olfm4 gene expression  (Santa Cruz Biotechnology)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity

doi: 10.1016/j.jcmgh.2022.12.004

Figure Lengend Snippet: IEC-specific Gp96 deletion leads to a continuous loss of proliferating cells, ISCs, and Paneth cells, and alters the morphology and location of secretory cell types. Small intestinal tissue sections of GP96 fl/fl (WT) and GP96 fl/fl -Villin creERT2 ( Gp96 KO) mice were collected on days 2–6 after first tamoxifen-injection and stained for ( A ) OLFM4 (green) and Ki67 (red); ( B ) lysozyme (green); or ( C ) lysozyme (green), Ki67 (red), and MUC2 (red) in combination. Scale bars : 20 μm ( B ), 50 μm ( A and C ). ( D ) Quantitative reverse-transcription PCR analysis in small intestinal epithelial cells, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and WT control mice on days 1, 3, and 5. N = 2–7 mice/group. ( E ) Western blot analysis on lysates from small intestinal epithelial cells collected on days 3 and 5 after first tamoxifen injection, normalized to β-actin. Graphs show representative Western blot images from each experimental group. N = 6 mice/group. ( F ) Schematic representation of the main findings of this figure showing the loss of the stem cell niche and the appearance of morphologically altered and mislocated Paneth and goblet cells as a consequence of GP96 depletion. Created by BioRender. Significance was calculated using 2-way analysis of variance with a Tukey correction (reverse-transcription PCR) or a Kruskal–Wallis test (1-way analysis of variance, nonparametric) (Western blot). Bars represent mean with SD. Asterisks indicate significant differences, as follows: ∗ P ≤ .05, ∗∗ P ≤ .01, ∗∗∗ P ≤ .001, and ∗∗∗∗ P ≤ .0001. DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA; NICD, Notch intracellular domain.

Article Snippet: Mouse OLFM4 real-time PCR gene expression assay FAM-MGB , Thermo Fisher Scientific , 4331182/Mm01320260_m1.

Techniques: Injection, Staining, Reverse Transcription, Control, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity

doi: 10.1016/j.jcmgh.2022.12.004

Figure Lengend Snippet: I nduction of Gp96 KO in small intestinal organoids reduces stemness and increases expression of differentiated cell type marker. Organoids were generated from duodenal crypts of GP96 fl/fl (WT) and GP96-Villin creERT2 mice. Cre-mediated recombination was induced by adding 4-OHT (2 μmol/L) or an equal volume of dimethyl sulfoxide (DMSO) to the culture medium for 48 hours. ( A ) Light microscope images on day 4. Images are representative of 3 independent experiments. Blue arrow highlights immature spheroids with complete absence of normal crypt–villi structure, green arrow highlights budds. Original magnification, ×10. ( B ) Western blot analysis of cell lysates collected on day 4 after Gp96 KO induction, normalized to β-actin. Representative protein bands from each experimental group are shown, originating from the same gel/blot (cropped regions are marked by dividing lines ). ( C ) Quantitative reverse-transcription PCR analysis, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 4-OHT–treated WT organoids collected on the same day. ( D ) GP96 (red) ( upper panels ), OLFM4 (green) and Ki67 (red) ( lower panels ) immunofluorescence on GP96-Villin creERT2 small intestinal organoids fixed on day 4 after Gp96 deletion. Scale bars : 50 μm (×20) or 20 μm (×40). Statistics were performed using 2-way analysis of variance with a Tukey correction (reverse-transcription PCR) and by a Kruskal–Wallis test (1-way analysis of variance, nonparametric) with a post hoc correction for multiple comparisons using the Dunn test (Western blot). Bars represent means with SD. Biological replicates, N = 6. Asterisks indicate significant differences, as follows: ∗ P ≤ .05, ∗∗ P ≤ .01, ∗∗∗ P ≤ .001, and ∗∗∗∗ P ≤ .0001. adj, adjusted; DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA.

Article Snippet: Mouse OLFM4 real-time PCR gene expression assay FAM-MGB , Thermo Fisher Scientific , 4331182/Mm01320260_m1.

Techniques: Expressing, Marker, Generated, Light Microscopy, Western Blot, Reverse Transcription, Immunofluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity

doi: 10.1016/j.jcmgh.2022.12.004

Figure Lengend Snippet: Gene expression signature of Wnt- and Notch-inhibited WT organoids is similar to Gp96 KO organoids. Intestinal organoids were generated from jejunal crypts of GP96 fl/fl (WT) and GP96-Villin creERT2 (VilCreERT2) mice. Cre-mediated recombination was induced by adding 4-OHT (1 μmol/L) or an equal volume of dimethyl sulfoxide (DMSO) to the culture medium for 48 hours. Wnt signaling was inhibited with tankyrase inhibitor XAV939 (XAV, 10 μmol/L) and Notch signaling with γ-secretase inhibitor DAPT (10 μmol/L) for 96 hours starting on the day of 4-OHT treatment. ( A ) Representative images on day 4 (1 of 3 independent experiments). Original magnification, ×10. ( B ) Quantitative reverse-transcription PCR analysis of relative messenger RNA (mRNA) expression, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and DMSO-treated WT control organoids on day 4. ( C ) Immunofluorescence staining of organoids fixed on day 4 after Gp96 KO induction (4-OHT) and Wnt (XAV)/Notch (DAPT) signaling inhibition, stained for OLFM4 (green) and Ki67 (red). Scale bars : 50 μm. Statistical analysis was performed using 2-way analysis of variance with the Dunnett method for multiple comparisons (reverse-transcription PCR). Bars represent means with SD. Biological replicates N = 3. Asterisks indicate significant differences, as follows: ∗ P ≤ .05, ∗∗ P ≤ .01, and ∗∗∗∗ P ≤ .0001. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Mouse OLFM4 real-time PCR gene expression assay FAM-MGB , Thermo Fisher Scientific , 4331182/Mm01320260_m1.

Techniques: Gene Expression, Generated, Reverse Transcription, Expressing, Control, Immunofluorescence, Staining, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity

doi: 10.1016/j.jcmgh.2022.12.004

Figure Lengend Snippet: Antibodies

Article Snippet: Mouse OLFM4 real-time PCR gene expression assay FAM-MGB , Thermo Fisher Scientific , 4331182/Mm01320260_m1.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity

doi: 10.1016/j.jcmgh.2022.12.004

Figure Lengend Snippet: TaqMan Assays

Article Snippet: Mouse OLFM4 real-time PCR gene expression assay FAM-MGB , Thermo Fisher Scientific , 4331182/Mm01320260_m1.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Control

Journal: International Journal of Cancer

Article Title: Olfactomedin 4 downregulation is associated with tumor initiation, growth and progression in human prostate cancer

doi: 10.1002/ijc.32535

Figure Lengend Snippet: Reduced OLFM4 expression is associated with higher Gleason scores and lower recurrence‐free survival in human primary prostate adenocarcinoma. ( a ) Representative images of HE staining and immunohistochemistry (IHC) analysis of OLFM4 protein expression in adjacent normal and tumor regions of whole‐mount section human prostate cancer tissue specimens (obtained from the Laboratory of Pathology, National Cancer Institute). All micrographs shown are for tissues obtained from the same case. LT, lower grade tumor (Gleason grade 3, GL. 3); HT, higher grade tumor Gleason grade 4, GL. 4). Scale bars: 50 μm. ( b ) Quantitation of OLFM4 protein expression from immunohistochemical analyses using human prostate cancer tissue slides obtained from CHTN and US Biomax. Data represent the mean ± standard deviation (SD) of 3,3′‐diaminobenzidine (DAB) intensity normalized to the number of nuclei. Adjacent Nor., normal tissue adjacent to primary tumor; Gleason score ≤4 + 3; Gleason score ≥4 + 4. We excluded CHTN and US Biomax cases with quality issues for which we could not obtain immunohistochemistry data. *** p ≤ 0.001 (ANOVA). ( c ) OLFM4 mRNA expression in prostate cancer specimens in data downloaded from the GSE21032 dataset. Data represent the mean ± SD. Normal, normal tissue adjacent to primary tumor; P‐tumor, primary tumor; M‐PCs; prostate tumor with distant metastasis. ** p ≤ 0.01; *** p ≤ 0.001 (ANOVA). ( d ) Kaplan–Meier plot of recurrence‐free survival for OLFM4 mRNA higher‐expressing (red line) and lower‐expressing (blue line) prostate adenocarcinoma patient cohorts in the GSE21032 dataset at 25% thresholds ( p = 0.0000154; log‐rank test).

Article Snippet: Lentiviral particles containing 3–5 expression constructs, each encoding a target‐specific 19–25 nucleotide (plus hairpin) short hairpin RNA (shRNA) designed to knock down OLFM4 gene expression (GC‐1 shRNA (h) lentiviral particles; Cat# sc‐75,113‐V), were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Expressing, Staining, Immunohistochemistry, Quantitation Assay, Immunohistochemical staining, Standard Deviation

Journal: International Journal of Cancer

Article Title: Olfactomedin 4 downregulation is associated with tumor initiation, growth and progression in human prostate cancer

doi: 10.1002/ijc.32535

Figure Lengend Snippet: Relationship between OLFM4 mRNA expression and clinicopathological parameters in 333 primary prostate adenocarcinoma patients

Article Snippet: Lentiviral particles containing 3–5 expression constructs, each encoding a target‐specific 19–25 nucleotide (plus hairpin) short hairpin RNA (shRNA) designed to knock down OLFM4 gene expression (GC‐1 shRNA (h) lentiviral particles; Cat# sc‐75,113‐V), were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Expressing, Translocation Assay

Journal: International Journal of Cancer

Article Title: Olfactomedin 4 downregulation is associated with tumor initiation, growth and progression in human prostate cancer

doi: 10.1002/ijc.32535

Figure Lengend Snippet: Reduced OLFM4 expression is associated with CpG site increased methylation of the OLFM4 gene promoter region in human prostate adenocarcinoma and prostate cell lines. ( a ) OLFM4 promoter region CpG site methylation status in DNA isolated from epithelial cells obtained from prostate cancer specimens using LCM. Methylation levels in adjacent normal prostate epithelial cells (N), lower grade prostate cancer cells (L) and higher‐grade prostate cancer cells (H) were measured using pyrosequencing. Data represent the mean ± SD (N = 5–8; L = 5–6; H = 6–8). The difference between pairs of groups was analyzed using Student's t ‐test. * p ≤ 0.05; ** p ≤ 0.01; NS, not significant. ( b and c ) Methylation status (percentage) of the eight CpG sites (−681, −666, −562, −486, −446, −91, +4 and +34, relative to the OLFM4 transcription start site) in the OLFM4 gene promoter region. RWPE1 cells were treated with Aza (5 μM) for 24, 48, 72 or 96 hr ( b ) and PC‐3 cells were treated with Aza (5 μM) for 4 and 7 days. ( d ) Relative expression of OLFM4 mRNA in RWPE1 cells determined by qRT‐PCR after treatment with Aza (5 μM) or control (DMSO) for 24, 48, 72 or 96 hr. ( e ) Relative expression of OLFM4 mRNA in PC‐3 cells determined by qRT‐PCR after treatment with Aza (5 μM) or control (DMSO) for 48 or 96 hr. ( d , e ) Data represent the mean ± SD of three experiments performed in triplicate. The difference between Aza treatment and vehicle at each time point was analyzed using the Student's t ‐test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. ( f ) OLFM4 promoter reporter activity. pGL3 basic vector control, −101 pGL3‐OLFM4 reporter plasmid, and −945 pGL3‐OLFM4 reporter plasmid were treated without (Met −) or with CpG methyltransferase (Met +). Data represent the mean (±SD, n = 3) relative luciferase activities of these plasmids after their transient transfection into PC‐3 cells. The difference between treated without (Met −) or with CpG methyltransferase (Met +) was analyzed using the Student's t ‐test. *** p ≤ 0.001.

Article Snippet: Lentiviral particles containing 3–5 expression constructs, each encoding a target‐specific 19–25 nucleotide (plus hairpin) short hairpin RNA (shRNA) designed to knock down OLFM4 gene expression (GC‐1 shRNA (h) lentiviral particles; Cat# sc‐75,113‐V), were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Expressing, Methylation, Isolation, Quantitative RT-PCR, Control, Activity Assay, Plasmid Preparation, Luciferase, Transfection

Journal: International Journal of Cancer

Article Title: Olfactomedin 4 downregulation is associated with tumor initiation, growth and progression in human prostate cancer

doi: 10.1002/ijc.32535

Figure Lengend Snippet: Knockdown of OLFM4 expression is associated with enhanced EMT‐marker expression in RWPE cells. ( a ) Expression of OLFM4 mRNA in RWPE1 and RWPE2 cells determined by qRT‐PCR. Values relative to β‐actin (ACTB) represent the mean ± SD of three experiments performed in triplicate. ( b ) RWPE1 or RWPE2 cells were knocked down with control shRNA (C‐shRNA) or OLFM4 shRNA (O‐shRNA). Expression of OLFM4 mRNA determined by qRT‐PCR in knockdown RWPE1 and RWPE2 cells. Values normalized to ACTB represent the mean ± SD of three experiments performed in triplicate compared to control shRNA‐knocked‐down cells (value set at 100%). ( c ) Relative expression of E‐cadherin (CDH1) and vimentin (VIM) mRNA determined by qRT‐PCR in OLFM4‐ or control‐knockdown RWPE1 and RWPE2 cells. Data represent the mean ± SD of three experiments performed in triplicate compared to control shRNA‐knocked‐down cells (value set at 1). * p ≤ 0.05 (Student's t ‐test). ( d ) Western‐blot analysis of OLFM4, E‐cadherin (Ecd), vimentin (Vim) and TWIST1 in OLFM4 (O) or control (C) knockdown RWPE1 and RWPE2 cells. β‐actin was used as a loading control. ( e ) Representative images of HE staining and immunohistochemistry analysis of E‐cadherin and vimentin expression in xenograft tissues that emerged in mice after subcutaneous inoculation with OLFM4‐ or control shRNA‐knockdown RWPE cells. Arrow indicates EMT cells; arrow head indicates Epithelial cells; Star indicates stromal cells. Scale bar: 100 μm. ( f ) Representative images of immunohistochemistry analysis of OLFM4, TWIST1, SNAIL1 and BMI1 expression in xenograft tissues that emerged in mice after subcutaneous inoculation with OLFM4‐knockdown RWPE2 cells. Scale bar: 50 μm.

Article Snippet: Lentiviral particles containing 3–5 expression constructs, each encoding a target‐specific 19–25 nucleotide (plus hairpin) short hairpin RNA (shRNA) designed to knock down OLFM4 gene expression (GC‐1 shRNA (h) lentiviral particles; Cat# sc‐75,113‐V), were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Knockdown, Expressing, Marker, Quantitative RT-PCR, Control, shRNA, Western Blot, Staining, Immunohistochemistry

Journal: International Journal of Cancer

Article Title: Olfactomedin 4 downregulation is associated with tumor initiation, growth and progression in human prostate cancer

doi: 10.1002/ijc.32535

Figure Lengend Snippet: Restoration of OLFM4 expression in prostate cancer cells inhibited tumor cell growth in vitro and xenograft tumor growth in mice. The stably expressing prostate cancer cell clones PC‐3V (vector‐GFP tag), PC‐3O (OLFM4‐GFP tag), DU145V (vector‐GFP tag) and DU145O (OLFM4‐GFP tag) were established. ( a , b ) Stably expressing prostate cancer cell clones were subjected to prostate sphere‐formation assays. Spheres were photographed after culturing for 12 days with Matrigel in 12‐well plates. Photographs were taken of identical fields under light (Light) and fluorescent (GFP) conditions. Squares in the top row of PC‐3 cell micrographs indicate GFP‐positive spheres. Scale bars: 200 μm. Bar graphs represent the mean number (±SD) of spheres (>50 μm in diameter) per well counted from six wells for each cell clone. ( c ) Stably expressing PC‐3 prostate cancer cell clones were grown in soft agar to support colony formation. PC‐3V and PC‐3O cell colonies were photographed after 14 days in culture. Photographs were taken of identical fields under light (Light) and fluorescent (GFP) conditions. Scale bar: 200 μm. Bar graph represents the mean number (±SD) of colonies formed ( n = 6). ( d , e ) Stably expressing prostate cancer cell clones were used to inoculate mice in tumor xenograft studies. The xenograft tumors that emerged after inoculation were dissected, photographed, and weighed; PC‐3 tumors were dissected 19 days after inoculation, and DU145 tumors were dissected 42 days after inoculation. Scott plot graphs represent the weights of the xenograft tumors shown in the photographs ( n = 5). The difference between pairs of groups in all panels was analyzed using Student's t ‐test. ( f ) Representative HE‐stained images from PC‐3 xenograft tumors. Box indicates area with fewer cells. Scale bar: 50 μm.

Article Snippet: Lentiviral particles containing 3–5 expression constructs, each encoding a target‐specific 19–25 nucleotide (plus hairpin) short hairpin RNA (shRNA) designed to knock down OLFM4 gene expression (GC‐1 shRNA (h) lentiviral particles; Cat# sc‐75,113‐V), were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Expressing, In Vitro, Stable Transfection, Clone Assay, Plasmid Preparation, Staining

Journal: International Journal of Cancer

Article Title: Olfactomedin 4 downregulation is associated with tumor initiation, growth and progression in human prostate cancer

doi: 10.1002/ijc.32535

Figure Lengend Snippet: Restoration of OLFM4 expression in prostate cancer cells inhibited EMT‐marker expression. ( a ) Relative expression of vimentin (VIM) mRNA determined by qRT‐PCR in stably expressing OLFM4 ‐GFP tag‐expressing (O) or vector‐GFP tag‐transfected control (V) PC‐3 and DU145 prostate cancer cell clones. Data represent mean ± SD percent expression in OLFM4‐GFP tag‐expressing cell clones compared to vector‐GFP tag‐expressing cell clones (value set at 100%; n = 3). The difference between pairs of groups was analyzed using Student's t ‐test. * p ≤ 0.05. ( b ) Western‐blot analysis of OLFM4, E‐cadherin, vimentin and β‐catenin in total cell lysates from prostate cancer cell clones. β‐actin was used as a loading control. ( c ) Western‐blot analysis of ZEB1 and TWIST1 in nuclear extracts from prostate cancer cell clones. Histone H3 was used as a loading control. ( d ) Proposed model of relationship between OLFM4 and EMT‐regulating proteins in benign and cancer stem‐cell‐like cells in the prostate.

Article Snippet: Lentiviral particles containing 3–5 expression constructs, each encoding a target‐specific 19–25 nucleotide (plus hairpin) short hairpin RNA (shRNA) designed to knock down OLFM4 gene expression (GC‐1 shRNA (h) lentiviral particles; Cat# sc‐75,113‐V), were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX).

Techniques: Expressing, Marker, Quantitative RT-PCR, Stable Transfection, Plasmid Preparation, Transfection, Control, Clone Assay, Western Blot