005, **** p<0.001). Data are representative of two independent experiments.

Article Snippet: For immunofluorescence, the following primary antibodies were incubated overnight in blocking buffer at 4°C: CD68 (KP1, Invitrogen, Invitrogen, Carsbad, CA), CD3 (A0452, Dako, Glostrup, Denmark), CXCR3 (1C6, BD Biosciences), Mac-2 (M3/38, Cedarlane, Burlington, Canada), CXCR3 (Bioss Antibodies, Woburn, MA), MHCII (sc-59322, Santa Cruz), Fibrin/Fibrinogen (A0080, Dako), CollagenIV (1340-01, SouthernBiotech).

Techniques: Gene Expression, Marker, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining, Control, In Vitro, Cell Culture, Recombinant, Filtration, Immunohistochemistry

Journal: Oncotarget

Article Title: Chronic obstructive sleep apnea promotes aortic remodeling in canines through miR-145/Smad3 signaling pathway

doi: 10.18632/oncotarget.17144

Figure Lengend Snippet: (A) Representative bands of CollagenI and III and ratio of these proteins to GAPDH. (B) Representative bands of TGF-β1 and MMP9, data from these proteins were normalized to GAPDH. (C) Representative bands of phospho-Smad3 and Smad3 as well as the ratio of these proteins to GAPDH. * P < 0.05, * P < 0.01, ** * P <0.001 vs. sham group, n=6 in each group.

Article Snippet: After blocking with 5% nonfat dry milk, the membranes were then incubated with anti-CollagenI (bs-0578R, Bioss, China), anti-Collagen III (bs-0549R, Bioss, China), anti-TGFβ1 (ab92486, Abcam, USA), anti-α-SMA (ab5694, Abcam, USA), anti-MMP9 (bs-4593R, Bioss, China), anti-Smad3 (#9523, Cell Signaling Technology, USA), p-Smad3 (#9520, Cell Signaling Technology, USA), anti-AIF (ab1998, Abcam, USA), anti-cleaved caspase 3 (#9664, Cell Signaling Technology, USA), anti-caspase 9 (#9508, Cell Signaling Technology, USA), anti-Bcl-2 (ab32124, Abcam, USA), anti-Bax (ab32503, Abcam, USA), anti-GAP43 (ab12274, Abcam, USA), anti-TH (ab112, Abcam, USA), anti-NLRP3 (bs-10021R, Bioss, China), anti-NF-κB p65 (10745-1-AP, Proteintech, USA), anti-p-NF-κB p65 (#3033, Cell Signaling Technology, USA), anti-CD68 (ab76308, Abcam, USA), HIF-1α (R12-2180, Assaybiotech, USA), anti-GAPDH (KC-5G4, Kangchen, China) and anti-β-actin (TA-09, Zhongshan, China) antibodies in 4°C overnight.

Techniques: