Journal: iScience

Article Title: Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages

doi: 10.1016/j.isci.2023.108484

Figure Lengend Snippet: The transcription factor SP1 is closely associated with fibrosis mediated by TGF-β1 (A and B) Thin sections of ligament from patients with lumbar spinal stenosis involving hypertrophic ligamentum flavum (Disease, n = 4) and healthy controls (Ctrl, n = 6). Sections were stained with hematoxylin and eosin or Masson stain (A) and immunostained to label collagen I and III (B). Quantitation of the collagen volume fraction and level of immunostaining are shown on the right. Scale bar, 20 μm. (C) Scanning electron micrographs of ligaments from patients (Disease, n = 4) and controls (Ctrl, n = 6). Scale bar, 50 μm or 10 μm. (D) Levels of mRNAs expressing the TGF-β1 and SP1 from ligaments of patients and controls (n = 3 per group). (E) Western blotting of total lysates from ligaments of patients and controls to detect SP1, pro-TGF-β1, and mature TGF-β1. Quantitative analysis of western blots is provided below. N = 3. (F) Western blotting of total lysates from ligaments of patients and controls to detect LOXL2, Smad2/3, p-Smad2/3, Smad4, and Smad7. Quantitative analysis of western blots is on the right. N = 3. (G) Immunostaining of thin sections of ligament from patients (Disease, n = 4) and controls (Ctrl, n = 6) to detect TGF-β1 and SP1. The images on the right are “zoomed” images of the areas encircled by a dotted black line in the images on the left. Quantitation of TGF-β1 and SP1 immunostaining is shown below. Scale bar, 20 μm (left) or 10 μm (right). (H) Dual immunostaining of thin sections of ligament from patients and controls to detect SP1 and the M2 macrophage marker CD163. Nuclei were counterstained with DAPI. The correlation analysis results are placed on the right. Scale bar, 20 μm. Data are mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Then the M0 macrophages were differentiated into M2 macrophages by incubating them for 48 h in medium containing IL-4 (20 ng/ml, cat# P00021, Solarbio) and IL-13 (20 ng/ml, cat# P00131, Solarbio).

Techniques: Staining, Quantitation Assay, Immunostaining, Expressing, Western Blot, Marker

Journal: iScience

Article Title: Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages

doi: 10.1016/j.isci.2023.108484

Figure Lengend Snippet: SP1 in M2 macrophages activates fibroblasts via TGF-β1 (A) Transwell assay in which M2 macrophages were co-cultured with ligamentum flavum fibroblasts (LGF), lung fibroblasts (LF), and skin fibroblasts (SF) and the migration of the fibroblasts was measured. As controls, LGF, LF, and SF were cultured on their own. Photomicrographs were taken after 4 days of co-culture, followed by staining with crystal violet. Scale bar, 50 μm. N = 3. (B) Proliferation of LGF was evaluated at indicated time points (0, 2, and 4 days) in monoculture and co-culture with M2 macrophages using the Cell Counting Kit-8, with detection performed at 450 OD. Each experiment was independently repeated three times. (C) Immunostaining of LGF for the fibroblast markers vimentin and collagen I and III, monoculture or co-culture with M2 macrophages. Quantitative analysis is on the right. Scale bar, 200 μm. N = 3. (D) Concentration of collagen I and III in the medium of cultures of LGF alone or together with M2 macrophages. N = 4. (E) Levels of protein Smad4, Smad2/3, p-Smad2/3, and Smad7 in LGF, LF, and SF after 4 days in monoculture or co-culture with M2 macrophages. Quantitative analysis of western blots is below. N = 3. (F) Levels of protein SP1 and pro-TGF-β1 in M2 macrophages after 4 days in monoculture or co-culture with LGF, LF, and SF, respectively. Quantitative analysis of western blots is below. N = 3. (G) Levels of SP1 and pro-TGF-β1 in M2 macrophages that had been transfected and treated as described after 4 days co-culture with LGF. Quantitative analysis of western blots is below. N = 3. (H) Proliferation of LGF was evaluated at indicated time points (0, 2, and 4 days) in co-culture with M2 macrophages that had been transfected and treated as described using the Cell Counting Kit-8, with detection performed at 450 OD. Each experiment was independently repeated three times. (I) Concentration of collagen I and III in the medium of cultures of LGF together with M2 macrophages that had been transfected and treated as described. N = 3. KRFK (50 μM) alone or together with CJJ300 (40 μM) was added to the co-culture medium of M2 macrophages and fibroblasts. Ligamentum flavum fibroblasts (LGF), lung fibroblasts (LF), and skin fibroblasts (SF). Data are mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Then the M0 macrophages were differentiated into M2 macrophages by incubating them for 48 h in medium containing IL-4 (20 ng/ml, cat# P00021, Solarbio) and IL-13 (20 ng/ml, cat# P00131, Solarbio).

Techniques: Transwell Assay, Cell Culture, Migration, Co-Culture Assay, Staining, Cell Counting, Immunostaining, Concentration Assay, Western Blot, Transfection

Journal: iScience

Article Title: Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages

doi: 10.1016/j.isci.2023.108484

Figure Lengend Snippet: The activation of fibroblasts by M2 macrophages is associated with the direct binding of SP1 and TGF-β1 gene promoter-binding elements (A) Potential SP1-binding sites (P1, P2, P3) in the TGF-β1 gene promoter and two SP1-binding elements (BE) in the P3 sequence. The box encloses the wild-type (WT) and mutant (mut) sequences used in dual-luciferase reporter assays. TSS, transcription start site. (B) The ability of SP1 to bind each of the potential binding regions P1–P3 in the TGF-β1 promoter was assessed in a dual-luciferase reporter assay, which was conducted in 293T cells expressing normal endogenous levels of SP1 (NC) or overexpressing SP1 off a plasmid (OE). N = 3. (C) Levels of chromatin containing the BE1 or BE2 sequence within region P3 of the TGF-β1 promoter were quantified using ChIP-qPCR after immunoprecipitation of chromatin from 293T cells with anti-SP1 antibody or control antibody (IgG). Values represented enrichment relative to input DNA. N = 3. (D) Binding of SP1 to wild-type (WT) or mutated (mut) BE1 sequences within region BE1 of the TGF-β1 promoter in 293T cells, based on the dual-luciferase reporter assay. Cells expressed normal endogenous levels of SP1 (SP1 NC), reduced endogenous levels (sg-SP1) or higher levels of SP1 off a plasmid (SP1 OE). N = 3. (E) Levels of chromatin containing the BE1 sequence within region P3 of the TGF-β1 promoter were quantified using ChIP-qPCR after immunoprecipitation of chromatin from 293T cells with anti-SP1 antibody or control antibody (IgG). Macrophages, which were cultured alone, expressed higher levels of SP1 off a plasmid (SP1 OE), or reduced endogenous levels (sg-SP1). Values represented enrichment relative to input DNA. N = 3. (F) Experiments analogous to those in panel (D), but involving M2 macrophages cultured alone. N = 3. (G) M2 macrophages stably expressing BE1 mutant sequences were co-cultured with ligamentum flavum fibroblasts (LGF), and the proliferation activity of LGF was detected using the Cell Counting Kit-8 at 0, 2, and 4 days, with detection performed at 450 OD, respectively. The SP1-OE plasmid was used to overexpress endogenous SP1 in M2 macrophages. TGF-β1 (5 ng/mL) was used to stimulate fibroblasts. Each experiment was independently repeated three times. (H) Transwell assay in which M2 macrophages were co-cultured with LGF and the migration of the fibroblasts was measured. Photomicrographs were taken after 4 days of co-culture, followed by staining with crystal violet. Quantitative analysis is presented below, with cell counts determined by randomly selecting three fields of view at the same magnification and averaging the results. Each experimental group underwent three independent replications. Scale bar, 100 μm. (I) The expression of collagen I and III in LGF was detected by immunofluorescence, and the mean fluorescence intensity was quantified on the right. Random selection of three fields of view at the same magnification. Each experimental group underwent three independent replications. Scale bar, 200 μm. (J) Levels of protein LOXL2, Smad4, Smad2/3, p-Smad2/3, and Smad7 in LGF after 4 days co-culture with M2 macrophages that had been transfected and treated as described. Quantitative analysis of western blots is on the right. N = 3. Ligamentum flavum fibroblasts (LGF). Data are mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Then the M0 macrophages were differentiated into M2 macrophages by incubating them for 48 h in medium containing IL-4 (20 ng/ml, cat# P00021, Solarbio) and IL-13 (20 ng/ml, cat# P00131, Solarbio).

Techniques: Activation Assay, Binding Assay, Sequencing, Mutagenesis, Luciferase, Reporter Assay, Expressing, Plasmid Preparation, ChIP-qPCR, Immunoprecipitation, Control, Cell Culture, Stable Transfection, Activity Assay, Cell Counting, Transwell Assay, Migration, Co-Culture Assay, Staining, Immunofluorescence, Fluorescence, Selection, Transfection, Western Blot

Journal: iScience

Article Title: Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages

doi: 10.1016/j.isci.2023.108484

Figure Lengend Snippet: Truncated mutation in SP1 cannot induce activation of fibroblasts by binding with the BE1 sequence of TGFB1 (A) Levels of chromatin containing the BE1 sequence within the TGF-β1 promoter were quantified using ChIP-qPCR after immunoprecipitation of chromatin from M2 macrophages cells using anti-SP1 antibody or control antibody (IgG). In SP1 ΔZn group, SP1 protein was missing three zinc fingers. Full-length SP1 protein was used as a normal control. Values represented enrichment relative to input DNA. N = 3. (B) Analysis of binding efficiency of truncated mutated SP1 to BE1 sequences, based on a dual-luciferase reporter assay. The full-length SP1 protein was used as a control. N = 3. (C) Concentration of TGF-β1 in the medium of cultures of LGF together with M2 macrophages transfected with truncated mutant SP1. N = 3. (D) Transwell assay in which M2 macrophages that transfected with truncated mutant SP1 were co-cultured with LGF, and the migration of the fibroblasts was measured. Photomicrographs were taken after 4 days of co-culture, followed by staining with crystal violet. Quantitative analysis is presented on the right, with cell counts determined by randomly selecting three fields of view at the same magnification and averaging the results. Each experimental group underwent three independent replications. Scale bar, 50 μm. (E and F) The expression of collagen I and III in LGF was detected by immunofluorescence. Quantitative analysis is presented on the right below, randomly selecting three fields of view at the same magnification and averaging the results. Each experimental group underwent three independent replications. Scale bar, 200 μm. (G) M2 macrophages stably expressing truncated mutant SP1 were co-cultured with ligamentum flavum fibroblasts (LGF), and the proliferation activity of LGF was detected using the Cell Counting Kit-8 at 0, 2, and 4 days, with detection performed at 450 OD, respectively. Full-length SP1 protein was used as a normal control. Each experiment was independently repeated three times. (H) Levels of protein LOXL2, Smad4, Smad2/3, p-Smad2/3, and Smad7 in LGF after 4 days co-culture with M2 macrophages that had been transfected and treated as described. Quantitative analysis of western blots is on the right. N = 3. Ligamentum flavum fibroblasts (LGF). Data are mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Then the M0 macrophages were differentiated into M2 macrophages by incubating them for 48 h in medium containing IL-4 (20 ng/ml, cat# P00021, Solarbio) and IL-13 (20 ng/ml, cat# P00131, Solarbio).

Techniques: Mutagenesis, Activation Assay, Binding Assay, Sequencing, ChIP-qPCR, Immunoprecipitation, Control, Zinc-Fingers, Luciferase, Reporter Assay, Concentration Assay, Transfection, Transwell Assay, Cell Culture, Migration, Co-Culture Assay, Staining, Expressing, Immunofluorescence, Stable Transfection, Activity Assay, Cell Counting, Western Blot

Journal: iScience

Article Title: Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages

doi: 10.1016/j.isci.2023.108484

Figure Lengend Snippet: The specific degradation of SP1 protein can significantly inhibit the fibrosis of ligamentum flavum in rats (A) Representative micro-computed tomographic images from rats with acupuncture-induced fibrosis of the ligamentum flavum (“Model”, n = 3) or control (n = 3). The red dashed line indicates the area of the ligamentum flavum. Contrast was normalized using ImageJ. (B) Thin sections of L5/6 ligamentum flavum from rats involving healthy controls (Ctrl); injured ligamentum flavum (Model) and injured ligamentum flavum treated with momordicoside G (MDG, 40 μM, 0.5mL) and plicamycin (Pli, 2.3 mM, 0.5mL). Sections were stained with hematoxylin-eosin. Scale bar, 50 μm. N = 6. (C) Scanning electron micrographs of ligaments from rats treated as in panel (B). Representative picture from three separate experiments. Scale bar, 10 μm. (D) Masson stain of ligaments from rats treated as in panel (B). Quantitation of the collagen volume fraction is shown on the right. Scale bar, 50 μm. N = 6. (E) Immunostaining of thin sections of ligament from rats to detect collagen Ⅰ and Ⅲ. The staining of the primary antibody dilution served as a negative control. Scale bar, 50 μm. N = 6. (F) Immunofluorescent staining of SP1 and M2 macrophage marker CD163. Representative picture from 3 separate experiments. Scale bar, 50 μm. (G) Levels of protein LOXL2, Smad4, Smad2/3, p-Smad2/3, and Smad7 in ligaments from rats treated as in panel (B). Quantitative analysis of western blots is below. N = 3. Data are mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Then the M0 macrophages were differentiated into M2 macrophages by incubating them for 48 h in medium containing IL-4 (20 ng/ml, cat# P00021, Solarbio) and IL-13 (20 ng/ml, cat# P00131, Solarbio).

Techniques: Control, Staining, Quantitation Assay, Immunostaining, Negative Control, Marker, Western Blot

Journal: iScience

Article Title: Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages

doi: 10.1016/j.isci.2023.108484

Figure Lengend Snippet: Schematic diagram of crosstalk between M2 macrophages and fibroblasts The transcription factor SP1 directly binds to the binding element of the TGF-β1 promoter to promote the expression of TGF-β1. TGF-β1 further activates fibroblasts to perform tissue remodeling functions, thereby promoting fibrosis.

Article Snippet: Then the M0 macrophages were differentiated into M2 macrophages by incubating them for 48 h in medium containing IL-4 (20 ng/ml, cat# P00021, Solarbio) and IL-13 (20 ng/ml, cat# P00131, Solarbio).

Techniques: Binding Assay, Expressing