Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 drives tumor-educated M2-like polarization of macrophages and promotes tumor-cell aggressiveness. (A) Workflow for generating TC-hMDMs and TC-BMDMs, circSMAD4 knockdown, and downstream functional assays. (B) RT–qPCR analysis of M1-associated markers (MHC-II [HLA-DRA in TC-hMDMs; H2-Ab1 in TC-BMDMs], NOS2, and CD86) and M2-associated markers (CD163, CD206, and ARG1) in TC-hMDMs and TC-BMDMs. (C) Representative flow-cytometry histograms for HLA-DR, iNOS, CD86, CD163, CD206, and ARG1 in TC-hMDMs. Gating strategy and marker thresholds were defined based on FMO controls (see ). (D) Flow-cytometry quantification of marker-positive cells in TC-hMDMs and TC-BMDMs. (E) ELISA of IL-10, TGF-β, and iNOS in culture supernatants. (F) CCK-8 assays of A549 and LLC cells. (G) Colony-formation assays of A549 and LLC cells with quantification. (H) Bioluminescence-based growth readouts of patient-derived LUAD organoids (PDO #1 and PDO #2) after co-culture with TC-hMDMs. (I) Immunoblot analysis of EMT-related proteins (E-cadherin, N-cadherin, Vimentin) in A549 and LLC cells. (J) Transwell migration and invasion assays of A549 and LLC cells with quantification. Scale bar, 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Sections were incubated with primary antibodies against Ki-67 (Servicebio, Cat# GB111499 ), E-cadherin (Proteintech, Cat# 20874-1-AP), and Vimentin (Proteintech, Cat# 10366-1-AP).

Techniques: Knockdown, Functional Assay, Quantitative RT-PCR, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Derivative Assay, Co-Culture Assay, Western Blot, Migration

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 depletion in macrophages restrains LUAD growth and metastasis in vivo. (A) Schematic of orthotopic lung implantation and experimental metastasis models using LLC cells mixed with BMDMs expressing shNC or sh-circSMAD4. (B) Representative images of orthotopic lung tumors. (C) Tumor weight of orthotopic implants. (D) Overall survival of mice bearing orthotopic tumors. (E) Immunofluorescence showing F4/80 and circSMAD4 signals in tumor tissues. Scale bar, 50 μm. (F, G) Representative Ki-67 IHC staining and quantification in orthotopic tumors. Scale bar, 50 μm. (H) Representative bioluminescence images of lung tumor burden in the metastasis model. (I) Tumor weight in the metastasis model. (J) Overall survival of mice in the metastasis model. (K–M) Representative IHC staining and quantification of E-cadherin and vimentin in tumors. Scale bar, 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Sections were incubated with primary antibodies against Ki-67 (Servicebio, Cat# GB111499 ), E-cadherin (Proteintech, Cat# 20874-1-AP), and Vimentin (Proteintech, Cat# 10366-1-AP).

Techniques: In Vivo, Expressing, Immunofluorescence, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Hyperglycemia increased the gingival senescent burden and induced serum SASP in diabetic mice. A, all protocols were performed strictly according to the procedure. C57 mice were rendered diabetic by STZ injections and sacrificed every 2 weeks. B, fasting glucose levels were determined every 2 weeks from weeks 5 to 17. The p value between control mice (N group) and diabetic mice (D group) is shown. **, p < 0.01. C, Western blotting analysis showing p16 and p21 specific immunoreactivity in the gingival tissue of the N group and the D group. The optical density (O.D.) values of p16 and p21 levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus N group. Panel C here and panels A and E in Fig. 3 use the same band of β-actin because of the identical protein sample in same Western blotting experiment. D, immunohistochemistry using antibody against p16 and p21 was analyzed in the gingival tissue of N group and D group. Scale bar, 50 μm. The percentage of positive cells is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus N group. E, the SASP factors in the serum of N group and D group were determined every 2 weeks by a Luminex assay customization tool and shown in the heat map. F, gingival tissues of N group and D group were stained for immunofluorescence using an F4/80 antibody targeting macrophages (red) and a p16 antibody (green). The nuclei were stained with DAPI (blue). Scale bar, 50 μm.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Control, Western Blot, Immunohistochemistry, Luminex, Staining, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: High glucose induced cellular senescence and SASP in macrophage derived from RAW 264.7 cell. A, the activity of SA–β-gal was determined in macrophage exposed to 5–30 mm glucose for 6 or 24 h. Scale bar, 100 μm. The rate of SA–β-gal–positive (blue-stained) cells is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus 30 mm glucose for 6 h. ##, p < 0.01 versus 30 mm glucose for 24 h. B, the expression levels of p16 and p21 in macrophage were analyzed by Western blotting. The O.D. values of p16 and p21 levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus 30 mm glucose for 6 h. #, p < 0.05; ##, p < 0.01 versus 30 mm glucose for 24 h. Panel B here and panels C and F in Fig. 3 use the same band of β-actin because of the identical protein sample in same Western blotting experiment. C, the SASP factors in the supernatant of macrophage were determined by a Luminex assay customization tool and shown in the heat map. D, cell proliferation was detected using EdU detection kits to analyze the incorporation of EdU during DNA synthesis. Scale bar, 100 μm. The percentage of proliferating cells is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus 30 mm glucose for 6 h. ##, p < 0.01 versus 30 mm glucose for 24 h.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Derivative Assay, Activity Assay, Staining, Expressing, Western Blot, Luminex, DNA Synthesis

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: High glucose was incapable of inducing cellular senescence and SASP in in IRF8−/− or NLRC4−/− macrophage exposed to 30 mm glucose for 24 h. A, the activity of SA–β-gal was determined in control, NLRC4−/−, and IRF8−/− macrophage exposed to 5 and 30 mm glucose for 24 h. Scale bar, 100 μm. The rate of SA–β-gal–positive cells is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose). B, p16 and p21 levels were measured by immunofluorescence staining. Scale bar, 50 μm. The fluorescence intensity is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose). C, the change of SASP-associated factors is displayed in the heat map. D, Western blotting analysis showing IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 specific immunoreactivity. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose).

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Activity Assay, Control, CRISPR, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Metformin ameliorated the burden of senescent cells in gingival tissue and the SASP in serum of diabetic mice. A, all protocols were performed strictly according to the procedure. The diabetic mice were treated with the metformin (300 mg/kg body weight, everyday) from weeks 9 to 17. B, fasting blood glucose levels were determined at sacrifice (week 17) among control mice (N group), diabetic mice (D group), and diabetic mice treated with metformin (DM group). The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus D group. C, representative of immunohistochemical staining of p16, p21, IRF8, and NLRC4 on the gingival sections from the N, D, and DM groups. Scale bar, 50 μm. The percentage of positive cells was calculated and is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus the D group. D, the SASP factors in the serum of the N, D, and DM groups were determined at sacrifice (week 17) by a Luminex assay customization tool and shown in the heat map. E, the gingival tissues of the N, D, and DM group mice were stained for immunofluorescence using an F4/80 antibody targeting macrophages (red) and a p16 antibody (green). The nuclei were stained with DAPI (blue). Scale bar, 50 μm. F, IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 in the gingival tissue of the N, D, and DM groups were analyzed by Western blotting. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus D group.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Control, Immunohistochemical staining, Staining, Luminex, Immunofluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Metformin attenuated high glucose–induced cellular senescence and SASP in macrophage exposed to high glucose (30 mm) for 24 h. A, the activity of SA–β-gal were determined in macrophage treated with low glucose (5 mm) (N), high glucose (30 mm) (HG), and high glucose (30 mm) + metformin (10 mm) (HGM). Scale bar, 100 μm. The rate of SA–β-gal–positive cells was calculated and is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus HG. B and E, p16, p21, IRF8, and NLRC4 levels were measured by immunofluorescence staining. Scale bar, 50 μm. The fluorescence intensity is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus HG. C, the change of SASP factors are displayed in the heat map. D, the expression levels of IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 in N, HG, and HGM were analyzed by Western blotting. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus HG.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Activity Assay, Immunofluorescence, Staining, Fluorescence, Expressing, Western Blot

Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: IL‐17A NAb inhibited EMT in SHR renal tissues. (A) Representative IHC images showing the expression of E‐cadherin, α‐SMA, and Collagen III, with quantitative analysis of their positive areas. (B) Representative immunoblots and relative expression levels of E‐cadherin, α‐SMA, and Collagen III proteins. (C) mRNA expression levels of E‐cadherin, α‐SMA, and Collagen III. Data are expressed as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Expressing, Western Blot