Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A) Transcriptome analysis of indicated genes in testes of ZIKV-infected AG6 mice at 5 dpi. Control mice were injected with PBS. (n = 3 mice for each group). (B and C) Dynamic changes of S100a4 gene level and S100A4+ cells in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at different time points as indicated and subjected to RT-qPCR or immunofluorescence staining (IFA) with anti-S100A4 antibody. (B) Change of S100a4 gene level was expressed as relative expression to β-actin, and shown as means ± SEM. (n = 3–4 mice for each time point). (C) The number of S100A4+ cells were quantified by method described in the experimental procedures, and the number of S100A4+ cells were recorded as cells/mm 2 and was shown as means ± SEM. (n = 5 mice for each time point). (D) IFA for S100A4+ cells. Testes from ZIKV-infected A6 mice were collected at 14 dpi and PBS-injected A6 mice served as controls. S100A4+ cells were detected by anti-S100A4 antibody and nuclei were stained with DAPI. Scale bar, 25 μm. (E-G) Flow cytometry assay for S100A4+ cells. Testicular cells from ZIKV-infected (14 dpi) or PBS-injected A6 mice were subjected to flow cytometry analysis with anti-S100A4 antibody and anti-CD11b antibody. (E) A representative result. (F) Percentage of CD11b+ cells in S100A4+ cells. Results were shown as means ± SEM. (n = 4 mice for each group). (G) Proportion of S100A4+ or S100A4- cells in CD11b+ cells. Results were shown as means ± SEM. (n = 4 mice for each group). (H and I) Co-immunofluorescence staining for S100A4+ cells. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescence staining with anti-S100A4 antibody and (H) anti-F4/80 antibody, or (I) anti-CD45 antibody. Nuclei were shown with DAPI. Scale bar, 50 μm. Relative mRNA expression of S100a4 and proportion in S100A4+ cells or CD11b+ cells in ZIKV-infected testes were analyzed using the Student’s t test, number of S100A4+ cells were analyzed using the Mann-Whitney U test. *p < 0.05 versus Ctrl, **p < 0.01 versus Ctrl.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Injection, Isolation, Quantitative RT-PCR, Immunofluorescence, Staining, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A and B) Distribution of S100A4+ macrophages in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to immunohistochemistry (IHC) staining with anti-S100A4 antibody. (A) Representative images at each time point. Scale bar, 50 μm. (B) Dynamic change of S100A4+ macrophages in seminiferous tubules (STs). The number of S100A4+ macrophages in seminiferous tubules were quantified by method described in the experimental procedures and expressed as cells/mm 2 STs, its number was shown as means ± SEM. (n = 3 mice for each time point). (C-E) Susceptibility of S100A4+ macrophages to ZIKV infection in vitro . Peritoneal macrophages were isolated as described in experimental procedures. (C) Peritoneal macrophages isolated from ZIKV-infected A6 mice at 7 dpi were doubly stained with anti-S100A4 and anti-F4/80 antibodies. (D) Peritoneal macrophages isolated from ZIKV-infected A6 mice at 7 dpi were doubly stained with anti-ZIKV and anti-S100A4 antibodies. Scale bar, 40 μm. (E) Peritoneal macrophages isolated from A6 mice treated with pristane were infected with ZIKV (MOI = 10), and then harvested at different time points as indicated and viral loads was determined by RT-qPCR (n = 3). (F) Susceptibility of S100A4+ macrophages to ZIKV infection in vivo . Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescence staining with anti-S100A4 antibody and anti-ZIKV antibody. Nuclei were shown with DAPI. Scale bar, 25 μm.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Isolation, Immunohistochemistry, In Vitro, Staining, Quantitative RT-PCR, In Vivo, Immunofluorescence

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A-C) Distribution and dynamic of CD8+ cells and their relationship with S100A4+ macrophages in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to IHC staining with anti-CD8α antibody, Scale bar, 50 μm (A) , or co-immunofluorescence staining with anti-CD8α and anti-S100A4 antibodies (B) . A typical seminiferous tubule was outlined with dotted line. Scale bar, 25 μm. (C) The number of CD8+ cells and S100A4+ macrophages in seminiferous tubules were quantified as described in experimental procedures, and expressed as cells/mm 2 STs. (n = 3 mice for each time point). (D) The number of intraluminal S100A4+ macrophages expressing caspase-8 or caspase-3 was quantified as described in experimental procedures, and expressed as cells/mm 2 STs. (n = 3 mice for each time point. See for the representative images). (E-H) Expression of GZMB in S100A4+ macrophages (E) or ZIKV-infected cells (G) . Testicular sections from ZIKV-infected A6 mice at 14 dpi were analyzed with co-immunofluorescence staining using anti-GZMB antibody and anti-S100A4 antibody or anti-ZIKV antibody. Scale bar, 25 μm. GZMB positive cell number of (F) S100A4+ macrophages or (H) ZIKV-infected cells were quantified inside and outside the seminiferous tubules, respectively, and expressed as cells/mm 2 . Results were shown as means ± SEM. (n = 3 mice for each group). Number of GZMB+ S100A4+ macrophages or GZMB+ ZIKV+ cells inside or outside of seminiferous tubules in ZIKV-infected testes were analyzed using the Student’s t test. *p < 0.05, **p < 0.01.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Isolation, Immunohistochemistry, Immunofluorescence, Staining, Expressing

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A) Susceptibility of spermatogenic cells to ZIKV infection. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescence staining with anti-DDX4 antibody and anti-ZIKV antibody. Nuclei were shown with DAPI. Scale bar, 25 μm. (B) Serial sections from ZIKV-infected testes at 14 dpi were subjected to IFA with anti-ZIKV (green), anti-DDX4 (red) or anti-S100A4 (indigo blue) antibodies. Nuclei were visualized with DAPI. Scale bar, 25 μm. (C) The numbers of ZIKV-infected testicular cells by type at 0–28 dpi were quantified as mentioned in experimental procedures and expressed as cells /mm 2 . (n = 3mice for each time point. See for the representative images.) (D-I) Ultrastructure morphological changes of seminiferous tubules from ZIKV-infected A6 mice. (D) A seminiferous tubule was attached by peripheral macrophage-like cells. (E) Peripheral macrophage-like cells in high magnification. (F) Macrophage-like cells accumulated in interstitial space. (G) Macrophage-like cells in outer layer lining seminiferous tubules. (H) Intraluminal macrophage-like cells were surrounded by cell debris. (I) Seminiferous tubules from uninfected A6 mice. M: macrophage-like cells, Sp: spermatogenic cells, SC: sperm cells, Se: Sertoli cells.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Isolation, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A-E) Testes from PBS-injected or ZIKV-infected A6 mice were collected at 7 dpi and distribution of CLDN1 were analyzed with co-immunofluorescence staining. (A) Distribution of CLDN1 in ZIKV-infected testes revealed by IFA using anti-CLDN1 and anti-S100A4 antibodies. Nuclei were stained with DAPI. Scale bar, 25 μm. (B-E) CLDN1 translocated into nuclei in various testicular cells using antibodies as indicated. (B) and (C) showing CLDN1 translocated into nuclei of spermatogenic cells (B) and their percentage in total of spermatogenic cells (Sps) (C). Scale bar, 25 μm. (D) and (E) showing CLDN1 translocated into nuclei of Sertoli cells (D) and their percentage in total of Sertoli cells (Ses) (E). Scale bar, 25 μm. All data were shown as means ± SEM. (n = 3 mice for each group). (F) Distribution of CLDN1 in ZIKV-infected testes from SA6 mice at 7 dpi. Nuclei were stained with DAPI. Scale bar, 25 μm. (G) The percentage of indicated cells with CLDN1 translocated into nuclei was quantified as described in Experimental Procedure and shown as means ± SEM. (n = 3 mice for each group). All small figures listed at the right side of the corresponding image showing area indicated by arrow in high magnification. Scale bar, 5 μm. Percentage of CLDN1 translocated into nuclei of spermatogenic cells and Sertoli cells in ZIKV-infected testes and control mice were analyzed using the Student’s t test. *p < 0.05, **p < 0.01.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Injection, Infection, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A) Dynamic of IFN-γ concentration in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at indicated time points and concentration of IFN-γ in testes was measured with Bio-Plex multiplex immunoassays (n = 4 mice for each time point). (B) Percentage of iNOS+ S100A4+ macrophages in total S100A4+ macrophages. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescent staining with anti-S100A4 and anti-iNOS or anti-CD163 antibodies. See for representative images. The percentage of iNOS+ S100A4+ macrophages was quantified as described in experimental procedure. (n = 3 mice for each time point). (C) Expression of IFN-γ in S100A4+ macrophages. Testis sections from PBS-injected or ZIKV-infected A6 mice at 14 dpi were analyzed using co-immunofluorescent staining with anti-S100A4 and anti-IFN-γ antibodies. Nuclei were stained with DAPI. Scale bar, 25 μm. (D) Effect of IFN-γ on CLDN1 distribution in vitro . Sertoli cells were treated with 50 ng IFN-γ at 32°C and were collected at 0, 24 and 48h. Distribution of CLDN1 was visualized by immunofluorescent staining. Nuclei were stained with DAPI. Scale bar, 25 μm. (E) Effect of IFN-γ on CLDN1 redistribution in vivo . A6 mice were intravenously injected with IFN-γ (4 μg per mouse daily) or PBS for 10 days. At 10 day after treatment, testes were collected and distribution of CLDN1 in testicular cells was analyzed by IFA. Scale bar, 25 μm. (F) Expression of CLDN1 and Occludin in testes from ZIKV-infected A6 and AG6 mice as well as their corresponding controls. Mice were challenged with ZIKV or injected with PBS, and testes were isolated at 7 dpi and subjected to Western Blot. (n = 3 for each group). (G) Distribution of CLDN1 in ZIKV-infected AG6 testes. CLDN1 distribution in testes from PBS-injected or ZIKV-infected AG6 mice (7 dpi) were analyzed using co-immunofluorescent staining. Nuclei were stained with DAPI. Scale bar, 25 μm. (H-J) Susceptibility of testicular cells to ZIKV in A6 or AG6 mice. Co-localization of ZIKV antigens with various cell marker molecules in testes from ZIKV-infected A6 or AG6 mice (7 dpi) was analyzed using co-immunofluorescent staining with anti-ZIKV antibody and (H) anti-DDX4 antibody, (I) anti-SOX9 antibody or (J) anti-α-SMA antibody. Nuclei were stained with DAPI. Scale bar, 50 μm. IFN-γ concentration in ZIKV-infected testis tissues were analyzed using the Student’s t test. *p < 0.05, **p < 0.01.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Concentration Assay, Infection, Isolation, Multiplex Assay, Staining, Expressing, Injection, In Vitro, In Vivo, Western Blot, Marker

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: Schematic diagram to show that S100A4+ macrophages assist ZIKV to infect mice testes and persist in seminiferous tubules.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques:

Journal: JCI Insight

Article Title: FGF-2 signaling in nasopharyngeal carcinoma modulates pericyte-macrophage crosstalk and metastasis

doi: 10.1172/jci.insight.157874

Figure Lengend Snippet: ( A ) Cross–data set quantitative heatmap of selected genes of various types of cancer and their adjacent control healthy tissues. Arrow points to distinctively upregulated genes in NPC. Log 2 fold changes were used for quantification. ( B ) Transcriptomic expression levels of FGF2 in human LUAD tissues, BRCA tissues and their adjacent healthy tissues. Sample number: control-LUAD/LUAD/control-BRCA/BRCA=347/483/291/1085. ( C ) Transcriptomic expression levels of FGF2 in various stages of human NPC tissues and their adjacent healthy tissues. Sample number: control/StageT1/StageT2/StageT3=10/16/11/4. ( D ) Human normal nasopharyngeal tissues (NNT), rhinitis tissues, and NPC tissues were stained with H&E and an anti–FGF-2 antibody (brown). Sample number: NNT/Rhinitis/NPC=3/10/6. Scale bar in upper panel: 500 μm. Scale bar in middle and lower panels: 50 μm. Quantification of FGF-2 + signals and FGF-2 + signals in stromal and epithelial components ( n = 8 random fields per group). ( E ) NPC cancer cells were sorted by MACS from freshly tissues. qPCR quantification of FGF2 mRNA ( n = 3 samples per group). ( F ) NNT rhinitis tissues and NPC tissues were stained. Sample number: NNT/Rhinitis/NPC=3/10/6. Scale bar in upper and middle panels: 50 μm. Scale bar in lower panel: 100 μm. Quantification of FSP1 + (brown), CD163 + (brown), CD31 + (red), and NG2 + (green) and coverage rate of NG2 + pericytes ( n = 8 random fields per group). ( G ) qPCR quantification of FGF2 , CD163 , CD31 , NG2 , and FSP1 mRNA in freshly collected tissues. Sample number: Rhinitis/NPC=5/6. ( H ) Correlation of FGF2 and CD163 expression of human NPCs and their control healthy tissues. Sample number: Control/NPC=10/31. * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired 2-tailed Student’s t test ( B , D , E , G , and H ) or 1-way ANOVA with Tukey’s multiple-comparison analysis ( C , D , and F ). Data are presented as mean ± SD.

Article Snippet: For IHC staining of tumor tissues, paraffin-embedded tissue sections were stained with a rabbit anti–FGF-2 antibody (catalog A0235, ABclonal, 1:100); a mouse anti-FSP1 antibody (catalog 66489-1, Proteintech, 1:100); a rabbit anti-CD163 antibody (catalog A8383, ABclonal, 1:100); a rabbit anti-F4/80 antibody (catalog 70076, Cell Signaling Technology, 1:1000); and a goat anti-CD206 antibody (catalog AF2535, R&D system, 1:400).

Techniques: Expressing, Staining

Journal: Molecular Medicine

Article Title: ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy

doi: 10.1186/s10020-021-00339-7

Figure Lengend Snippet: Occurrence of EndMT and increase in PFN2 expression in DN patients. A HE staining and Masson staining in renal biopsy specimens of DN patients and control participants. Magnification: ×20. Scale bar: 20 μM. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 20 for the control group, n = 7 for the DN2 group, n = 7 for the DN3 group, n = 6 for the DN4 group) B Immunostaining of CD31, vimentin, αSMA and S100A4 and PFN2 in renal biopsy specimens of DN patients and control participants. Magnification: ×20. Scale bar: 20 μM. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 20 for the control group, n = 7 for the DN2 group, n = 7 for the DN3 group, n = 6 for the DN4 group)

Article Snippet: The membranes were incubated in a 5% skimmed milk solution at room temperature for 1 h. Then, all membranes were incubated with the corresponding primary antibodies at 4 °C for 12 h. The primary antibodies used in the present study were as follows: monoclonal antibodies against β-actin (Proteintech, Wuhan, China), KMT5A (Proteintech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), ets1 (Proteintech, Wuhan, China), PFN2 (Proteintech, Wuhan, China), αSMA (Proteintech, Wuhan, China), S100A4 (Proteintech, Wuhan, China), CD31 (Proteintech, Wuhan, China) and vimentin (Proteintech, Wuhan, China).

Techniques: Expressing, Staining, Immunostaining

Journal: Molecular Medicine

Article Title: ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy

doi: 10.1186/s10020-021-00339-7

Figure Lengend Snippet: High-glucose treatment mediated EndMT via the upregulation of PFN2 expression in HUVECs. A Results from the Western blot analysis of CD31, vimentin, αSMA, S100A4 and PFN2 levels in HUVECs with the corresponding treatment. B Compared with the control group, the mRNA expression of CD31 was decreased in hyperglycemic HUVECs. C Compared with the control group, the mRNA expression of vimentin was increased in hyperglycemic HUVECs. D Compared with the control group, the mRNA expression of αSMA was increased in hyperglycemic HUVECs. E Compared with the control group, the mRNA expression of S100A4 was increased in hyperglycemic HUVECs. F Compared with the control group, the mRNA expression of PFN2 was increased in hyperglycemic HUVECs. G Results from the Western blot analysis of PFN2, CD31, vimentin, αSMA and S100A4 levels in HUVECs with the corresponding treatment. H The effects of si-PFN2 were confirmed by qPCR. I Compared with high-glucose treatment, si-PFN2 increased CD31 mRNA expression in hyperglycemic HUVECs. J Compared with high-glucose treatment, si-PFN2 decreased vimentin mRNA expression in hyperglycemic HUVECs. K Compared with high-glucose treatment, si-PFN2 decreased αSMA mRNA expression in hyperglycemic HUVECs. L Compared with high-glucose treatment, si-PFN2 decreased S100A4 mRNA expression in hyperglycemic HUVECs. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 5/group)

Article Snippet: The membranes were incubated in a 5% skimmed milk solution at room temperature for 1 h. Then, all membranes were incubated with the corresponding primary antibodies at 4 °C for 12 h. The primary antibodies used in the present study were as follows: monoclonal antibodies against β-actin (Proteintech, Wuhan, China), KMT5A (Proteintech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), ets1 (Proteintech, Wuhan, China), PFN2 (Proteintech, Wuhan, China), αSMA (Proteintech, Wuhan, China), S100A4 (Proteintech, Wuhan, China), CD31 (Proteintech, Wuhan, China) and vimentin (Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot

Journal: Molecular Medicine

Article Title: ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy

doi: 10.1186/s10020-021-00339-7

Figure Lengend Snippet: ets1 participated in high glucose-induced EndMT by augmenting PFN2 expression in HUVECs. A Results from the Western blot analysis of ets1, PFN2, CD31, vimentin, αSMA and S100A4 in the HUVECs with the corresponding treatment. B The effects of si-ets1 were confirmed by qPCR. C Compared with high-glucose treatment, si-ets1 decreased PFN2 mRNA expression in hyperglycemic HUVECs. D Compared with high-glucose treatment, si-ets1 increased CD31 mRNA expression in hyperglycemic HUVECs. E Compared with high-glucose treatment, si-ets1 decreased vimentin mRNA expression in hyperglycemic HUVECs. F Compared with high-glucose treatment, si-ets1 decreased αSMA mRNA expression in hyperglycemic HUVECs. G Compared with high-glucose treatment, si-ets1 decreased S100A4 mRNA expression in hyperglycemic HUVECs. H Results from the Western blot analysis of ets1, PFN2, CD31, vimentin, αSMA and S100A4 in the HUVECs with the corresponding treatment. I The effects of ets1 overexpression were confirmed by qPCR. J The effects of si-PFN2 were confirmed by qPCR. K ets1 overexpression inhibited CD31 mRNA expression, which was reversed by si-PFN2 treatment. L ets1 overexpression increased vimentin mRNA expression, which was reversed by si-PFN2 treatment. M ets1 overexpression increased αSMA mRNA expression, which was reversed by si-PFN2 treatment. N ets1 overexpression increased S100A4 mRNA expression, which was reversed by si-PFN2 treatment. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 5/group)

Article Snippet: The membranes were incubated in a 5% skimmed milk solution at room temperature for 1 h. Then, all membranes were incubated with the corresponding primary antibodies at 4 °C for 12 h. The primary antibodies used in the present study were as follows: monoclonal antibodies against β-actin (Proteintech, Wuhan, China), KMT5A (Proteintech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), ets1 (Proteintech, Wuhan, China), PFN2 (Proteintech, Wuhan, China), αSMA (Proteintech, Wuhan, China), S100A4 (Proteintech, Wuhan, China), CD31 (Proteintech, Wuhan, China) and vimentin (Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot, Over Expression

Journal: Molecular Medicine

Article Title: ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy

doi: 10.1186/s10020-021-00339-7

Figure Lengend Snippet: KMT5A suppression participated in high glucose-mediated EndMT by augmenting PFN2 expression in HUVECs. A Results from the Western blot analysis of KMT5A, PFN2, CD31, vimentin, αSMA and S100A4 in the HUVECs with the corresponding treatment. B The effects of KMT5A overexpression were verified by qPCR. C The high glucose-mediated increase in PFN2 mRNA expression was inhibited by KMT5A overexpression. D The high glucose-mediated decrease in CD31 mRNA expression was counteracted by KMT5A overexpression. E The high glucose-mediated increase in vimentin mRNA expression was inhibited by KMT5A overexpression. F The high glucose-mediated increase in αSMA mRNA expression was inhibited by KMT5A overexpression. G The high glucose-mediated increase in S100A4 mRNA expression was inhibited by KMT5A overexpression. H Results from the Western blot analysis of KMT5A, PFN2, CD31, vimentin, αSMA and S100A4 in the HUVECs with the corresponding treatment. I si-PFN2 did not affect KMT5A mRNA expression. J The effect of si-PFN2 was verified by qPCR. K si-PFN2 upregulated CD31 mRNA expression in sh-KMT5A-treated HUVECs. L si-PFN2 decreased vimentin mRNA expression in sh-KMT5A-treated HUVECs. M si-PFN2 decreased αSMA mRNA expression in sh-KMT5A-treated HUVECs. N si-PFN2 decreased S100A4 mRNA expression in sh-KMT5A-treated HUVECs. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 5/group)

Article Snippet: The membranes were incubated in a 5% skimmed milk solution at room temperature for 1 h. Then, all membranes were incubated with the corresponding primary antibodies at 4 °C for 12 h. The primary antibodies used in the present study were as follows: monoclonal antibodies against β-actin (Proteintech, Wuhan, China), KMT5A (Proteintech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), ets1 (Proteintech, Wuhan, China), PFN2 (Proteintech, Wuhan, China), αSMA (Proteintech, Wuhan, China), S100A4 (Proteintech, Wuhan, China), CD31 (Proteintech, Wuhan, China) and vimentin (Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot, Over Expression

Journal: Inflammation and Regeneration

Article Title: Imatinib inhibits pericyte-fibroblast transition and inflammation and promotes axon regeneration by blocking the PDGF-BB/PDGFRβ pathway in spinal cord injury

doi: 10.1186/s41232-022-00223-9

Figure Lengend Snippet: Pericyte transition into fibroblasts after SCI. a – d Representative immunofluorescence images taken in the spinal cords of uninjured mice and injured mice at 3, 7, 14, and 28 days post-injury (dpi) showing that fibrotic scarring PDGFRβ + fibroblasts (red) lose the expression of the pericyte marker NG2 (green, a and b ) but robustly express the fibroblast markers FSP1 (green, c ) and vimentin (green, d ) after SCI. The nuclei are stained with DAPI (blue). The high magnification z-stack images of the dotted area in a are shown below as the region of interest in b. e Quantification of the percentage of NG2 + PDGFRβ + cells out of the total PDGFRβ + cells in the lesion core. f , g Quantification of the percentage of FSP1 + area ( f ) and vimentin + area ( g ). The asterisks indicate the lesion core. Data are shown as mean ± s.e.m. n = 4 mice per time point. Scale bars: 100 μm ( a ) and 10 μm ( b–d ). All images are from sagittal sections. NS, no significance; * p < 0.05, *** p < 0.001 by one-way ANOVA followed by Tukey’s post hoc test in e , f . 3, 7, 14, and 28 dpi versus 0 dpi in e

Article Snippet: The primary antibodies used were as follows: goat anti-CD31(1:100, AF3625, R&D Systems), rat anti-PDGFRβ (1:100, 14-1402-82, Invitrogen), goat anti-PDGFRβ (1:100, AF1042, R&D Systems), rabbit anti-NG2 (1:100, AB5320, Sigma-Aldrich), rabbit anti-FSP1 (1:100, 16105-1-AP, Proteintech), rabbit anti-Vimentin (1:300, ab92547, Abcam), rabbit anti-PDGF-BB (1:50, NBP1-58279, Novus), rat anti-GFAP (1:200, 13-0300, Invitrogen), rabbit anti-GFAP (1:100, 16825-1-AP, Proteintech), rat anti-CD68 (1:300, MCA1957, AbD Serotec), goat anti-5-HT (1:5000, #20080, Immunostar), rabbit anti-neurofilament-heavy polypeptide (NF-H, 1:500, ab207176, Abcam), rabbit anti-growth-associated protein 43 (GAP43, 1:100, 16971-1-AP, Proteintech), rabbit anti-NeuN (1:500, ab177487, Abcam), rabbit anti-fibronectin (1:100, 15613-1-AP, Proteintech), rabbit anti-Laminin (1:100, 23498-1-AP, Proteintech), rat anti-Ki67 (1:100, 14-5698-80, Invitrogen), rabbit anti-cleaved caspase-3 (Asp175) (C-Cas3,1:250, 9661, Cell Signaling Technology), and rabbit anti-fibrinogen (1:100, 15841-12-AP, Proteintech).

Techniques: Immunofluorescence, Expressing, Marker, Staining

Journal: Inflammation and Regeneration

Article Title: Imatinib inhibits pericyte-fibroblast transition and inflammation and promotes axon regeneration by blocking the PDGF-BB/PDGFRβ pathway in spinal cord injury

doi: 10.1186/s41232-022-00223-9

Figure Lengend Snippet: Endothelial PDGF-BB induces pericyte-fibroblast transition and extracellular matrix deposition in vitro. a The PDGF-BB expression levels in bEnd.3 cells treated with 1 mg/ml myelin debris at the indicated time points were measured by ELISA. b , c Western blot analysis ( b ) and quantification ( c ) of PDGF-BB in bEnd.3 cells treated as described above in a . d The PDGF-BB expression levels in bEnd.3 cells treated with the indicated concentrations of myelin debris for 72 h were measured by ELISA. e , f Western blot analysis ( e ) and quantification ( f ) of PDGF-BB in bEnd.3 cells treated as described above in d . g Experimental schematic diagram of pericyte phenotypic transition induced by culture medium (empty, control), EC-CM treated with PBS, Mye-CM treated with myelin debris, and PDGF-BB. h Immunostaining of PDGFRβ (red), pericyte marker NG2 (green, upper panel), and fibroblast markers FSP1 (green, middle panel) and vimentin (green, lower panel) in primary pericytes treated as in g . i Quantification of the percentage of NG2 + , FSP1 + , and vimentin + pericytes. j , k Immunostaining and quantification of extracellular matrix collagen I (green, upper panel) and laminin (green, lower panel) in primary pericytes treated as described in g . The nuclei are stained blue with DAPI. l , m Western blot analysis ( l ) and quantification ( m ) of NG2, FSP1, and vimentin in primary pericytes treated as described above. n , o Western blot analysis ( n ) and quantification ( o ) of extracellular matrix collagen I and laminin in primary pericytes treated as described above. Scale bar: 25 μm ( h and j ). Data are expressed as mean ± s.e.m. n = 3 independent cultures. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus 0 h, 0 mg/ml or control by one-way ANOVA followed by Tukey’s post hoc test in a , c , d , f , i , k , m , and o

Article Snippet: The primary antibodies used were as follows: goat anti-CD31(1:100, AF3625, R&D Systems), rat anti-PDGFRβ (1:100, 14-1402-82, Invitrogen), goat anti-PDGFRβ (1:100, AF1042, R&D Systems), rabbit anti-NG2 (1:100, AB5320, Sigma-Aldrich), rabbit anti-FSP1 (1:100, 16105-1-AP, Proteintech), rabbit anti-Vimentin (1:300, ab92547, Abcam), rabbit anti-PDGF-BB (1:50, NBP1-58279, Novus), rat anti-GFAP (1:200, 13-0300, Invitrogen), rabbit anti-GFAP (1:100, 16825-1-AP, Proteintech), rat anti-CD68 (1:300, MCA1957, AbD Serotec), goat anti-5-HT (1:5000, #20080, Immunostar), rabbit anti-neurofilament-heavy polypeptide (NF-H, 1:500, ab207176, Abcam), rabbit anti-growth-associated protein 43 (GAP43, 1:100, 16971-1-AP, Proteintech), rabbit anti-NeuN (1:500, ab177487, Abcam), rabbit anti-fibronectin (1:100, 15613-1-AP, Proteintech), rabbit anti-Laminin (1:100, 23498-1-AP, Proteintech), rat anti-Ki67 (1:100, 14-5698-80, Invitrogen), rabbit anti-cleaved caspase-3 (Asp175) (C-Cas3,1:250, 9661, Cell Signaling Technology), and rabbit anti-fibrinogen (1:100, 15841-12-AP, Proteintech).

Techniques: In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining, Marker, Staining

Journal: Inflammation and Regeneration

Article Title: Imatinib inhibits pericyte-fibroblast transition and inflammation and promotes axon regeneration by blocking the PDGF-BB/PDGFRβ pathway in spinal cord injury

doi: 10.1186/s41232-022-00223-9

Figure Lengend Snippet: Microvascular endothelial cells induce pericyte-fibroblast transition via the PDGF-BB/PDGFRβ signaling pathway in vitro. a , b Western blot analysis ( a ) and quantification ( b ) of PDGF-BB in bEnd.3 cells transfected with siNC or siRNAs targeting Pdgfb. c The expression levels of PDGF-BB in bEnd.3 cells transfected with siNC or siPdgfb#2 followed by myelin debris treatment were detected by ELISA. d , e Western blot analysis ( d ) and quantification ( e ) of PDGF-BB in bEnd.3 cells treated as described above in c . f Experimental schematic diagram of pericyte phenotypic transition transfected with siNC or siPdgfb#2 followed by myelin debris treatment. g Immunostaining of NG2 (green, upper panel), FSP1 (green, middle panel), and vimentin (green, lower panel) in primary pericytes treated as described above in f . h Quantification of the percentage of NG2 + , FSP1 + , and vimentin + pericytes in g . i Experimental schematic diagram of pericyte phenotypic transition blocked with the PDGFRβ inhibitor imatinib (a selective PDGFRβ inhibitor) or Su16f (a specific PDGFRβ inhibitor) followed by Mye-CM. j Immunostaining of NG2 (green, upper panel), FSP1 (green, middle panel), and vimentin (green, lower panel) in primary pericytes treated as described in i . k Quantification of the percentage of NG2 + , FSP1 + , and vimentin + pericytes in j . Scale bars: 25 μm ( g and j ). Data are expressed as mean ± s.e.m. n = 3 independent cultures. ** p < 0.01 and *** p < 0.001 by one-way ANOVA followed by Tukey’s post hoc test in b versus siNC, and k . * p < 0.05, ** p < 0.01, and ***p < 0.001 versus siNC by unpaired two-tailed Student’s t test in c , e , and h

Article Snippet: The primary antibodies used were as follows: goat anti-CD31(1:100, AF3625, R&D Systems), rat anti-PDGFRβ (1:100, 14-1402-82, Invitrogen), goat anti-PDGFRβ (1:100, AF1042, R&D Systems), rabbit anti-NG2 (1:100, AB5320, Sigma-Aldrich), rabbit anti-FSP1 (1:100, 16105-1-AP, Proteintech), rabbit anti-Vimentin (1:300, ab92547, Abcam), rabbit anti-PDGF-BB (1:50, NBP1-58279, Novus), rat anti-GFAP (1:200, 13-0300, Invitrogen), rabbit anti-GFAP (1:100, 16825-1-AP, Proteintech), rat anti-CD68 (1:300, MCA1957, AbD Serotec), goat anti-5-HT (1:5000, #20080, Immunostar), rabbit anti-neurofilament-heavy polypeptide (NF-H, 1:500, ab207176, Abcam), rabbit anti-growth-associated protein 43 (GAP43, 1:100, 16971-1-AP, Proteintech), rabbit anti-NeuN (1:500, ab177487, Abcam), rabbit anti-fibronectin (1:100, 15613-1-AP, Proteintech), rabbit anti-Laminin (1:100, 23498-1-AP, Proteintech), rat anti-Ki67 (1:100, 14-5698-80, Invitrogen), rabbit anti-cleaved caspase-3 (Asp175) (C-Cas3,1:250, 9661, Cell Signaling Technology), and rabbit anti-fibrinogen (1:100, 15841-12-AP, Proteintech).

Techniques: In Vitro, Western Blot, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Immunostaining, Two Tailed Test

Journal: Inflammation and Regeneration

Article Title: Imatinib inhibits pericyte-fibroblast transition and inflammation and promotes axon regeneration by blocking the PDGF-BB/PDGFRβ pathway in spinal cord injury

doi: 10.1186/s41232-022-00223-9

Figure Lengend Snippet: Pharmacologically inhibiting the PDGF-BB/PDGFRβ signaling pathway reduces fibrotic scarring and fibroblasts after SCI. a Representative immunofluorescence images of GFAP (green) and PDGFRβ (red) in mice treated with imatinib or PBS (control) at 14 and 28 days post-injury (dpi). b Representative immunofluorescence images of PDGFRβ (green) and extracellular matrix fibronectin (red, upper panel) and laminin (red, lower panel) at 28 dpi. c , d Quantification of fibrotic scar area ( c ) and extracellular matrix area ( d ) in a and b . e , f Representative immunofluorescence images of PDGFRβ (green) and FSP1 (red) in mice treated as described above at 14 dpi ( e ) and 28 dpi ( f ). The nuclei are stained with DAPI (blue). High magnification images of the dotted area in the left panel are shown in the right panel. All images are from sagittal sections. g Quantification of the fibroblast area occupied by FSP1 in e and f . Scale bars: 100 μm ( a , b and left panel in e and f ) and 20 μm (right panel in e and f ). Data are expressed as mean ± s.e.m. n = 4–6 per group. ** p < 0.01 and *** p < 0.001 versus control by unpaired two-tailed Student’s t test in c , d , and g

Article Snippet: The primary antibodies used were as follows: goat anti-CD31(1:100, AF3625, R&D Systems), rat anti-PDGFRβ (1:100, 14-1402-82, Invitrogen), goat anti-PDGFRβ (1:100, AF1042, R&D Systems), rabbit anti-NG2 (1:100, AB5320, Sigma-Aldrich), rabbit anti-FSP1 (1:100, 16105-1-AP, Proteintech), rabbit anti-Vimentin (1:300, ab92547, Abcam), rabbit anti-PDGF-BB (1:50, NBP1-58279, Novus), rat anti-GFAP (1:200, 13-0300, Invitrogen), rabbit anti-GFAP (1:100, 16825-1-AP, Proteintech), rat anti-CD68 (1:300, MCA1957, AbD Serotec), goat anti-5-HT (1:5000, #20080, Immunostar), rabbit anti-neurofilament-heavy polypeptide (NF-H, 1:500, ab207176, Abcam), rabbit anti-growth-associated protein 43 (GAP43, 1:100, 16971-1-AP, Proteintech), rabbit anti-NeuN (1:500, ab177487, Abcam), rabbit anti-fibronectin (1:100, 15613-1-AP, Proteintech), rabbit anti-Laminin (1:100, 23498-1-AP, Proteintech), rat anti-Ki67 (1:100, 14-5698-80, Invitrogen), rabbit anti-cleaved caspase-3 (Asp175) (C-Cas3,1:250, 9661, Cell Signaling Technology), and rabbit anti-fibrinogen (1:100, 15841-12-AP, Proteintech).

Techniques: Immunofluorescence, Staining, Two Tailed Test