anti-rabbit olfm4 14369s  (Cell Signaling Technology Inc)

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Screening and validation of downstream target genes of miR-192-5p. A. Volcano plot of differentially expressed genes. B. Heatmap of differentially expressed genes with P < 0.05, |log 2 FC| > 1, and FPKM >1. C. Venn diagram showing the intersection of differentially expressed genes and predicted miR-192-5p target genes from the miRWalk database. D. Predicted binding sites of wild-type and mutated OLFM4 with miR-192-5p. E. Luciferase reporter assays in HEK-293 T cells after cotransfection with wild-type (WT) or mutant (MUT) CDS OLFM4 plasmids and miRNA mimics. (n = 3). F. RT-qPCR detection of OLFM4 expression changes in the HaCaTs oxidative stress model (n = 3). G-H. RT-qPCR analysis of OLFM4 mRNA expression changes in HaCaTs after overexpression and knockdown of miR-192-5p (n = 3). I-L. Immunofluorescence detection of OLFM4 protein expression changes in HaCaTs after overexpression and knockdown of miR-192-5p (scale bar: 50 μm, n = 3). M-P. Western blot analysis of OLFM4 protein expression changes in HaCaTs after overexpression and knockdown of miR-192-5p. Data are shown as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, and ns, no significance, by two-tailed unpaired Student's t -test (F-H, J, L, N and P), and by one-way ANOVA followed by the Tukey-Kramer's post hoc test (E).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Biomarker Discovery, Binding Assay, Luciferase, Cotransfection, Mutagenesis, Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Immunofluorescence, Western Blot, Two Tailed Test

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Overexpression of OLFM4 protected HaCaTs from dysfunction induced by miR-192-5p overexpression. A-C. RT-qPCR and immunofluorescence validation of the transfection efficiency of the OLFM4 overexpression plasmid (scale bar: 50 μm, n = 3). D. CCK8 assay to assess the effect of miR-192-5p and OLFM4 overexpression on HaCaTs cell viability (n = 3). E and F. Scratch assay to evaluate the effect of miR-192-5p and OLFM4 overexpression on HaCaTs migration ability (scale bar: 500 μm, n = 3). G and H. Flow cytometry analysis of the effect of miR-192-5p and OLFM4 overexpression on HaCaTs apoptosis (n = 3). I. ROS staining fluorescence images after overexpressing miR-192-5p and OLFM4(scale bar: 50 μm). J-K. Flow cytometry assessment of ROS levels in HaCaTs after overexpressing miR-192-5p and OLFM4 (n = 3). Data are shown as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001, and ns, no significance, by two-tailed unpaired Student's t -test (A and C), and by one-way ANOVA followed by the Tukey-Kramer's post hoc test (D, F, H and K).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Over Expression, Quantitative RT-PCR, Immunofluorescence, Biomarker Discovery, Transfection, Plasmid Preparation, CCK-8 Assay, Wound Healing Assay, Migration, Flow Cytometry, Staining, Fluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Knockdown of OLFM4 reversed the protective effect of miR-192-5p knockdown on HaCaTs function under oxidative stress model. A-C. RT-qPCR and immunofluorescence validation of the transfection efficiency of OLFM4 small interfering RNA (siRNA) (scale bar: 50 μm, n = 3). D. CCK8 assay to assess the effect of miR-192-5p and OLFM4 knockdown on HaCaTs cell viability (n = 3). E and F. Scratch assay to evaluate the effect of miR-192-5p and OLFM4 knockdown on HaCaTs migration ability (scale bar: 500 μm, n = 3). G and H. Flow cytometry analysis of the effect of miR-192-5p and OLFM4 knockdown on HaCaTs apoptosis (n = 3). I. ROS staining fluorescence images after miR-192-5p and OLFM4 knockdown (scale bar: 50 μm). J-K. Flow cytometry assessment of ROS levels in HaCaTs after knockdown of miR-192-5p and OLFM4 (n = 3). Data are shown as the mean ± SD. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, and ns, no significance, by two-tailed unpaired Student's t -test (A and C), and by one-way ANOVA followed by the Tukey-Kramer's post hoc test (D, F, H and K).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Knockdown, Quantitative RT-PCR, Immunofluorescence, Biomarker Discovery, Transfection, Small Interfering RNA, CCK-8 Assay, Wound Healing Assay, Migration, Flow Cytometry, Staining, Fluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Mechanistic exploration of the effect of OLFM4 on HaCaTs proliferation and apoptosis. A. Volcano plot of differentially expressed genes in HaCaTs overexpressing OLFM4. B. KEGG pathway enrichment analysis. C. Violin Plot of selected genes in the "Cell growth and death" pathway. D. Heatmap of enriched genes in the "Cell growth and death" pathway. E. RT-qPCR validation of the expression changes of genes enriched in the "Cell growth and death" pathway (n = 3). Data are shown as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, by two-tailed unpaired Student's t -test (E).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: TL1A-activated T cells remodel the rectal mucosa in Crohn’s disease patients with perianal fistulizing disease

doi: 10.1101/2025.06.26.657455

Figure Lengend Snippet: A: Dot plot representing the activity of the “epithelial-to-mesenchymal transition” (EMT) process (Gene Ontology) in each epithelial cell type comparing CDun and CD+PFDun patients. Dot size indicates adjusted p-value, while color scale indicates the arbitrary value of the pathway generated by the AddModuleScore function in the Seurat package. The Wilcoxon-rank sum test (two-sided) with Holm correction was applied to compare CDun and CD+PFDun patients. B: Whole-tissue qRT-PCR analysis of DUOXA2 and OLFM4 expression in rectal biopsies from CDun (n=8) and CD+PFDun (n=12) patients; Mann-Whitney test, * = q < 0.05, AU: arbitrary units. C: Uniform Manifold Approximation and Projection (UMAP) plots of the whole object, showing the expression of IL2RA , IL2RB , IL13RA1 , IL13RA2 , IL22RA1 , and IL22RA2 . D: Violin plot of IL13 expression in myeloid cells. None of the comparisons were significant by DEG analysis. E: Bar plot indicating the expression of IL22 in each of the T-cell subtypes. Bars in red indicate those cell types whose IL22 expression was significantly higher than the rest. Adjusted p-value was obtained by FindAllMarkers function. * = adjusted p-value < 0.05. F: qRT-PCR of epithelial organoids seeded as a 2D monolayer upon stimulation with IL-22 (5ng/ml) for 24 hours. Data is represented as the fold change (FC) of stimulated organoids relative to the unstimulated condition. One sample T test, parametric, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. n= 5. G: Correlation analysis of DUOX2 and OLFM4 expression to the levels of IL-22 protein production in the tissue explants stimulated with TL1A for 24 hours. Data is represented as the logarithm in base 2 of the FC (log2fc) relative to the unstimulated condition. r and p-values are indicated. H: Protein production of IL-22 in the supernatants of peripheric CD3+ T cells cultured for 48h and stimulated with anti-CD3, anti-CD3+TL1A or anti-CD3+TNF. Paired T test analysis compared to the anti-CD3 condition, * = p < 0.05, ns = p > 0.05.

Article Snippet: Sections were blocked with 20% horse serum and incubated overnight at 4C with anti-rabbit OLFM4 (14369S, Cell Signaling, concentration 1/100).

Techniques: Activity Assay, Generated, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Cell Culture

Journal: bioRxiv

Article Title: TL1A-activated T cells remodel the rectal mucosa in Crohn’s disease patients with perianal fistulizing disease

doi: 10.1101/2025.06.26.657455

Figure Lengend Snippet: A and B: On the left, volcano plot representing the differentially expressed genes (DEGs) in the AQP8+ enterocytes ( A ) or the transit amplifying cells (TA) ( B ) between CDun and CD+PFDun patients. A two-sided Wilcoxon rank sum test was applied. Genes with an adjusted p-value < 0.05, and an avgLog2FC ˃ log2 (1.2) or avgLog2FC ˂ -log2 (1.2) were considered up-(UPP) or down-regulated (DWW), respectively. Genes with a nominal p-value ˂ 0.05 are also indicated in light colors if avgLog2FC ˃ log2 (1.2) (UP) or avgLog2FC ˂ -log2 (1.2) (DW). On the right, dot plot indicating pathways upregulated in the AQP8+ enterocytes ( A ) or TA ( B ) of CD+PFDun patients. Dot size indicates adjusted p-value in logarithm base 10, while color scale indicates the arbitrary value of each biological process generated by the AddModuleScore function in the Seurat package. The Wilcoxon-rank sum test (two-sided) with Holm correction was applied to compare CDun and CD+PFDun patients. C: Violin plot of OLFM4 expression in epithelial cells. P-value was obtained by DEGs analysis. * = adjusted p-value < 0.05 and avgLog2FC ˃ log2 (1.2), # = nominal p-value ˂ 0.05 and avgLog2FC ˃ log2 (1.2). D: Olfactomedin 4 (OLFM4) immunohistochemistry staining of CDun and CD+PFDun formalin-fixed rectum biopsies. Representative images of tissue sections per each group are shown. Percentage of OLFM4+ cells were scored with the software QuPath. Unpaired t-Test, *** = p < 0.001. Scale bar: 100µm. E: qRT-PCR of organoid-derived monolayer (left) and cultured tissue explants (right) after 24 hours cultured with or without TL1A (300ng/ml). Data are represented as the fold change (FC) relative to the unstimulated condition. One sample t-Test, * = p < 0.05, ns = p > 0.05. F: Dot plot indicating top 5 cytokines explained by CytoSig for undifferentiated and absorptive cells. Dot size indicates adjusted p-value in logarithm base 10, while color scale indicates the difference in Z-score between CD+PFDun and CDun. G: Violin plot of IL22 expression in the IL22 + expressing cell types (ILC3, Th17, CD4 effector memory and CD4 effector TNF). P-value was obtained by DEG analysis considering only IL22 expressing cells. H: Heatmap indicating the difference score of IL-22 pathway activity by AddModuleScore of CD+PFDun relative to CDun patients. The Wilcoxon-rank sum test (two-sided) with Holm correction was applied to compare CDusn and CD+PFDun patients. Asterisks indicate significant differences (adjusted p-value < 0.05).

Article Snippet: Sections were blocked with 20% horse serum and incubated overnight at 4C with anti-rabbit OLFM4 (14369S, Cell Signaling, concentration 1/100).

Techniques: Generated, Expressing, Immunohistochemistry, Staining, Software, Quantitative RT-PCR, Derivative Assay, Cell Culture, Activity Assay

Journal: Nature Communications

Article Title: Epithelial GREMLIN1 disrupts intestinal epithelial-mesenchymal crosstalk to induce a wnt-dependent ectopic stem cell niche through stromal remodelling

doi: 10.1038/s41467-025-60364-6

Figure Lengend Snippet: A T-SNE plot of Epcam -expressing epithelial cells in the proximal small bowel of wild-type, Vil1 - Grem1 , and UCB Ab7326-treated Vil1 - Grem1 mice, classified by cell type, with accompanying subplots separated by mouse genotype/treatment and tissue compartment B The number of cells of each epithelial cell type, as well as the corresponding percentage of total epithelial cells of each cell type for each sample. The number of stem progenitor cells in either the crypt or villi/polyps show a substantial expansion of the stem progenitor cell population in both the crypt and villi of the Vil1 - Grem1 mice, which is reversed after subjecting the Vil1 - Grem1 mice to 10 weeks of treatment with UCB Ab7326. C The expanded stem progenitor cell population in the Vil1 ; Grem1 mice is characterised by increased expression of Mki67 , Sox9 , Olfm4 and Fgfbp1 .

Article Snippet: All antibodies used in this work are as follows: Anti-Ki-67 (D3B5) Rabbit mAb (1:200, Cell Signalling Technology, Cat#CS12202S); Anti-lysozyme Rabbit pAb (1:1000, DAKO, Cat#EC3.2.1.17); Anti-mCherry (TdTomato) mouse mAb (1:500, Novus Bio, Cat#NBP1-96752); Anti-SOX9 Rabbit pAb (1:1000, Sigma Aldrich, Cat#AB5535); Human/Mouse EphB2 Antibody (1:125, R&D, Cat#AF467; Phospho-SMAD1/5 (1:200, Ser463/465) Rabbit mAb (Cell Signalling Technology, Cat#41D10); Anti-β-Catenin Clone 14 (1:50, BD Biosciences, Cat#610154); Olfm4 (D6Y5A) XP® Rabbit mAb (1:200, Cell Signalling Technology, Cat#39141), anti-GFP/YFP Rabbit polyclonal Ab (1:1000, ThermoFisher Scientific, Cat#A6455), anti-Cytokeratin 20 Rabbit polyclonal Ab (1:500, Abcam, ab118574).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: miR-181a-5p mediates the effects of BMP4 on intestinal cell proliferation and differentiation

doi: 10.1038/s41419-025-07730-w

Figure Lengend Snippet: A – D Human duodenum organoids were cultured in the presence of BMP4 (500 ng/ml) or control PBS for 4 d. Total RNA was extracted, and KRT20, APOA4, FABP1, and VILLIN mRNA expression was assessed by qPCR ( A ). Data are normalized to GAPDH; n = 3 biological repeats. Representative image of organoids from control PBS and BMP4 treatment groups is shown in B with quantification of the organoid area shown in ( C ) (98 organoids per PBS group and 105 organoids per BMP4 group were measured). qPCR analysis of OLFM4 expression ( D ). E – H Mouse SI organoids were cultured in the presence of BMP4 (500 ng/ml) or control PBS for 4 d. qPCR analysis of selected enterocyte markers in mouse SI organoids differentiated in the absence or presence of BMP4 ( E ). Data are normalized to GAPDH; n = 3 biological repeats. Representative image of organoids from control PBS and BMP4 treatment is shown in ( F ). Quantification of the organoid area is shown in ( G ) (21 organoids per PBS group and 25 organoids per BMP4 group were measured). OLFM4, Ki67, and CCND1 mRNA expression were assessed by qPCR ( H ). Data are normalized to GAPDH; n = 3 biological repeats. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005.

Article Snippet: Antibodies to Cyclin D1 (ab134175) and KRT20 (ab854) from Abcam (Cambridge, UK), OLFM4 (#39141), APOA4 (#5700), FABP1 (#13368), HK1 (#2024), Na-K ATPase (#3010) (all from Cell Signaling, Danvers, MA) and HK2 (GeneTex, Irvine, CA; GTX124375), VILLIN (sc-7672, Santa Cruz Biotechnology, Dallas, TX), and β-actin (A1978, Sigma) were used, and following blotting with a horseradish peroxidase-conjugated secondary antibody, protein expression was visualized using an enhanced chemiluminescence (ECL) detection system.

Techniques: Cell Culture, Control, Expressing

Journal: Cell Death & Disease

Article Title: miR-181a-5p mediates the effects of BMP4 on intestinal cell proliferation and differentiation

doi: 10.1038/s41419-025-07730-w

Figure Lengend Snippet: Mouse SI organoids were cultured in the presence of LNA miR-181a-5p inhibitor or control oligos (0.75 μM) for 5 d. A , B Organoids were stained with anti-Ki67 antibody to detect proliferating cells and visualized as shown in ( A ). The percentage of Ki67 positive cells was quantified comparing organoids treated with LNA miR-181a-5p inhibitor or control oligos ( B ) ( n = 20). C , D IHC staining for proliferation marker cyclin D1. Cyclin D1 positive cells were visualized as shown in ( C ). The percentage of cyclin D1 positive cells was quantified ( D ) ( n = 20). E qPCR analysis of stem cell marker OLFM4 mRNA expression; n = 3 biological repeats. F Western blot analysis of cyclin D1 protein expression. The images are representative of three independent experiments. Cyclin D1 signals from three separate experiments were quantitated by densitometer and expressed as fold change with respect to β -actin. G –J C57BL/6 mice were injected IP with control oligos or miR-181a-5p inhibitor. Seven d after injection, mouse SI was harvested and analyzed. Western blot analysis ( H ). Cyclin D1 signals from five mice were quantitated by densitometer and expressed as fold change with respect to β -actin. Cyclin D1 mRNA levels were determined by qPCR ( H ). IHC staining for cyclin D1 ( I ) and Ki67 ( J ) demonstrated that the number of positive cells was decreased after in SI treated with miR-181a-5p inhibitor. ( n = 5 mice, 20 crypts per mouse). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005.

Article Snippet: Antibodies to Cyclin D1 (ab134175) and KRT20 (ab854) from Abcam (Cambridge, UK), OLFM4 (#39141), APOA4 (#5700), FABP1 (#13368), HK1 (#2024), Na-K ATPase (#3010) (all from Cell Signaling, Danvers, MA) and HK2 (GeneTex, Irvine, CA; GTX124375), VILLIN (sc-7672, Santa Cruz Biotechnology, Dallas, TX), and β-actin (A1978, Sigma) were used, and following blotting with a horseradish peroxidase-conjugated secondary antibody, protein expression was visualized using an enhanced chemiluminescence (ECL) detection system.

Techniques: Cell Culture, Control, Staining, Immunohistochemistry, Marker, Expressing, Western Blot, Injection

Journal: Cell Death & Disease

Article Title: miR-181a-5p mediates the effects of BMP4 on intestinal cell proliferation and differentiation

doi: 10.1038/s41419-025-07730-w

Figure Lengend Snippet: A , B Mouse SI organoids were infected with lentivirus expressing the HK1 shRNA or NTC shRNA and cultured in the presence of puromycin (2 μg/ml) to select for organoid cells with stable knockdown of HK1. The organoids with stable knockdown of HK1 were incubated for 5 d followed by extraction of cell lysates to measure IAP activity as an indication of differentiation ( A ). Total RNA was extracted, and qPCR performed; n = 3 biological repeats ( B ). C , D Mouse SI organoids were treated with 2-DG (5 mM) for 4 d followed by assessment of IAP activity ( C ). Expression of selective enterocyte markers was analyzed by qPCR; n = 3 biological repeats ( D ). E – H Human duodenum organoids were treated with 2-DG (5 mM) for 4 d and then analyzed for the expression of enterocyte markers ( E ) and the stem cell marker OLFM4 ( F ) by qPCR; n = 3 biological repeats. Representative image of organoid morphology following treatment with either 2-DG or control ( G ). Organoid numbers ( n = 10 fields per group) ( H , left panel)) and organoid size ( n = 66 organoids per control group; n = 38 organoids per 2-DG treatment group) ( H , right panel) were assessed. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005. ( I ) Summary model illustrating the proposed role of BMP4/miR-181a-5p signaling pathway in intestinal cell proliferation and differentiation.

Article Snippet: Antibodies to Cyclin D1 (ab134175) and KRT20 (ab854) from Abcam (Cambridge, UK), OLFM4 (#39141), APOA4 (#5700), FABP1 (#13368), HK1 (#2024), Na-K ATPase (#3010) (all from Cell Signaling, Danvers, MA) and HK2 (GeneTex, Irvine, CA; GTX124375), VILLIN (sc-7672, Santa Cruz Biotechnology, Dallas, TX), and β-actin (A1978, Sigma) were used, and following blotting with a horseradish peroxidase-conjugated secondary antibody, protein expression was visualized using an enhanced chemiluminescence (ECL) detection system.

Techniques: Infection, Expressing, shRNA, Cell Culture, Knockdown, Incubation, Extraction, Activity Assay, Marker, Control