Fig 1: Upregulation of the SASP by ROS and Padi2 knockdown leads to cellular senescence and the inhibition of the SASP ameliorates cellular senescence and osteogenic dysfunction. A Representative images of SA-ß-Gal staining. MC3T3-E1 cells were cultured in the conditioned medium (CM) from MC3T3-E1 transfected with siCont or siPadi2 for 8 days and SA-ß-Gal staining was performed. Percentage of SA-ß-Gal-positive cells in each group was calculated as the percentage of SA-ß-Gal-positive cells relative to the total cells stained with DAPI in the same field (8–10 fields/group). Magnification ×20, Scale bars 100 µm. Bars, statistical significance was determined using one-way ANOVA for multiple comparisons, *P < 0.05, ***P < 0.001. Three independent experiments were performed. B MC3T3-E1 cells were cultivated under Padi2 KD-CM for 8 days or siCont-CM, followed by performing RT-qPCR of Cdkn1a. Data for RT-qPCR are presented as the mean of three replicates; bars, ± SD. Statistical significance was determined using one-way ANOVA for multiple comparisons, **P < 0.01, ***P < 0.001; n.s., not significant. Two independent experiments were performed. C Analysis of the relative expression levels of the SASP genes by RNA-seq data (black bar) and RT-qPCR (gray bar). Data are the fold change of 4 day-H2O2-treated group relative to the non-treated control. The red and blue-dotted lines mean fold change 1 and - 1, respectively. Data for RT-qPCR are presented as the mean of three replicates; bars, ± SD. statistical significance was determined using two-tailed Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant. Three independent experiments were performed for RT-qPCR. D MC3T3-E1 cells were transfected with siCont, siPadi2 #2 or siPadi2 #3 and then incubated for additional 4 days in the osteogenic medium. The expression level of each gene was measured by RT-qPCR and the fold change in mRNA levels was calculated by normalization to Gapdh. Data are presented as the mean of three replicates; bars, ± SD; statistical significance was determined using one-way ANOVA for multiple comparisons, *P < 0.05, ***P < 0.001, ****P < 0.0001. Three independent experiments were performed. E Five days-cell culture soup from MC3T3-E1 cells transfected with siCont, siPadi2 #2, or siPadi2 #3 was collected and the levels of CCL2, CCL5, and CCL7 in each cell culture soup were measured by ELISA. Data are presented as the mean of three replicates; bars, ± SD; statistical significance was determined using one-way ANOVA for multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Three independent experiments were performed. F Representative images of SA-ß-Gal staining. MC3T3-E1 cells knocked down with siCont or siPadi2 were treated with IgG or each neutralizing antibody for 4 days and SA-ß-Gal staining was performed. Quantity of SA-ß-Gal-positive cells was expressed as the percentage of SA-ß-Gal-positive cells relative to the total cells stained with DAPI in the same field (8–10 fields/group). Magnification ×20, Scale bars 100 µm. bars, mean ± SD. statistical significance was determined using two-way ANOVA for multiple comparisons, ***P < 0.001. Three independent experiments were performed. G When MC3T3-E1 cells transfected with siCont, siCcl2, siCcl5, or siCcl7 were fully confluent, cells were treated with 100 µM H2O2 for 4 days in osteogenic medium and ALP staining was performed. Quantification of ALP staining was performed by ImageJ. Bars, mean ± SD; statistical significance was determined using one-way or two-way ANOVA for multiple comparisons, *P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant. H When MC3T3-E1 cells knocked down with siPadi2 in combination with or without siCcl2, 5, or 7 were fully confluent, cells were changed with osteogenic medium and incubated for 4 days and ALP staining was performed. Quantification of ALP staining (right). Bars, mean ± SD; statistical significance was determined using one-way or two-way ANOVA for multiple comparisons, *P < 0.05, **P < 0.01,; ns not significant. Three independent experiments were performed
Fig 2: BRG1 interacts with AP-1 to activate CCL7 transcription in a redox-sensitive manner. (A) CCL7 promoter-luciferase constructs were transfected into HepG2 cells with or without BRG1 followed by treatment with LPS or PA. Luciferase activities were normalized by protein concentration and GFP fluorescence. (B) HepG2 cells were transfected with siRNA targeting AP-1 or scrambled siRNA (SCR) followed by treatment with LPS or PA. ChIP assays were performed with anti-BRG1 or IgG. (C) HepG2 cells were treated with LPS or PA in the presence or absence of NAC. ChIP assays were performed with anti-c-Jun, anti-c-Fos, or anti-BRG1. (D) HepG2 cells were treated with LPS or PA in the presence or absence of NAC. Immunoprecipitation was performed with anti-BRG1 or IgG. (E) C57/BL6 mice were injected with LPS in the presence or absence of NAC for 12 h. Immunoprecipitation was performed with anti-BRG1. (F) C57/BL6 mice were fed an MCD diet in the presence or absence of NAC for 4wk. Immunoprecipitation was performed with anti-BRG1. (G) HepG2 cells were treated with LPS or PA for 12 h. Re-ChIP assay was performed with indicated antibodies.
Fig 3: BRG1-dependent hepatocyte-derived CCL7 promotes macrophage migration. (A) HepG2 cells were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with LPS for 6 h. Conditioned media were collected and chemotaxis assay was performed as described in Methods. (B) HepG2 cells were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with palmitate for 12 h. Conditioned media were collected and chemotaxis assay was performed as described in Methods. (C) Primary hepatocytes were isolated from WT or LKO mice and treated with or without LPS for 6 h. Conditioned media were collected and chemotaxis assay was performed as described in Methods. (D) Primary hepatocytes were isolated from WT or LKO mice and treated with palmitate for 12 h. Conditioned media were collected and chemotaxis assay was performed as described in Methods. (E) HepG2 cells were infected with BRG1 adenovirus (Ad-BRG1) or the control adenovirus (Ad-GFP) followed by treatment with LPS for 6 h. Conditioned media were collected and chemotaxis assay was performed in the presence or absence of a CCL7-neutralizing antibody. (F) HepG2 cells were infected with BRG1 adenovirus (Ad-BRG1) or the control adenovirus (Ad-GFP) followed by treatment with palmitate for 12 h. Conditioned media were collected and chemotaxis assay was performed in the presence or absence of a CCL7-neutralizing antibody. (G) Primary hepatocytes were infected with BRG1 adenovirus (Ad-BRG1) or the control adenovirus (Ad-GFP) followed by treatment with LPS for 6 h. Conditioned media were collected and chemotaxis assay was performed in the presence or absence of a CCL7-neutralizing antibody. (H) Primary hepatocytes were infected with BRG1 adenovirus (Ad-BRG1) or the control adenovirus (Ad-GFP) followed by treatment with palmitate for 12 h. Conditioned media were collected and chemotaxis assay was performed in the presence or absence of a CCL7-neutralizing antibody.
Fig 4: CCL7 promoted macrophages migration and M1 polarization. (A) Migration of BMDMs in response to rmCCL7(100 ng/ml) stimulation. Scale bars represented 100 µm(n = 3). (B and C), Expression of (B) M1 markers (iNOS, IL-6, IL-12A, IL-12B, TNF-a) and (C) M2 markers (Arg 1, CD206)in response to rmCCL7 in BMDMs examined by qRT-PCR(n = 3). (D) Protein abundance of iNOS and CD206 in abdominal aortas of saline-infused (n = 7) and Ang II-infused (n = 6) mice. (E) Immunofluorescence co-staining of CD68 and iNOS in abdominal aortas of saline-infused (n = 3) and Ang II-infused (n = 3) mice. Values were represented as mean ± SEM; Student's t test was used in (A-D). *P < .05; **P < .01; ***P < .001, respectively
Fig 5: Ccl7 is a HIF1a direct target gene.a qPCR analysis of Ccl7 mRNA expression in Hif1afl/fl and Hif1a?SMC VSMCs treated with vehicle, CoCl2, normoxia, or hypoxia (2% O2) for 6, 12, and 24 h. *P < 0.05, **P < 0.01, ***P < 0.001, n = 6 (independent experiments) per group. VSMCs isolated from Hif1afl/fl mice and Hif1a?SMC mice were infected with oxygen-stable HIF1a-expressing lentivirus, and then treated with Ang II for 24 h, b Ccl7 mRNA was measured by qPCR and c CCL7 protein was detected by ELISA. d qPCR analysis of Ccl7 mRNA expression in vehicle or Ang II-treated Hif2afl/fl and Hif2a?SMC VSMCs. e Schematic diagram of the mouse Ccl7 promoter illustrating the HREs in the regulatory region; the upstream regions were numbered in relation to the transcription initiation site. f Luciferase-reporter constructs under the control of the mouse Ccl7 promoter. HEK293T human embryonic kidney cells transiently transfected with the luciferase construct, and cotransfected with empty vector or HIF1a expression plasmids. Standard dual-luciferase assays were performed. EV, empty vector. **P < 0.01, n = 3. g, h ChIP assays of vehicle or Ang II-treated wild-type VSMCs using HIF1a or HIF2a antibodies. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. i, j ChIP assays of vehicle or Ang II-treated Hif1afl/fl and Hif1a?SMC VSMCs using HIF1a antibody. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. Statistical significance was determined by one-way ANOVA test followed by the unpaired t-test
Supplier Page from Abcam for Mouse MCP3 ELISA Kit (CCL7)