Journal: Cell Death & Disease

Article Title: Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

doi: 10.1038/s41419-022-04607-0

Figure Lengend Snippet: a Confocal image of IF staining on sections of the small intestine (SI) of Cd44 Δie and Cd44 +/+ mice using anti-lysozyme antibodies. Scale bar: 50 µm. b Quantification of lysozyme-stained PCs in the crypts of Cd44 Δie and Cd44 +/+ mice. Error bars, ±SE. c qPCR of lysozyme and α-defensin5 (Lyz and Defa5) expression in intestinal crypts from Cd44 Δie and Cd44 +/+ mice. Gapdh and β-Actin were used as reference genes. Error bars, ±SE. d Nuclear β-catenin (arrowheads) IHC staining. Scale bar: 25 μm. e In situ hybridization (ISH) (brown, red) of Lgr5 and Axin2 expression in the SI. Positive control: Ppib . Error bars, ± SE. Scale bar: 50 µm. f , g Quantification of ISH. Error bars, ±SE. h qPCR of Wnt target genes and stem cell marker expression in SI crypts isolated from Cd44 Δie and Cd44 +/+ mice. Non-Wnt-regulated control: Cxcr4 . Error bars, ±SE. Student’s t test; * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. N = number of mice, n = number of crypts.

Article Snippet: Primary antibodies directed against CD44-ICD (1:200; Trans Genic Inc. Cat# KO601, RRID: AB_2833239), LRP6 (1:100; abcam Cat# ab75358, RRID: AB_2139308), DVL2 (1:100; LSBio Cat# LS-C340131, RRID: AB_2833240), AXIN (1:100; antikörper-online.de) or IgG control (Cell Signaling Cat# 011–01, RRID: AB_1550038, Cell Signaling Cat# 5415 S, RRID: AB_10829607) were diluted in Duolink® Antibody Diluent and incubated overnight at 4 °C in blocking solution.

Techniques: Staining, Expressing, Immunohistochemistry, In Situ Hybridization, Positive Control, Marker, Isolation

Journal: Cell Death & Disease

Article Title: Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

doi: 10.1038/s41419-022-04607-0

Figure Lengend Snippet: a IHC staining of OLFM4. Scale bar: 50 μm. b Average number of OLFM4-positive stem cells per crypt. Error bars, ±SE. N = number of mice, n = number of crypts. c SI organoids observed 5 days after Cd44 deletion and controls. Scale bar: 200 µm. d Budded vs. round organoid numbers after deletion of Cd44 and passaging (P) every 5 days. n = number of organoids. Error bars, ±SE. e Representative images of f . Scale bar: 200 µm. f Relative organoid number along passages in Cd44 fl/fl ;VillinCreER T2 organoid cultured with or without 4-OHT treatment. N = 3 mice. Scale bar: 500 µm. g Organoid vs. cystic organoid numbers after deletion of Cd44 and treatment with Wnt3a CM, control CM or CHIR. N = 3 experiments. Error bars, ±SE. h Representative images of g CHIR and Wnt3a conditions. Scale bar: 200 µm. Student’s t test; * p < 0.05, ** p < 0.01, ns = not significant. 4-OHT 4-hydroxytamoxifen. The dotted line in g indicates that the CHIR and Wnt3a conditions are two independent but related experiments.

Article Snippet: Primary antibodies directed against CD44-ICD (1:200; Trans Genic Inc. Cat# KO601, RRID: AB_2833239), LRP6 (1:100; abcam Cat# ab75358, RRID: AB_2139308), DVL2 (1:100; LSBio Cat# LS-C340131, RRID: AB_2833240), AXIN (1:100; antikörper-online.de) or IgG control (Cell Signaling Cat# 011–01, RRID: AB_1550038, Cell Signaling Cat# 5415 S, RRID: AB_10829607) were diluted in Duolink® Antibody Diluent and incubated overnight at 4 °C in blocking solution.

Techniques: Immunohistochemistry, Passaging, Cell Culture

Journal: Cell Death & Disease

Article Title: Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

doi: 10.1038/s41419-022-04607-0

Figure Lengend Snippet: a Experimental design. b Daily body weight change (% of initial body weight) of Cd44 Δie and Cd44 +/+ mice subjected to 2.5% DSS or normal drinking water. The mean weight difference of the two DSS treated groups ( Cd44 Δie vs. Cd44 +/+ mice) was compared for every timepoint separately. Error bars, ±SE. c Representative images of colons from DSS-treated Cd44 Δie and Cd44 +/+ mice. d Colon lengths from Cd44 Δie and Cd44 +/+ mice +/− DSS. Error bars, ±SE. e Disease activity index (DAI) of the mice described in b . Error bars, ±SE. f H&E staining of colon sections of the mice described in b . Scale bar: 200 µm. Asterisk: submucosa. Arrowheads: mucosa (a’–d’ is an enlargement of a–d). g Histological colitis score (HCS) of the mice described in b . Error bars, ±SE. Student’s t test; * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, except Fig. 3e, g: Mann–Whitney U -test. IP intraperitoneal, TAM tamoxifen, N = number of mice.

Article Snippet: Primary antibodies directed against CD44-ICD (1:200; Trans Genic Inc. Cat# KO601, RRID: AB_2833239), LRP6 (1:100; abcam Cat# ab75358, RRID: AB_2139308), DVL2 (1:100; LSBio Cat# LS-C340131, RRID: AB_2833240), AXIN (1:100; antikörper-online.de) or IgG control (Cell Signaling Cat# 011–01, RRID: AB_1550038, Cell Signaling Cat# 5415 S, RRID: AB_10829607) were diluted in Duolink® Antibody Diluent and incubated overnight at 4 °C in blocking solution.

Techniques: Activity Assay, Staining, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

doi: 10.1038/s41419-022-04607-0

Figure Lengend Snippet: a IHC staining of F4/80 (macrophages) on colon sections. Arrowheads: mucosa, Arrows: submucosa. Scale bar: 50 µm. b IF staining of myeloperoxidase (MPO) (neutrophils) on colon sections. Scale bar: 50 µm. c qPCR of inflammation marker Tnfα expression in colon samples from Cd44 Δie and Cd44 +/+ mice. Reference genes: Gapdh and β-Actin . Error bars, ±SE. Inflammation: Mann–Whitney U -test. Regeneration: Student’s t test with unequal variances; * p < 0.05. N = number of mice.

Article Snippet: Primary antibodies directed against CD44-ICD (1:200; Trans Genic Inc. Cat# KO601, RRID: AB_2833239), LRP6 (1:100; abcam Cat# ab75358, RRID: AB_2139308), DVL2 (1:100; LSBio Cat# LS-C340131, RRID: AB_2833240), AXIN (1:100; antikörper-online.de) or IgG control (Cell Signaling Cat# 011–01, RRID: AB_1550038, Cell Signaling Cat# 5415 S, RRID: AB_10829607) were diluted in Duolink® Antibody Diluent and incubated overnight at 4 °C in blocking solution.

Techniques: Immunohistochemistry, Staining, Marker, Expressing, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

doi: 10.1038/s41419-022-04607-0

Figure Lengend Snippet: a IHC staining using antibodies against β-catenin. Arrowheads: nuclear β-catenin ( N = 3 mice). Scale bar: 50 μm. b In situ hybridization (ISH) (brown, red) of Lgr5 Wnt target gene expression in the SI of Cd44 Δie and Cd44 +/+ mice treated with DSS. Positive control: Ppib . Scale bar: 50 µm. c Quantification of ISH staining. Error bars, ±SE. Student’s t test, (* p < 0.05). TAM tamoxifen, N = number of mice, n = number of crypts. d Kaplan–Meier analysis of Crohn’s disease patients (GSE137344) relapse-free survival and expression of CD44 above or below the median. Hazard ratio (HR) = 1.4; p = 0.038; n = 112; dotted lines = SD. e Corrplot package analysis for visualization of the correlation matrix between CD44 and the indicated genes in Crohn’s disease patients (GSE171244). The values in the circles are the adjusted p values and the color intensities correspond to Spearman correlation values. Adjusted p values < 0.01 are annotated as 0. Sizes of the circle are proportional to the p value.

Article Snippet: Primary antibodies directed against CD44-ICD (1:200; Trans Genic Inc. Cat# KO601, RRID: AB_2833239), LRP6 (1:100; abcam Cat# ab75358, RRID: AB_2139308), DVL2 (1:100; LSBio Cat# LS-C340131, RRID: AB_2833240), AXIN (1:100; antikörper-online.de) or IgG control (Cell Signaling Cat# 011–01, RRID: AB_1550038, Cell Signaling Cat# 5415 S, RRID: AB_10829607) were diluted in Duolink® Antibody Diluent and incubated overnight at 4 °C in blocking solution.

Techniques: Immunohistochemistry, In Situ Hybridization, Expressing, Positive Control, Staining

Journal: Cell Death & Disease

Article Title: Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

doi: 10.1038/s41419-022-04607-0

Figure Lengend Snippet: a Mean fluorescence of chromobodies (CB) against active β-catenin in the nucleus of DMSO, CHIR, control CM, or Wnt3a CM-treated HeLa_BC1-TagGFP2 or CD44 −/− _HeLa_BC1-TagGFP2 cells 24 h after induction. Control CM vs. Wnt3a CM: Mann–Whitney U -test. DMSO vs. CHIR: Student’s t test. Error bars, ±SE. b , c CD44/LRP6, CD44/DVL, and CD44/AXIN d , e in situ PLA complexes (dots) in HeLa cells treated with Wnt3a CM for 15 and 45 min, compared to control CM-treated cells. Cells were incubated with primary antibodies against CD44 and LRP6, DVL, or AXIN. Error bars, ±SE. Scale bar: b 20 μm, d 10 μm. c , e Fold induction of average interactions per cell, compared to control CM treatment. Error bars, ±SE. Student’s t test. f , g Co-immunoprecipitation of AXIN/CD44 ( f ) and CD44/DVL ( g ) on lysates of HeLa cells induced with Wnt3a CM for the indicated time points. Student’s t test/Mann–Whitney U -test: * p < 0.05, ** p < 0.01, ns not significant. n = number of analyzed cells.

Article Snippet: Primary antibodies directed against CD44-ICD (1:200; Trans Genic Inc. Cat# KO601, RRID: AB_2833239), LRP6 (1:100; abcam Cat# ab75358, RRID: AB_2139308), DVL2 (1:100; LSBio Cat# LS-C340131, RRID: AB_2833240), AXIN (1:100; antikörper-online.de) or IgG control (Cell Signaling Cat# 011–01, RRID: AB_1550038, Cell Signaling Cat# 5415 S, RRID: AB_10829607) were diluted in Duolink® Antibody Diluent and incubated overnight at 4 °C in blocking solution.

Techniques: Fluorescence, MANN-WHITNEY, In Situ, Incubation, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

doi: 10.1038/s41419-022-04607-0

Figure Lengend Snippet: Since CD44 is a Wnt target gene and a positive modulator of signal transduction of the Wnt pathway, it establishes a positive feedback loop to amplify Wnt signaling in the intestine acting at the level of the signalosome. Removal of Cd44 from the intestinal epithelium leads to decreased Wnt signaling still sufficient in homeostatic conditions but fails to increase Wnt activity as required in the regeneration of the intestinal epithelium after DSS damage.

Article Snippet: Primary antibodies directed against CD44-ICD (1:200; Trans Genic Inc. Cat# KO601, RRID: AB_2833239), LRP6 (1:100; abcam Cat# ab75358, RRID: AB_2139308), DVL2 (1:100; LSBio Cat# LS-C340131, RRID: AB_2833240), AXIN (1:100; antikörper-online.de) or IgG control (Cell Signaling Cat# 011–01, RRID: AB_1550038, Cell Signaling Cat# 5415 S, RRID: AB_10829607) were diluted in Duolink® Antibody Diluent and incubated overnight at 4 °C in blocking solution.

Techniques: Transduction, Activity Assay

Journal: Nature Communications

Article Title: Longitudinal plasma proteome profiling reveals the diversity of biomarkers for diagnosis and cetuximab therapy response of colorectal cancer

doi: 10.1038/s41467-024-44911-1

Figure Lengend Snippet: A Association of proteomic subtypes with therapy response (two-sided Fisher’s exact test). B Correlation of the pathways of the G-III subtype (two-sided Pearson’s correlation test). C , E Boxplots for immune score among G-I ( N = 24), G-II ( N = 34), and G-III ( N = 31) ( P = 0.042) ( C ), and CD8+Tem score between S and NS groups in plasma samples (N (S) = 16, N (NS) = 15; P = 0.035) and tissue samples (N (S) = 12, N (NS) = 19; P = 0.005) (two-sided Student’s t test). D Differential cell types between G-III and other subtypes (two-sided Student’s t test). F Qualification of CD44 ( P = 1.0E-5) and GZMK ( P = 8.0E-6) stained by IHC in representative examples (two-sided Student’s t test). Data are shown as mean ± SD ( n = 3 independent experiments). G Association between CD8+Tem score (Z score) with tumor size (cm), and S/NS group. H Correlation of CD8+Tem score and tumor size (two-sided Pearson’s correlation test). Data are presented as Pearson r with 95%CI. I Diagram showing the potential mechanism of cetuximab sensitivity. The little heatmap depicted the log-transformed fold-change of S versus NS groups. J Correlation of CD8+Tem score and RPTOR/IMPDH2 (two-sided Pearson’s correlation test). K Kaplan–Meier curves of Overall survival (OS) in the CPTAC cohort (two-sided log rank test). L Differential expression of RPTOR ( P = 8.9E-5, 2.9E-5) and IMPDH2 ( P = 4.2E-8, 3.4E-6) in discovery cohort (N (S) = 31, N (NS) = 45) and validation cohort (N (S) = 16, N (NS) = 15) (two-sided Wilcoxon rank-sum test). PRM, parallel reaction monitoring. Boxplots show median (central line), upper and lower quartiles (box limits), 1.5×interquartile range (whiskers) ( C , E , and L ). Source data are provided as a file.

Article Snippet: A standard IHC protocol was followed to stain the tumor tissue samples of the CD8+ Tem markers by using the rabbit monoclonal antibody against CD44 (1:200, Signalway Antibody, catalog No: 48911-1), and the rabbit polyclonal antibody against GZMK (1:300, Signalway Antibody, catalog No: 40985-1).

Techniques: Clinical Proteomics, Staining, Transformation Assay, Quantitative Proteomics, Biomarker Discovery, Targeted Proteomics