Journal: Gastroenterology

Article Title: The origin and contribution of cancer-associated fibroblasts in colorectal carcinogenesis

doi: 10.1053/j.gastro.2021.11.037

Figure Lengend Snippet: (A, B) Immunohistochemistry (IHC) for ACTA2 in human colorectal samples. (A) Representative pictures. (B) ACTA2 positivity in total stromal cells (visualized by hematoxylin counterstaining). 3 high power fields (HPFs, 400x)/patient, 4–5 patients each. (C) Violin plots depict ACTA2 transcripts in normal fibroblasts (n = 2053 cells) and CRC CAFs (n = 1854 cells) assessed by single-cell RNA-sequencing (scRNA-seq) from human colorectal tissues. (D) Kaplan-Meier survival curves in The Cancer Genome Atlas (TCGA) dataset. (E) Violin plots showing ACTA2 expression level in four consensus molecular subtypes (CMS). n = 76, 220, 72, and 143 patients (CMS1-4). (F) Scheme for the experimental course of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colorectal carcinogenesis. (G) Representative endoscopic images of the normal colon mucosa and AOM/DSS tumors. T, tumors. (H, I) Immunohistochemistry for ACTA2 in the normal mucosa and AOM/DSS tumors. (H) Representative pictures. (I) ACTA2 positivity in total stromal cells. 3 HPFs/mouse, 3 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison test (B), Wilcoxon rank-sum test (C), Log-rank test (D), Kruskal-Wallis test followed by Dunn’s multiple comparisons test (E), and two-tailed unpaired Student’s t-test (I). ****, P < 0.0001; **, P = 0.00299; *, P = 0.0451. Scale bars, 50 μm. In all figures, box plots have whiskers of maximum and minimum values; the boxes represent first, second (median), and third quartiles. In all violin plots, solid and dotted black lines denote median and quartiles, respectively.

Article Snippet: Then, to examine for genes expressed at the protein level in human CRC stroma, we interrogated human CRC immunohistochemistry data in the Human Protein Atlas database and selected 6 proteins that were highly expressed in the CRC stroma ( Figure 4B , Step 4 ; Supplementary Figure 14 ).

Techniques: Expressing, Immunohistochemistry, Single Cell, RNA Sequencing, Comparison, Two Tailed Test

Journal: Gastroenterology

Article Title: The origin and contribution of cancer-associated fibroblasts in colorectal carcinogenesis

doi: 10.1053/j.gastro.2021.11.037

Figure Lengend Snippet: (A) Experimental schematic for isolating colonic fibroblasts from the normal adult colon, AOM/DSS tumors, and postnatal day 14 colon. Gating strategy to isolate CD45 − Ter119 − CD31 − EPCAM − fibroblasts by fluorescence-activated cell-sorting (FACS) is shown for one mouse adult normal colon. n = 4 mice each. (B) Strategy to identify a colonic stromal gene upregulated in development and carcinogenesis, which is associated with human CRC survival. (1) Venn diagram showing 342 genes upregulated in AOM/DSS tumors and postnatal day 14 colon, compared with the normal adult colon fibroblasts. (2) Survival analysis using TCGA dataset. (3) Using our RNA-seq data, genes upregulated in EPCAM − CD31 − CD45 − Ter119 − fibroblasts compared with EPCAM + epithelial cells, both in the normal adult colon and AOM/DSS tumors, were selected. Mean ± s.e.m. (4) The Human protein atlas data were used to select genes whose protein expression was restricted to the CRC stroma. Mcam is highlighted in red. (C, D) Immunohistochemistry for MCAM. (C) Representative images. Blue, red, and green arrowheads denote MCAM expression in the normal adult colon, AOM/DSS tumor, and postnatal day 14 colon, respectively. (D) The ratio of MCAM + cells in total stromal cells (visualized by hematoxylin counterstaining). 3 HPFs/mouse, 3 mice each. (E) Co-Immunofluorescence for MCAM and ACTA2 using AOM/DSS tumors from Lepr -Cre; Rosa26-tdtomato mice. Yellow arrowheads denote Lepr -lineage MCAM + ACTA2 + CAFs. See Supplementary Figure 17C and D for quantification and separate channel images. (F, G) Co-immunofluorescence for MCAM and BrdU. (F) Representative images. Yellow arrowheads denote proliferating MCAM + cells. (G) The ratio of MCAM + BrdU + cells in total MCAM + cells. 3 HPFs/mouse, 2–3 mice each. Scale bars, 50 μm. ****, P < 0.0001. Log-rank test (B(2)), one-way ANOVA followed by Tukey’s post-hoc multiple comparison test (B(3) and D), and two-tailed unpaired Student’s t-test (G)

Article Snippet: Then, to examine for genes expressed at the protein level in human CRC stroma, we interrogated human CRC immunohistochemistry data in the Human Protein Atlas database and selected 6 proteins that were highly expressed in the CRC stroma ( Figure 4B , Step 4 ; Supplementary Figure 14 ).

Techniques: Marker, Fluorescence, FACS, RNA Sequencing, Expressing, Immunohistochemistry, Immunofluorescence, Comparison, Two Tailed Test

Journal: Gastroenterology

Article Title: The origin and contribution of cancer-associated fibroblasts in colorectal carcinogenesis

doi: 10.1053/j.gastro.2021.11.037

Figure Lengend Snippet: (A, B) Immunohistochemistry for MCAM in human colorectal samples. (A) Representative pictures. (B) MCAM positivity in total stromal cells (visualized by hematoxylin counterstaining). 3 HPFs (400x)/patient, 4–5 patients each. (C) Violin plots showing MCAM expression levels in four CMSs. n = 76 (CMS1), 220 (CMS2), 72 (CMS3), and 143 patients (CMS4). (D) A mouse colonic fibroblast cell line, YH2, was incubated with vehicle, recombinant TGFβ1, or recombinant TGFβ1 + TGFβ1-receptor inhibitor (Galunisertib) for 24 hours, followed by quantitative reverse-transcription PCR (qRT-PCR). mean ± s.e.m. n = 3. (E) ScRNA-seq data show MCAM transcript levels are positively correlated with ACTA2 expression in colorectal CAFs. n = 1854 CAFs. Solid line, linear regression (F, G) MCAM immunohistochemistry in a CRC tissue microarray (F) Representative images and scoring system. (G) Kaplan-Meier survival curves. Scale bars, 50 μm. ****, P < 0.0001; *, P = 0.0124. Kruskal-Wallis test followed by Dunn’s multiple comparisons test (B and C), one-way ANOVA followed by Tukey’s post-hoc multiple comparison test (D), Spearman correlation (E), and Log-rank test (G).

Article Snippet: Then, to examine for genes expressed at the protein level in human CRC stroma, we interrogated human CRC immunohistochemistry data in the Human Protein Atlas database and selected 6 proteins that were highly expressed in the CRC stroma ( Figure 4B , Step 4 ; Supplementary Figure 14 ).

Techniques: Expressing, Immunohistochemistry, Incubation, Recombinant, Reverse Transcription, Quantitative RT-PCR, Microarray, Comparison

Journal: Gastroenterology

Article Title: The origin and contribution of cancer-associated fibroblasts in colorectal carcinogenesis

doi: 10.1053/j.gastro.2021.11.037

Figure Lengend Snippet: (A) Experimental scheme showing orthotopic injection of Apc Δ/Δ , Kras G12D/ Δ , Trp53 Δ/Δ CRC organoids (AKP tumoroids) into the colon. WT, wild type; KO, knockout; IVIS, in vivo imaging system. (B) Kaplan-Meier survival curves. (C, D) Luciferase signals from AKP tumoroids were assessed by IVIS. 18 Mcam -WT and 16 KO mice. (E, F) Macroscopic evaluation of colon tumors. Mice were harvested 3 weeks after tumoroid injection. (E) Representative pictures. Dotted lines indicate tumors. (F) Quantification of tumor volumes. 2 injections/mouse, 8 Mcam -WT and 6 KO mice (G, H) Immunohistochemistry for CD68 and CD11b. (G) Representative pictures. M, macrophages as assessed by morphology. (H) 3,3’-Diaminobenzidine (DAB)-positive areas. A.U., arbitrary unit. (I) Venn diagram showing the overlap of 41 macrophage/monocyte chemoattractant genes identified by Gene Ontologies and 462 genes upregulated in MCAM high CAFs compared with MCAM low CAFs (scRNA-seq data from GSE132465 ). (J, K, L) Lentivirus-mediated overexpression of MCAM augments IL-1β-p65- Il34/Ccl8 signaling in YH2 cells. MCAM-overexpressing or empty YH2 cells were stimulated with recombinant IL-1β, followed by Western blotting (WB; J, K ) and qRT-PCR ( L ). mean ± s.e.m. n = 3 each. p-p65, phosphorylated p65. (M) Immunoprecipitation (IP) for MCAM-hemagglutinin (HA) tag with an anti-HA antibody, followed by western blotting. A green asterisk denotes the interaction of MCAM-HA with IL1R1. An anti-MYC antibody was used to detect IL1R1 protein tagged with MYC. Blue and red dotted boxes indicate mScarlet-HA and MCAM-HA proteins, respectively. (N, O) In situ hybridization (ISH) for Il34 , Ccl8 , and a negative control probe (bacterial DapB gene) (N) Representative pictures. Green dotted lines indicate borders between Stromal (S) and Epithelial (E) areas (visualized by hematoxylin counterstaining). Red arrowheads denote Il34 + or Ccl8 + stromal cells. (O) DAB + areas in the tumor stroma. Scale bars, 200 μm (A), 2 mm (E), 50 μm (G and N) All histopathological analyses were performed using mice harvested 3 weeks after tumoroid injection. 3 HPFs (400x)/tumor, 1–2 tumors/mouse, 5 mice each group (H and O). Log-rank test (B), two-tailed unpaired t-test with Welch’s correction (D), Mann-Whitney U-test (F, H, and O), and two-way ANOVA followed by Tukey’s post-hoc multiple comparison test (K and L). ****, P ≤ 0.0001; ***, P ≤ 0.001; *, P ≤ 0.05; n.s., P > 0.05

Article Snippet: Then, to examine for genes expressed at the protein level in human CRC stroma, we interrogated human CRC immunohistochemistry data in the Human Protein Atlas database and selected 6 proteins that were highly expressed in the CRC stroma ( Figure 4B , Step 4 ; Supplementary Figure 14 ).

Techniques: Injection, Knock-Out, In Vivo Imaging, Luciferase, Immunohistochemistry, Over Expression, Recombinant, Western Blot, Quantitative RT-PCR, Immunoprecipitation, In Situ Hybridization, Negative Control, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Scientific Reports

Article Title: Integrated machine learning and multi-omics analysis identifies ALOX5 as a potential therapeutic target for tubulointerstitial inflammation in diabetic kidney disease

doi: 10.1038/s41598-026-44445-0

Figure Lengend Snippet: Representative immunohistochemistry (IHC) images retrieved from the Human Protein Atlas (HPA) database. The panels display the baseline protein expression levels of ALOX5, ALOX5AP, CYSLTR1, and RELA (NF- \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\kappa$$\end{document} B p65) in normal renal tissue microarrays.

Article Snippet: To validate the protein expression levels of ALOX5, ALOX5AP, and other key markers in normal kidney tissue, we retrieved immunohistochemistry (IHC) images from the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Immunohistochemistry, Expressing

Journal: Scientific Reports

Article Title: Integrated machine learning and multi-omics analysis identifies ALOX5 as a potential therapeutic target for tubulointerstitial inflammation in diabetic kidney disease

doi: 10.1038/s41598-026-44445-0

Figure Lengend Snippet: Representative immunohistochemistry (IHC) images retrieved from the Human Protein Atlas (HPA) database. The panels illustrate the protein expression patterns of CD68 (pan-macrophage marker), iNOS (pro-inflammatory macrophage marker), and CD163 (anti-inflammatory macrophage marker) in normal renal tissue microarrays.

Article Snippet: To validate the protein expression levels of ALOX5, ALOX5AP, and other key markers in normal kidney tissue, we retrieved immunohistochemistry (IHC) images from the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Immunohistochemistry, Expressing, Marker

Journal: Scientific Reports

Article Title: Integrated machine learning and multi-omics analysis identifies ALOX5 as a potential therapeutic target for tubulointerstitial inflammation in diabetic kidney disease

doi: 10.1038/s41598-026-44445-0

Figure Lengend Snippet: Representative immunohistochemistry (IHC) images retrieved from the Human Protein Atlas (HPA) database. The panels display the baseline protein expression levels of ALOX5, ALOX5AP, CYSLTR1, and RELA (NF- \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\kappa$$\end{document} B p65) in normal renal tissue microarrays.

Article Snippet: Figure 7 Representative immunohistochemistry (IHC) images retrieved from the Human Protein Atlas (HPA) database.

Techniques: Immunohistochemistry, Expressing

Journal: Scientific Reports

Article Title: Integrated machine learning and multi-omics analysis identifies ALOX5 as a potential therapeutic target for tubulointerstitial inflammation in diabetic kidney disease

doi: 10.1038/s41598-026-44445-0

Figure Lengend Snippet: Representative immunohistochemistry (IHC) images retrieved from the Human Protein Atlas (HPA) database. The panels illustrate the protein expression patterns of CD68 (pan-macrophage marker), iNOS (pro-inflammatory macrophage marker), and CD163 (anti-inflammatory macrophage marker) in normal renal tissue microarrays.

Article Snippet: Figure 7 Representative immunohistochemistry (IHC) images retrieved from the Human Protein Atlas (HPA) database.

Techniques: Immunohistochemistry, Expressing, Marker

Journal: 3 Biotech

Article Title: Identification of a 9-gene autophagy-related signature for predicting prognosis and immune exhaustion features in breast cancer

doi: 10.1007/s13205-026-04756-5

Figure Lengend Snippet: Protein level validation of key signature genes. Representative immunohistochemistry (IHC) staining images of MTDH, HSP90AA1, and VDAC1 in normal breast tissues (upper panel) and breast cancer tissues (lower panel). The images were retrieved from the Human Protein Atlas (HPA) database. The results demonstrate that the protein expression of these risk genes is significantly higher in tumor tissues (strong brown staining) compared to normal tissues (weak or negative staining)

Article Snippet: To verify the expression of the identified prognostic genes at the translational level, immunohistochemistry (IHC) staining images were retrieved from the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Biomarker Discovery, Immunohistochemistry, Expressing, Staining, Negative Staining