Journal: Oxidative Medicine and Cellular Longevity

Article Title: Qinbaohong Zhike Oral Liquid Attenuates LPS-Induced Acute Lung Injury in Immature Rats by Inhibiting OLFM4

doi: 10.1155/2022/7272371

Figure Lengend Snippet: Functional analysis of QBH-regulated DEPs in the lung tissue determined by quantitative proteomics. (a) Functional GO enrichment analysis of the DEPs according to the classification of BP, CC and MF. (b) Functional KEGG enrichment analysis showing the top 20 signaling pathways. (c) The PPI network analysis generated by two different comparisons. (d) Abundances of OLFM4 reported by TMT-based quantitative proteomic result. Data are presented as mean ± SD ( n = 5/group). ∗ p < 0.05 vs. control group; # p < 0.05 vs. ALI group.

Article Snippet: After blocking with 5% non-fat milk at 4°C overnight, the membrane was incubated with primary antibody olfactomedin 4 (cat. No. bs-6558R, 1 : 1,000, Bioss, Beijing, China) overnight at 4°C. β -actin (cat. No. 3700, 1 : 1,000, Cell Signaling Technology, MA, USA) was used as an internal control.

Techniques: Functional Assay, Generated

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Qinbaohong Zhike Oral Liquid Attenuates LPS-Induced Acute Lung Injury in Immature Rats by Inhibiting OLFM4

doi: 10.1155/2022/7272371

Figure Lengend Snippet: Validation of QBH on the expression of OLFM4 in the lung tissue. (a) Representative images of OLFM4 expression determined by western blot. (b) Relative protein expression level of OLFM4. Data are presented as mean ± SD ( n = 3/group). ∗∗ p < 0.01 vs. control group; # p < 0.05 and ## p < 0.01 vs. ALI group.

Article Snippet: After blocking with 5% non-fat milk at 4°C overnight, the membrane was incubated with primary antibody olfactomedin 4 (cat. No. bs-6558R, 1 : 1,000, Bioss, Beijing, China) overnight at 4°C. β -actin (cat. No. 3700, 1 : 1,000, Cell Signaling Technology, MA, USA) was used as an internal control.

Techniques: Expressing, Western Blot

Journal: Clinical and Translational Gastroenterology

Article Title: Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

doi: 10.14309/ctg.0000000000000124

Figure Lengend Snippet: Expression and cellular distribution of OLFM4 in tumor and nontumor tissue samples. ( a , b ). Staining of OLFM4 in HCC cells and in nontumor cells: proportion of samples showing no staining (none), cytoplasmic staining (cytoplasm only), membrane staining (membrane only), or both (m + c). Representative cytoplasm staining pattern in HCC samples: ( c ) score 0, negative; ( d ) score 1, weak; ( e ) score 2, strong. Representative typical positive membrane staining in HCC tissues: ( f ) score 0, negative; ( g ) score 1, less than 30% of cells; ( h ) 30%–70% of cells; ( i ) over 70% of cells. Magnification, ×40. HCC, hepatocellular carcinoma.

Article Snippet: Anti-OLFM4 polyclonal rabbit antibody (BIOZOL GmbH, Eching, Germany), anti-MMP-7 monoclonal mouse antibody (Merck KgaA, Gernsheim, Germany), and anti-E-cadherin monoclonal mouse antibody (Cell Signaling Technology, Danvers, MA) were applied as primary antibodies.

Techniques: Expressing, Staining, Membrane

Journal: Clinical and Translational Gastroenterology

Article Title: Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

doi: 10.14309/ctg.0000000000000124

Figure Lengend Snippet: Assessment of the correlations between OLFM4 and E-cadherin ( a ) or MMP-7 ( b ) mRNA expression levels according to the analysis of an independent HCC cohort. HCC, hepatocellular carcinoma.

Article Snippet: Anti-OLFM4 polyclonal rabbit antibody (BIOZOL GmbH, Eching, Germany), anti-MMP-7 monoclonal mouse antibody (Merck KgaA, Gernsheim, Germany), and anti-E-cadherin monoclonal mouse antibody (Cell Signaling Technology, Danvers, MA) were applied as primary antibodies.

Techniques: Expressing

Journal: Clinical and Translational Gastroenterology

Article Title: Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

doi: 10.14309/ctg.0000000000000124

Figure Lengend Snippet: Prognostic significance of OLFM4 according to cellular distribution in patients undergoing hepatectomy. ( a ) Overall survival (OS) of the entire patients' collective as determined by the Kaplan-Meier method. (b) OS according to evidence of vessel invasion. OS according to cytoplasm staining for OLFM4 (positive or negative ( c )) or according to semiquantitative assessment of staining intensity ( d ). OS according to membrane staining for OLFM4 (positive or negative ( e )) or according to semiquantitative assessment of staining intensity ( f ). ( g ) OS according to OLFM4 staining in HCC tissues from LMU Munich. ( h ) Kaplan-Meier survival curves according to OLFM4 mRNA expression levels in a second independent cohort of patients with HCC from TCGA. +Censored cases. HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas.

Article Snippet: Anti-OLFM4 polyclonal rabbit antibody (BIOZOL GmbH, Eching, Germany), anti-MMP-7 monoclonal mouse antibody (Merck KgaA, Gernsheim, Germany), and anti-E-cadherin monoclonal mouse antibody (Cell Signaling Technology, Danvers, MA) were applied as primary antibodies.

Techniques: Staining, Membrane, Expressing

Journal: Clinical and Translational Gastroenterology

Article Title: Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

doi: 10.14309/ctg.0000000000000124

Figure Lengend Snippet: Overall survival according to OLFM4 staining in non-HCC tissues from LMU Munich cohort of hepatectomy. +Censored cases. HCC, hepatocellular carcinoma.

Article Snippet: Anti-OLFM4 polyclonal rabbit antibody (BIOZOL GmbH, Eching, Germany), anti-MMP-7 monoclonal mouse antibody (Merck KgaA, Gernsheim, Germany), and anti-E-cadherin monoclonal mouse antibody (Cell Signaling Technology, Danvers, MA) were applied as primary antibodies.

Techniques: Staining

Journal: Clinical and Translational Gastroenterology

Article Title: Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

doi: 10.14309/ctg.0000000000000124

Figure Lengend Snippet: In vitro assessment of the effect of OLFM4 silencing on MMP-7 and E-cadherin. ( a ) Effect of transfection of Huh7 and PLCPRF5 cells with siRNA specifically targeting OLFM4 or non-coding siRNA (Beta-gal, 50 nM) for 24 hours on the expression of the indicated molecules after immunoblotting. ( b ) Typical pattern of OLFM4 expression at immunofluorescence after transfection with control-targeting or OLFM4-targeting siRNA. Green: DAPI staining of the nuclei; Red: immunofluorescence of OLFM4.

Article Snippet: Anti-OLFM4 polyclonal rabbit antibody (BIOZOL GmbH, Eching, Germany), anti-MMP-7 monoclonal mouse antibody (Merck KgaA, Gernsheim, Germany), and anti-E-cadherin monoclonal mouse antibody (Cell Signaling Technology, Danvers, MA) were applied as primary antibodies.

Techniques: In Vitro, Transfection, Expressing, Western Blot, Immunofluorescence, Control, Staining

Journal: Clinical and Translational Gastroenterology

Article Title: Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

doi: 10.14309/ctg.0000000000000124

Figure Lengend Snippet: Effect of OLFM4 silencing on cell migration and invasion. ( a , b ) Assessment of cell motility by wound healing assay in Huh7 or PLCPRF5 cells transfected with siRNA-OLFM4 or control-si-RNA. ( c ) Quantitative analysis of healing area in percentages. ( d , e ) The migration and invasion of Huh7 and PLCPRF5 cells transfected with siRNA-OLFM4 compared with the controls by transwell assay after 24 hours. ( f , g ) Quantitative analysis of migration and invasion cells. ( h ) The E-cadherin expression of Huh7 and PLCPRF5 cells transfected with siRNA-OLFM4 compared with the controls by immunofluorescence after 24 hours. ( i – j ) Effects of OLFM4 silencing by siRNA on sub-G1 events, cell cycle distribution, and cell viability in Huh7 ( i ) and PLCPRF5 ( j ) cell lines. Values represented mean ± SD from 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, Student t -test. Blue: DAPI staining of nuclei; green, immunofluorescence of E-cadherin.

Article Snippet: Anti-OLFM4 polyclonal rabbit antibody (BIOZOL GmbH, Eching, Germany), anti-MMP-7 monoclonal mouse antibody (Merck KgaA, Gernsheim, Germany), and anti-E-cadherin monoclonal mouse antibody (Cell Signaling Technology, Danvers, MA) were applied as primary antibodies.

Techniques: Migration, Wound Healing Assay, Transfection, Control, Transwell Assay, Expressing, Immunofluorescence, Staining

Journal: Clinical and Translational Gastroenterology

Article Title: Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

doi: 10.14309/ctg.0000000000000124

Figure Lengend Snippet: Schematic diagram describing the hypothesized effect of OLFM4, MMP family members and on invasion and metastasis formation in HCC. HCC, hepatocellular carcinoma.

Article Snippet: Anti-OLFM4 polyclonal rabbit antibody (BIOZOL GmbH, Eching, Germany), anti-MMP-7 monoclonal mouse antibody (Merck KgaA, Gernsheim, Germany), and anti-E-cadherin monoclonal mouse antibody (Cell Signaling Technology, Danvers, MA) were applied as primary antibodies.

Techniques:

Journal: Gut

Article Title: SETDB1 is required for intestinal epithelial differentiation and the prevention of intestinal inflammation

doi: 10.1136/gutjnl-2020-321339

Figure Lengend Snippet: Loss of epithelial SETDB1 is associated with DNA damage and cell death. (A) Percentage of histone H2AX phosphorylation (γH2AX)-positive cells per crypt as determined by immunohistochemical staining (left) and representative stainings (right) in the ileum of the indicated mouse strains. (B) Western blot analysis for p53 and GAPDH in small intestinal intestinal epithelial cells (IECs) of the indicated mouse strains. (C) Percentage of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells per crypt (left) and representative immunofluorescence stainings (right, TUNEL in green) in the ileum of the indicated mouse strains. Arrows show TUNEL-positive IECs. (D) Quantification of crypt necrosis and cellular swelling as well as of the number of necrotic cells per high power field in the ileum of the indicated mouse strains. (E–G) Representative electron microscopy images in the ileum of the indicated mouse strains. Arrows indicate normal mitochondria (E–F). Arrowheads indicated swelling and loss of the structural integrity of mitochondria (E) and loss of integrity of the nuclear membrane (F) as well as swelling of the cytoplasm and rupture of the plasma membrane (G). All data were obtained at day 10 after the first tamoxifen injection. Representative results of two independent experiments are shown (A–G). In (A, C–D), dots represent individual mice and the bar indicates the median. Size bars indicate 100 µm in (A, C), 2 µm (E, F), 10 µm (G). The Student’s t-test (A) or the Mann-Whitney U test (C–D) were applied. SETDB1, SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET.

Article Snippet: For immunohistochemistry, antibodies against SETDB1 (D4M8R, 1:1000, Cell Signaling Technology), phospho-histone H2A.X (1:200, Cell Signaling Technology), OLFM4 (D6Y5A, 1:400, Cell Signaling Technology), SGLT1 (1:100, Abcam), Ly6G (E6Z1T, 1:200, Cell Signaling Technology) and F4/80 (D2S9R, 1:300, Cell Signaling Technology) were used for staining overnight at 4°C.

Techniques: Immunohistochemical staining, Staining, Western Blot, TUNEL Assay, Immunofluorescence, Electron Microscopy, Injection, MANN-WHITNEY

Journal: Communications biology

Article Title: CD115 - monocytic myeloid-derived suppressor cells are precursors of OLFM4 high polymorphonuclear myeloid-derived suppressor cells.

doi: 10.1038/s42003-023-04650-3

Figure Lengend Snippet: Fig. 5 CD115−M-MDSCs express OLFM4 and differentiate into OLFM4hi PMN-MDSCs. a BM cells from three EL4 TB mice were pooled and CD115−M- MDSCs, CD115+ M-MDSCs, and PMN-MDSCs were sorted. Cells were stained for OLFM4 and OLFM4hi cells were counted from ten random fields. Data pooled from two to three separate experiments. Representative IHC analysis of OLFM4 expression and ratios of OLFM4hi cells. The letter “t” indicates that cells were isolated from TB mice. b Schematic diagram of experimental design. CD115−and CD115+ M-MDSCs were sorted from donor EL4 TB mice and labeled with CFSE, then CD115−and CD115+ M-MDSCs were injected into separate recipient EL4 TB mice (n = 3 per group). Two days later, BM cells from three recipient mice were pooled and OLFM4 expression in sorted PMN-MDSCs (CD11b+Ly6G+Ly6Clo) were analyzed by IHC to determine the ratio of OLFM4hi PMN-MDSC. c OLFM4hi cells were counted from seven to ten random fields. IHC analysis of OLFM4 expression and ratios of OLFM4hi cells. Data pooled from two separate experiments. Scale bars = 20 μm. One-way ANOVA with correction for multiple comparisons test was used: *p < 0.05. Data are mean ± SD.

Article Snippet: In brief, slides were incubated overnight with a rabbit anti-OLFM4 antibody (Novus, Littleton, CO, USA) or normal rabbit IgG (Santa Cruz, CA, USA) at a 1:300 dilution at 4 °C in a humidity chamber.

Techniques: Staining, Expressing, Isolation, Labeling, Injection

Journal: Communications biology

Article Title: CD115 - monocytic myeloid-derived suppressor cells are precursors of OLFM4 high polymorphonuclear myeloid-derived suppressor cells.

doi: 10.1038/s42003-023-04650-3

Figure Lengend Snippet: Fig. 6 Depletion of CD115+ M-MDSCs does not affect OLFM4hi PMN-MDSCs. CD115+ monocytic cells were transiently depleted in MMDTR mice by intraperitoneal injection of DT. a Ratio of M-MDSCs (% CD11c−CD11b+Ly6G−SiglecF−Ly6Chi) and their subpopulations (CD115−and CD115+) in BM of MMDTR mice with or without DT treatment (n = 4 per group). b Tumor volume of EL4 TB C57BL/6 and MMDTR mice with or without DT treatment (n = 7–9 per group). c–f BM cells were prepared from indicated mice (n = 3 per group) and neutrophilic cells (CD11b+Ly6G+Ly6Clo) were sorted from pooled BM cells. c Representative plots of neutrophilic cells in BM of naive and EL4 TB MMDTR mice with or without DT injection. Ratio and absolute numbers of neutrophilic cells in BM of naive and EL4 TB mice with or without DT injection are shown in graphs. Data shown are from two biological replicates of each group. d, e Heatmap of neutrophil-associated gene expression (d) and Olfm4 expression (e) in sorted neutrophilic cells from indicated mice. Data shown are from two biological replicates of each group. f IHC assessment of ratio of OLFM4hi neutrophilic cells in BM of indicated mice. OLFM4hi cells were counted from twenty random fields. Data pooled from two separate experiments. Statistical comparisons were performed using unpaired Student’s t test when only two groups were compared or by Bonferroni’s test-corrected ANOVA when more than two groups were compared: *p < 0.05. Data are mean or mean ± SD.

Article Snippet: In brief, slides were incubated overnight with a rabbit anti-OLFM4 antibody (Novus, Littleton, CO, USA) or normal rabbit IgG (Santa Cruz, CA, USA) at a 1:300 dilution at 4 °C in a humidity chamber.

Techniques: Injection, Gene Expression, Expressing

Journal: bioRxiv

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

doi: 10.1101/2024.04.28.591245

Figure Lengend Snippet: A. Schematic shows recombination and harvesting timepoint for acute Lgr4 knockout in 120 day old Rosa-CreER T2 , Lgr4 fl/fl ; Vil1-Grem1 mice. B. KI67 staining and C. quantification of proliferating crypt base and ectopic crypt cells following acute Lgr4 knockout (n=5 mice per group, t test, p values as stated). D. Change in wnt target and receptor staining in advanced Vil1-Grem1 polyps. E. Representative staining and quantification of Notch pathway ligands ( Dlk1, Jag1 ) and target gene (HES1) expression in wildtype and Vil1-Grem1 mice (n=5 mice per group, t test, p values as stated). F. Schematic shows recombination and harvesting timepoint for gamma secretase inhibition with Dibenzazepine in 120- day old Vil1-Grem1 mice. G. Representative staining and quantification of Alcian blue goblet cell staining and OLFM4 expression in untreated and Dibenzazepine-treated Vil1-Grem1 mice (n=5 mice per group, t test, p values as stated). Scale bars 200µm.

Article Snippet: All antibodies used in this work are as follows: Anti-Ki67 (D3B5) Rabbit mAb (Cell Signalling Technology, Cat#CS12202S); Anti-lysozyme Rabbit pAb (DAKO, Cat#EC3.2.1.17); Anti-mCherry (TdTomato) mouse mAb (Novus Bio, Cat#NBP1- 96752); Anti-SOX9 Rabbit pAb (Sigma Aldrich, Cat#AB5535); Human/Mouse EphB2 Antibody (R&D, Cat#AF467; Phospho-SMAD1/5 (Ser463/465) Rabbit mAb (Cell Signalling Technology, Cat#41D10); Anti-β-Catenin Clone 14 (BD Biosciences, Cat#610154); HES1 (D6P2U) Rabbit mAb (Cell Signalling Technology, Cat#11988); Olfm4 (D6Y5A) XP® Rabbit mAb (Cell Signalling Technology, Cat#39141); anti- RFP Rabbit pAb (Rockland, Cat#600-401-379).

Techniques: Knock-Out, Staining, Expressing, Inhibition

Journal: Journal of leukocyte biology

Article Title: Olfactomedin 4 defines a subset of human neutrophils.

doi: 10.1189/jlb.0811417

Figure Lengend Snippet: Figure 3. FACS of neutrophils confirms the existence of two subsets: OLFM4 high and OLFM4 low. (A) Isolated neutrophils from a healthy donor unstained or stained with a FITC-conjugated isotype control or with a FITC-conjugated anti-OLFM4 antibody [GC-1(N-20)], clearly showing two distinct populations of OLFM4, whereas staining for gelatinase (B) reveals only one population. (C) Sorting strategy for FACS of OLFM4 high and OLFM4 low populations depicting the gating of the two populations. FITC-A, FITC-area; FSC-A, forward-scatter-area. (D) Western blot of sorted populations. Samples representing 4 105 cells were applied in each well. Whereas the OLFM4 low population has no immunoreactivity for OLFM4, the amount of NGAL is equal in the two populations. (E) Cytospins of the sorted populations ac- quired in a LSM 700 (Zeiss) microscope.

Article Snippet: After blocking 10 min in TBS/1% BSA, cells were incubated 30 min with FITC-conjugated antibodies targeting OLFM4 [GC-1(N-20), Santa Cruz Biotechnology; 1:500], gelatinase (in-house; 1:100), or rabbit IgG (BD Biosciences, Franklin Lakes, NJ, USA; 1:1000) and analyzed on FACSsort (BD Biosciences) with CellQuest software.

Techniques: Isolation, Staining, Control, Western Blot, Microscopy

Journal: Journal of leukocyte biology

Article Title: Olfactomedin 4 defines a subset of human neutrophils.

doi: 10.1189/jlb.0811417

Figure Lengend Snippet: Figure 4. OLFM4 mRNA is present in all cells at the MY/MM stage of dif- ferentiation. Fluorescent in situ hybridization of MY/MM isolated from hu- man bone marrow using no probe, a probe for OLFM4 mRNA, a probe for NGAL mRNA, or a probe for hCAP18 mRNA. mRNA for OLFM4 is present in all cells at the MY/MM stage of differentiation and shows no differential expression compared with NGAL and hCAP18.

Article Snippet: After blocking 10 min in TBS/1% BSA, cells were incubated 30 min with FITC-conjugated antibodies targeting OLFM4 [GC-1(N-20), Santa Cruz Biotechnology; 1:500], gelatinase (in-house; 1:100), or rabbit IgG (BD Biosciences, Franklin Lakes, NJ, USA; 1:1000) and analyzed on FACSsort (BD Biosciences) with CellQuest software.

Techniques: In Situ Hybridization, Isolation, Quantitative Proteomics