Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage–myofibroblast transition

doi: 10.1073/pnas.1917663117

Figure Lengend Snippet: Pou4f1 is expressed by MMT cells in human kidney disease. (A) Confocal microscopy shows that CD68+ macrophages (red) and α-SMA+ myofibroblasts (green) are distinct cell populations in cases of end-stage renal disease (ESRD) and diabetic kidney disease (DKD). By contrast, many MMT cells coexpressing CD68 and α-SMA (yellow) can be seen in an area of active fibrosis in a case of chronic allograft dysfunction (CAD). (B) Opal multiplex IHC system identified that most CD68+ macrophages express Pou4f1 (purple) in an area of active fibrosis in CAD, including Pou4f1 expression by many α-SMA+CD68+ MMT cells (white). This is more clearly seen in the 3D illustration. (C) Graphs show quantification of staining from a cohort of 10 cases of kidney disease. A significant correlation is evident between Pou4f1+ macrophages and the contribution of the MMT cells in total α-SMA+ myofibroblasts. (C) *P < 0.05, **P < 0.01, ***P < 0.001 vs. ESRD; ##P < 0.001, ###P < 0.001 vs. ESRD. (Scale bars, 100 μm.)

Article Snippet: In brief, sections were labeled overnight with various combinations of directly conjugated primary antibodies as follows: fluorescein isothiocyanate (FITC)-conjugated anti-CD68 antibody (bs-0649R-FITC, Bioss), FITC-conjugated anti-F4/80 (11-4801-81, eBioscience), Cy3-conjugated α-SMA antibody (C6198, Sigma), or Pou4f1 (sc-8429, Santa Cruz) conjugated with Pacific Blue ( {"type":"entrez-protein","attrs":{"text":"P30013","term_id":"20141226","term_text":"P30013"}} P30013 , Invitrogen) or PE-conjugated Pou4f1 (sc-8429 PE, Santa Cruz).

Techniques: Confocal Microscopy, Multiplex Assay, Expressing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage–myofibroblast transition

doi: 10.1073/pnas.1917663117

Figure Lengend Snippet: A Pou4f1-dependent fibrogenic gene network at an early stage of MMT. (A) TGF-β1 (5 ng/mL) induced Pou4f1 transcription in BMDM at day 1 which rapidly declined to baseline as shown by real-time PCR. (B) Western blot shows a peak of Pou4f1 protein expression on day 1 after TGF-β1 stimulation of BMDMs which preceded the peak expression of fibrosis markers α-SMA and collagen I on day 5. siRNA-mediated silencing of Pou4f1 (siPou4f1) inhibited TGF-β1-driven MMT on day 5 in BMDMs compared to the nonsense siRNA control (NC) in terms of (C) a reduction in α-SMA+CD68+ MMT cells via immunofluorescence microscopy, and (D) a reduction in the fibrosis markers α-SMA and collagen I via Western blotting. For microarray analysis, siPou4f1 or NC treated BMDMs, with or without TGF-β1 stimulation, were collected at 24 h. The transcriptome similarity was clearly distinguishable in each group containing a pool of three independent replicates shown by (E) heatmap, and (F) a 3D principal component analysis (PCA) plot. (G) A total of 215 MMT-dependent genes were identified at 24 h of TGF-β1-driven MMT, whereas 51 genes were regulated in a Pou4f1-dependent fashion in BMDMs as shown in the Venn diagram. (H) The Pou4f1-dependent genes formed a regulatory network containing a number of fibrogenic effectors (red) by STRING network analysis. Data represent three independent in vitro experiments. (A) **P < 0.01 vs. control. (Scale bars, 20 μm.)

Article Snippet: In brief, sections were labeled overnight with various combinations of directly conjugated primary antibodies as follows: fluorescein isothiocyanate (FITC)-conjugated anti-CD68 antibody (bs-0649R-FITC, Bioss), FITC-conjugated anti-F4/80 (11-4801-81, eBioscience), Cy3-conjugated α-SMA antibody (C6198, Sigma), or Pou4f1 (sc-8429, Santa Cruz) conjugated with Pacific Blue ( {"type":"entrez-protein","attrs":{"text":"P30013","term_id":"20141226","term_text":"P30013"}} P30013 , Invitrogen) or PE-conjugated Pou4f1 (sc-8429 PE, Santa Cruz).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Microscopy, Microarray, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage–myofibroblast transition

doi: 10.1073/pnas.1917663117

Figure Lengend Snippet: Silencing of Pou4f1 in BMDM inhibits MMT in an adoptive transfer version of the UUO model. A day 5 UUO model was performed in LysM-Cre/DTR mice. Mice were treated with diptheria toxin (DT) for 3 d before UUO surgery to deplete macrophages. At 6 h after UUO surgery, mice were injected with either Pou4f1-knockdown (siPou4f1) or nonsense siRNA-treated (NC) BMDMs (2 × 106 cells/mouse) which had a 24-h stimulation with TGF-β1 (5 ng/mL) in culture prior to injection. (A) Immunostaining for Pou4f1 (Upper) and confocal microscopy for α-SMA (green) and F4/80 (red) (power panel), show that compared to the UUO control, DT treatment depleted both F4/80+ macrophages and Pou4f1+ interstitial cells. Both populations were restored by adoptive transfer with NC-BMDMs, while transfer of siPou4f1-BMDMs restored the F4/80+ macrophage population but not the interstitial Pou4f1+ cells on day 5 UUO. (B) Flow cytometry analysis shows that DT-induced macrophage depletion reduced both the total α-SMA+ myofibroblast population and SMA+CD68+ MMT cells. While NC-BMDM transfer restored both of these populations, the transfer of siPou4f1-BMDMs failed to achieve this. Data represent results from six mice/group. **P < 0.01, ***P < 0.001 vs. sham control; ##P < 0.01, ###P < 0.001 vs. UUO; @@P < 0.01, @@@P < 0.001 vs. adoptive transfer of NC-BMDMs under macrophage depletion (NC-BMDM). (Scale bars, 50 μm.)

Article Snippet: In brief, sections were labeled overnight with various combinations of directly conjugated primary antibodies as follows: fluorescein isothiocyanate (FITC)-conjugated anti-CD68 antibody (bs-0649R-FITC, Bioss), FITC-conjugated anti-F4/80 (11-4801-81, eBioscience), Cy3-conjugated α-SMA antibody (C6198, Sigma), or Pou4f1 (sc-8429, Santa Cruz) conjugated with Pacific Blue ( {"type":"entrez-protein","attrs":{"text":"P30013","term_id":"20141226","term_text":"P30013"}} P30013 , Invitrogen) or PE-conjugated Pou4f1 (sc-8429 PE, Santa Cruz).

Techniques: Adoptive Transfer Assay, Injection, Immunostaining, Confocal Microscopy, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage–myofibroblast transition

doi: 10.1073/pnas.1917663117

Figure Lengend Snippet: Silencing of Pou4f1 in BMDMs inhibits MMT in an adoptive transfer version of the IRI model. A day 7 IRI model was performed in LysM-Cre/DTR mice. Mice were treated with DT for 3 d before IRI surgery to deplete macrophages. At 24 h after IRI surgery, mice were injected with either Pou4f1-knockdown (siPou4f1) or nonsense siRNA treated (NC) BMDM (2 × 106 cells/mouse) which had a 24-h stimulation with TGF-β1 (5 ng/mL) in culture prior to injection. (A) Immunohistochemistry staining for Pou4f1 (Upper) and Col-I (Middle), and confocal microscopy for α-SMA (green) and F4/80 (red) (power panel). Compared to the IRI control, DT treatment reduced F4/80+ macrophages, α-SMA+ myofibroblasts, Pou4f1+ interstitial cells, and interstitial collagen I deposition. Adoptive transfer of NC-BMDMs restored all of these populations and collagen I deposition in the IRI model. However, transfer of siPou4f1-BMDMs restored only the F4/80+ macrophage population. (B) Flow cytometry analysis, and (C) real-time PCR show that DT-induced macrophage depletion reduced both total α-SMA expression and α-SMA+CD68+ MMT cells. Transfer of NC-BMDMs, but not siPou4f1-BMDMs, restored both α-SMA expression and α-SMA+CD68+ MMT cells. Data represent results of six mice/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham control; #P < 0.05, ###P < 0.001 vs. IRI; ^^^0.001 vs. macrophage depletion (DT); @@P < 0.01, @@@0.001 vs. adoptive transfer of NC-BMDMs under macrophage depletion (NC-BMDM). (Scale bars, 50 μm.)

Article Snippet: In brief, sections were labeled overnight with various combinations of directly conjugated primary antibodies as follows: fluorescein isothiocyanate (FITC)-conjugated anti-CD68 antibody (bs-0649R-FITC, Bioss), FITC-conjugated anti-F4/80 (11-4801-81, eBioscience), Cy3-conjugated α-SMA antibody (C6198, Sigma), or Pou4f1 (sc-8429, Santa Cruz) conjugated with Pacific Blue ( {"type":"entrez-protein","attrs":{"text":"P30013","term_id":"20141226","term_text":"P30013"}} P30013 , Invitrogen) or PE-conjugated Pou4f1 (sc-8429 PE, Santa Cruz).

Techniques: Adoptive Transfer Assay, Injection, Immunohistochemistry, Staining, Confocal Microscopy, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing

Journal: Nutrients

Article Title: A Mitochondrial Specific Antioxidant Reverses Metabolic Dysfunction and Fatty Liver Induced by Maternal Cigarette Smoke in Mice

doi: 10.3390/nu11071669

Figure Lengend Snippet: Markers of liver inflammation TNF-α, IL-1β ( a,b ), macrophage markers MCP-1, CD68 and F4/80 ( c–e ), stellate cell activation marker α- SMA ( f ) and fibrous deposition of collagen 1a1, collagen III, and total collagen ( g–i ) in 13 weeks old male offspring. Results are expressed as mean ± SEM ( n = 4–8). * p < 0.05, ** p < 0.01. Representative images of hepatic CD68 ( j , positive staining indicated by arrows) and collagen III ( k , positive staining indicated by arrows) immunohistochemistry staining in 13 weeks old male offspring, bar = 50 µm. ( l ) Representative images of SHG showing total collagen staining after gamma correction (collagen is green/yellow), bar = 1000 µm. α-SMA: alpha-smooth muscle actin, CD68: cluster of differentiation 68, F4/80: EGF-like module-containing mucin-like hormone receptor-like 1, IL-1β: interleukin 1 beta, MCP-1: monocyte chemoattractant protein 1, SE: cigarette smoke exposure, SE + MQ: SE with MitoQ supplementation, SHG: second harmonic generation, TNF-α: tumour necrosis factor-α.

Article Snippet: Antigen retrieval was performed [ ] before the sections were incubated with anti-rabbit collagen III (1:50, Cat# NB600-594, Novus Biotechnology, Centennial, CO, USA) or cluster of differentiation 68 (CD68) (1:600, Cat# OABB00472, Aviva Systems Biology, San Diego, CA, USA) primary antibody and horseradish peroxidase anti-rabbit Envision system (Dako Cytochemistry, Tokyo, Japan).

Techniques: Activation Assay, Marker, Staining, Immunohistochemistry

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase 4 Regulates Inflammation in Ischemic Heart Failure: Role of Soluble Epoxide Hydrolase

doi: 10.1089/ars.2018.7548

Figure Lengend Snippet: Cardiac remodeling and macrophage infiltration is reduced in Nox4−/− mice after LAD ligation. WT and Nox4−/− mice hearts were analyzed by immunofluorescence microscopy 2 weeks after sham or from the peri-infarct region after LAD ligation (A) Cardiomyocyte CSA was measured by WGA staining. (B) Capillary density was measured by vWF staining. (C) Cell death was measured in cardiomyocytes by co-staining with TUNEL and MYH7, a cardiomyocyte cell marker. (arrowheads indicate TUNEL+ nuclei). (D) Macrophages were identified by CD68 staining in sham-operated mice and in the border region of the infarct zone of LAD-ligated mice. MYH7 was used to identify the border region. (E) Quantification of cardiomyocyte CSA, (F) quantification of capillary density, (G) quantification of TUNEL+ cells, and (H) quantification of CD68+ cells. Data are the mean ± SEM, n = 9. Scale is 100 μm. *p < 0.05, **p < 0.01, and ****p < 0.0001. CSA, cross-sectional area; MYH7, myosin heavy chain beta; TUNEL, terminal deoxynucleotidyl transferase; vWF, von Willebrand Factor; WGA, wheat germ agglutinin.

Article Snippet: Macrophages were stained by using an Alexa Fluor conjugated anti-CD68 primary antibody (Cat No. bs-1432R-A594; Bioss, Woburn, MA).

Techniques: Ligation, Immunofluorescence, Microscopy, Staining, TUNEL Assay, Marker