Journal: bioRxiv

Article Title: CRISPR-mediated conditional mutagenesis of Smad1/5/8 reveals BMP/GDF signaling restricts postnatal bone overgrowth

doi: 10.64898/2026.01.22.701170

Figure Lengend Snippet: (A) Representative images of SCA-1 immunostaining of longitudinal radius sections at two months focused on the mid-diaphysis of Cas9 eGFP ; Prx1 -cre control and PCC mice (n=4 Cas9 eGFP ; Prx1 -cre and n=6 PCC). (B) Quantification of fibrous layer thickness (μm) at the mid-diaphysis, ROI represented in (A). (C) Representative images for Periostin (POSTN, magenta) and Ki67 (proliferation, green) immunostaining of longitudinal radius sections at two months (n=6 Cas9 eGFP ; Prx1 -cre and n=8 PCC). (D) Quantification of proliferation index (% Ki67+) at the mid-diaphysis, ROIs represented in (C). Significant periosteal expansion results from increased proliferation of cells in the cambium layer. (E) PECAM staining of longitudinal radius sections at two months (n=4 Cas9 eGFP ;Prx1- cre and n=5 PCC). (F) Representative images for RUNX2 (magenta) and Ki67 (green) immunostaining at the mid-diaphysis (n=4 Cas9 eGFP ; Prx1 -cre and n=4 PCC). (G) Quantification of RUNX2+ (magenta bars), Ki67+ (green bars), and RUNX2+Ki67+ double positive cells (magenta and green striped bars) in ROIs represented in (F). (H) Representative images for COL1A1 (magenta) and Ki67 (green) immunostaining at the mid-diaphysis (n=6 Cas9 eGFP ; Prx1 -cre and n=6 PCC). (I) Quantification of COL1A1+ (magenta bars), Ki67+ (green bars), and Col1a1+Ki67+ double positive cells (magenta and green striped bars) in ROIs represented in (H). B = bone. c = cambium layer. f = fibrous layer. Ma = marrow. Scale bars = 50 μm. Arrowheads indicate Ki67+ cells. Data represent mean +/-SD. * denotes Welch’s t-test <0.05. ** denotes Welch’s t-test <0.005. *** denotes Welch’s t-test <0.0005. ns = not significant.

Article Snippet: The following primary antibodies and dilutions were used for immunofluorescence of tissue sections: SCA-1(Invitrogen, 14-5981-82, 1: 250); POSTN (Abcam, ab14041, 1: 100) [citric acid]; PECAM (Invitrogen, 14-0311-82, 1: 250); GFP (Invitrogen, A10262, 1: 500); RUNX22 (Abcam, ab76956, 1:500); COL1A1 (Developmental Studies Hybridoma Bank, Sp1.D8, 1:20) [citric acid]; Ki67 rat (Invitrogen, 14-5698-82, 1: 150); Ki67 rabbit (Abcam, ab15580, 1:200); PRX1 (Invitrogen, PA5-106700, 1: 100).

Techniques: Immunostaining, Control, Staining

Journal: International Journal of Molecular Medicine

Article Title: Deciphering the CAF-LCN2 axis: Key to overcoming anti-PD-L1 immunotherapy resistance in lung cancer

doi: 10.3892/ijmm.2026.5735

Figure Lengend Snippet: Influence of LCN2 on the growth of lung cancer in mice. (A) Simplified flowchart outlining the establishment of an anti-PD-L1 immunotherapy tolerance model in mice with lung cancer. (B) Western blot analysis of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice. (C) Immunohistochemical detection of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice (scale bar, 100 µm). (D) Establishment of a model for anti-PD-L1 immunotherapy in LCN2-silenced mice. (E) Evaluation of LCN2 mRNA expression in tumor tissues after LCN2 gene intervention in mice. (F) Assessment of LCN2 protein expression in tumor tissues following LCN2 gene intervention in mice. (G) Tumor size in LCN2 gene intervention mice (n=10 in each group). *P<0.05, **P<0.01 and ***P<0.001. Unpaired two-tailed Student's t-test was used for (B) and (C) and one-way ANOVA with Tukey's multiple comparisons test was used for (E) and (G). LCN2, lipocalin 2; PD-L1, programmed death-ligand 1; sh-, short hairpin; NC, negative control; COL1A1, collagen α-1(I) chain; THY1, Thy-1 membrane glycoprotein; i.p., intraperitoneal.

Article Snippet: Following permeabilization, cells were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-collagen α-1(I) chain (COL1A1) (cat. no. PA5-101300; 1:250; Invitrogen; Thermo Fisher Scientific, Inc.), rabbit anti-Cytokeratin (cat. no. ab53280; 1:500; Abcam) and mouse anti-CD31 (cat. no. sc-376764; 1:50; Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Two Tailed Test, Negative Control, Membrane

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: Rabeprazole inhibits fibrosis. After treatment with or without rabeprazole stimulation for 24 h at indicated concertations in (A) GES-1 and (B) AGS-1 cells, reverse transcription-quantitative PCR was conducted to analyze gene expression. Data was displayed as the mean ± SD and quantified by one-way ANOVA, followed by the Dunnett's post hoc test for significance against 0 µM. * P<0.05, ** P<0.01 and **** P<0.0001; n=3. (C) GES-1 and (D) AGS cells were treated as previosuly described in (A and B). The total cells were harvested to detect the indicated protein levels. The quantitation of bands was analyzed using one-way ANOVA, followed by the Dunnett's post hoc test for significance against 0 µM. ** P<0.01, *** P<0.001 and **** P<0.0001; n=3. FN, fibronectin; Col1a1, collagen type I alpha 1 chain; α-SMA, α-smooth muscle actin.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Gene Expression, Quantitation Assay

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: TIF1γ is essential for rabeprazole-modulated ECM. (A) GES-1 and (B) AGS cells were treated with or without rabeprazole for 48 h, and the expression of TIF1γ mRNA was analyzed by reverse transcription-quantitative PCR. Data are shown as the mean ± SD and quantified by one sample t-test for significance against 0 µM. *** P<0.001; n=3. (C) Western blotting was used to detect the protein level of TIF1γ in AGS cells in the absence or presence of rabeprazole for 48 h, and the bands were quantified and analyzed using one sample t-test. Data are displayed as the mean ± SD. **** P<0.0001; n=3. (D) TIF1γ promoter plasmid combined with Renilla plasmid were co-transfected into AGS cell for 24 h, followed by treatment with or without rabeprazole for another 24 h. Relative light units were measured using the dual-luciferase reporter assay system according to the manufacturer's instructions. Data are displayed as the mean ± SD and quantified by two sample t-test for significance against 0 µM. ** P<0.01; n=3. (E) Following transfection with pooled shTIF1γ plasmids overnight, AGS cells were treated with or without rabeprazole for another 48 h, and the bands were quantified and analyzed using one sample t-test. ** P<0.01 and **** P<0.0001; n=3. TIF1γ, transcriptional intermediary factor 1γ; FN, fibronectin; Col1a1, collagen type I alpha 1 chain.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection, Luciferase, Reporter Assay