Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of key candidate biomarkers for severe influenza infection by integrated bioinformatical analysis and initial clinical validation

doi: 10.1111/jcmm.16275

Figure Lengend Snippet: Phase II validation: Candidate proteins evaluate disease severity and predict patient outcome. A, Concentration levels of RETN, MMP8, LCN2, HP, OLFM4, ELANE, TCN1, DEFα4, BPI and LTF in plasma obtained at the first sampling time‐point (T1) from healthy donors (n = 25) and influenza patients (n = 63) and presented as scatter diagram. HD: Healthy donors; M: Moderate patient (n = 34); S: Severe patients (n = 29). B, Severity prediction using LCN2, BPI, ELANE and MMP8 expression levels, and proportion of neutrophils and lymphocytes. Receiver‐operator characteristic (ROC) curves of four candidate proteins and two clinical indicators to predict disease severity: ROC‐AUC (LCN2: 0.646; BPI: 0.774; ELANE: 0.671; MMP8: 0.872; Proportion of neutrophils: 0.754; Proportion of Lymphocytes: 0.650). C, The protein levels of BPI, MMP8, DEFα4, OLFM4, LCN2 and ELANE in survivors (n = 49) and non‐survivors (n = 14) with influenza infection. Statistical significance is determined by unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The concentration levels of human resistin (RETN), human matrix metalloproteinase‐8 (MMP8), human lipocalin 2 (LCN2), human haptoglobin (HP) , human olfactomedin 4 (OLFM4), human neutrophil elastase (ELANE), human bactericidal/permeability‐increasing protein (BPI), human lactoferrin (LTF; Elabscience, Wuhan, China), human transcobalamin I (TCN1) and human defensin α4 (DEFA4) (mlbio, Shanghai, China) in the plasma samples were measured according to manufacturer's instructions, respectively.

Techniques: Concentration Assay, Sampling, Expressing, Infection

Journal: PLoS ONE

Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

doi: 10.1371/journal.pone.0226707

Figure Lengend Snippet: (a) Schematic of the procedure for NGS analysis. (b, c) NGS analysis for the GEM administration and GEM + nab-PTX administration groups. Treatment resistance score was defined as the NE value ratio (treated group / untreated group) × NE value difference (treated group–untreated group). (d, e) The NE value of OLFM4 mRNA. The ratio of NE values for treated and control groups were greater than 1.0 for all lines of PDXs. GEM, gemcitabine. nab-PTX, nab-paclitaxel.

Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

Techniques: Control

Journal: PLoS ONE

Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

doi: 10.1371/journal.pone.0226707

Figure Lengend Snippet: (a) Immunostaining for OLFM4 in PDXs. Control and chemotherapy-administered PDXs are shown at 200× each. (b) Analysis of the number of pixels of OLFM4-stained cells using Image J.

Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

Techniques: Immunostaining, Control, Staining

Journal: PLoS ONE

Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

doi: 10.1371/journal.pone.0226707

Figure Lengend Snippet: (a) Schematic representation of the procedure. Expression of the control vector and OLFM4 was induced in indicated cell lines. After 24 h (day 1), GEM was added at various concentrations. Cell viability assays were performed 48 h after GEM administration (day 3). (b and c) Rate of change of each measured OD value of the control vector and OLFM4-expressing HeLa cells (b) and MIA Paca2 cells (c) is shown. (d) Rate of change of each measured OD value of siRNA negative control and specific siRNA targeting OLFM4 induced in SUIT-2 cells is shown.

Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

Techniques: Expressing, Control, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

doi: 10.1371/journal.pone.0226707

Figure Lengend Snippet: Correlation between OLFM4 expression and clinicopathological features in 80 cases of pancreatic cancer.

Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

Techniques: Expressing, Adjuvant

Journal: PLoS ONE

Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

doi: 10.1371/journal.pone.0226707

Figure Lengend Snippet: (a) Immunohistochemical staining for OLFM4 in pancreatic cancer tissues (magnification: 100×). The left and right figures are the same sample tissue blocks and correspond to staining intensity. Left: HE staining. Right: immunohistochemical staining for OLFM4. (b) Criteria for determination of OLFM4 expression levels. OLFM4 expression levels for immunostaining were determined based on the intensity of staining and percentage of stained cells. Staining intensity and staining percentage criteria are shown. (c) Kaplan-Meier survival analysis in patients with pancreatic cancer (n = 80), showing overall survival according to OLFM4 protein expression. Red line: high expression group (n = 52); blue line: low expression group (n = 28).

Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunostaining

Journal: PLoS ONE

Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

doi: 10.1371/journal.pone.0226707

Figure Lengend Snippet: Univariate and multivariate analyses of prognostic factor for overall survival in 80 patients with pancreatic cancer.

Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway

doi: 10.1038/srep16974

Figure Lengend Snippet: ( a ) Representative lower-grade prostatic intraepithelial neoplasia (LG-PIN) in the anterior prostate (AP) and dorsal-lateral prostate (DLP) of HE-stained sections from littermate Olfm4 (+/+) and Olfm4 (−/−) mice at 18 months of age. Scale bar, 100 μm. Arrow indicates LG-PIN. ( b ) Representative prostate epithelial lesions (upper panels) and tumor (lower panels) in the DLP of HE-stained sections from Olfm4 (−/−) mice. Arrowhead indicates hyperplasia and arrow indicates higher-grade prostatic intraepithelial neoplasia (HG-PIN) (upper right panel), and asterisk indicates microinvasion in tumor (lower right panel) at 20 months (upper panels) and 23 months (lower panels) of age. Scale bar, 100 μm. ( c ) Identification of tumor type from DLP of Olfm4 (−/−) mice at 20 months of age using cell markers. Sections were stained with HE or with antibodies to specific cellular markers: androgen receptor (AR); the basal cell marker p63 (P63); and the neuron endocrine cell marker synaptophysin SY38 (Syn). Scale bar, 50 μm. ( d ) The percentage of prostatic epithelial lesions in 13–24-month-old Olfm4 (+/+), (+/−), and (−/−) mice. HP, hyperplasia; LG-PIN, lower-grade prostatic intraepithelial neoplasia; HG-PIN, higher-grade prostatic intraepithelial neoplasia. ( e ) The Kaplan-Meier plot for tumor-free 13–24-month-old Olfm4 (+/+), (+/−), and (−/−) mice. The significance of differences between experimental groups was determined by the log-rank test.

Article Snippet: The pCMV-6-AC-GFP tag-vector and pCMV-6- OLFM4 -GFP tag plasmids were purchased from Origene.

Techniques: Staining, Marker

Journal: Scientific Reports

Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway

doi: 10.1038/srep16974

Figure Lengend Snippet: ( a ) Representative images of Ki67 staining in sections of DLP tissue from Olfm4 (+/+), Olfm4 (+/−), and Olfm4 (−/−) mice at 12 months of age. Bar, 100 μm. ( b ) Quantitative results of Ki67 staining in DLP of Olfm4 (+/+) and Olfm4 (−/−) mice at 3–6, 10–12, or 18–24 months of age. Error bars represent the SD. The significance of differences between experimental groups was determined by the Student’s t-test. ( c ) Representative images of TUNEL assays of AP and DLP from littermate 18–24-month-old Olfm4 (+/+) and Olfm4 (−/−) mice. Scale bar, 50 μm. Bar graphs represent the quantitative results of TUNEL staining. NS, not significant. Error bars represent the SD. The significance of differences between experimental groups was determined by the Student’s t-test. ( d ) Western-blot analysis of protein expression for caspase 3 in prostate tissues from Olfm4 (+/+) and Olfm4 (−/−) mice at 3 months of age. β-actin was used as a loading control.

Article Snippet: The pCMV-6-AC-GFP tag-vector and pCMV-6- OLFM4 -GFP tag plasmids were purchased from Origene.

Techniques: Staining, TUNEL Assay, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway

doi: 10.1038/srep16974

Figure Lengend Snippet: ( a ) Cell-signaling pathways identified from GeneGo analyses of upregulated gene expression in prostate tissues of Olfm4 (−/−) mice at 3 months of age. ( b ) Mean fold-change in expression of hedgehog signaling-pathway target genes in microarray analyses of prostate tissues from wild-type (WT; n = 4 or 3) and Olfm4 -knockout (KO; n = 4 or 3) mice at 3 and 15 months of age. The significance of differences between experimental groups was determined by ANOVA. ( c ) Mean (±SD, n = 5) fold-change (knockout [KO] vs. wild-type [WT]) in expression of hedgehog signaling-pathway component genes in 3-month-old mouse prostate determined using qRT-PCR. The significance of differences between experimental groups was determined by the Student’s t-test. ( d ) Western-blot analysis of protein expression of hedgehog signaling-pathway components in 3-month-old Olfm4 (+/+) and Olfm4 (−/−) mouse prostate. β-actin was used as a loading control. ( e ) Mean fold-change in expression of upregulated (red text) and downregulated (blue text) genes for EMT, cytokeratin, and stem/progenitor-cell markers in microarray analyses of prostate tissues from Olfm4 (−/−) mice when compared with littermate Olfm4 (+/+) mice at 15 months of age. The significance of differences between experimental groups was determined by ANOVA. ( f ) Mean (±SD, n = 5) fold-change (knockout [KO] vs. wild-type [WT]) in expression of EMT genes in 15-month-old mouse prostate determined using qRT-PCR. The significance of differences between experimental groups was determined by the Student’s t-test.

Article Snippet: The pCMV-6-AC-GFP tag-vector and pCMV-6- OLFM4 -GFP tag plasmids were purchased from Origene.

Techniques: Protein-Protein interactions, Gene Expression, Expressing, Microarray, Knock-Out, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway

doi: 10.1038/srep16974

Figure Lengend Snippet: The OLFM4 stably expressing human metastatic prostate-cancer cell clones PC-3V (vector-GFP tag), PC-3O ( OLFM4 -GFP tag); DU145V (vector-GFP tag), DU145O ( OLFM4 -GFP tag); 22RV1V (vector-GFP tag clone 1 and 2), and 22RV1O ( OLFM4 -GFP tag clone 1and 2; clone 1 data are presented in panel a) were established. ( a ) qRT-PCR analysis of SHH , PTCH1 , and GLI1 in prostate-cancer cell clones. Data represent the mean (±SD) percent expression in OLFM4 -GFP tag-expressing cell clones compared with vector-GFP tag-expressing cell clones (value set at 100%) (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001. The significance of differences between experimental groups was determined by the Student’s t-test. ( b ) Western-blot analysis of protein expression for OLFM4, SHH, PTCH1, GLI1, and GLI2 in prostate-cancer cell clones. β-actin was used as a loading control.

Article Snippet: The pCMV-6-AC-GFP tag-vector and pCMV-6- OLFM4 -GFP tag plasmids were purchased from Origene.

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

Journal: Scientific Reports

Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway

doi: 10.1038/srep16974

Figure Lengend Snippet: ( a ) Immunofluorescent staining of PC-3 cells transfected with OLFM4 -V5 tag (PC-3 OLFM4 clones) or vector control (PC-3V), using anti-V5 (green) anti-SHH (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. ( b ) Coimmunoprecipitation analysis of OLFM4 and SHH. Cell lysates of PC-3 vector control-transfected cell clones (PC-3V), PC-3 cell clones stably expressing OLFM4 -V5 tag (PC-3W), or PC-3 cell clones expressing OLFM4 -N (a truncated deletion of OLFM4 )-Flag tag (PC-3N) were immunoprecipitated with anti-V5 or anti-Flag (or normal IgG) antibody. Immunoprecipitates were subjected to Western-blot analysis with anti-SHH (upper panel) or anti-OLFM4 (middle panel) antibody. Total lysate subjected to Western-blot analysis with anti-SHH antibody was used as a loading control (lower panel). IgG indicates a normal IgG used as a negative control in the immunoprecipitation assays presented. ( c ) Time course of SHH protein secretion into the culture media of vector control-transfected PC-3 (PC-3V) and OLFM4 -transfected PC-3 (PC-3O) cell clones. The cell-culture media (RPMI 1640 containing 0.5% FBS) was harvested from 3 individual wells of 12-well plates after culturing for 6, 18, 24, 30, 42, or 54 h. SHH secretion was determined by ELISA. Data represent the mean ± SD (n = 3). * P < 0.05, * * P < 0.01, *** P < 0.001. The significance of differences between experimental groups was determined by ANOVA.

Article Snippet: The pCMV-6-AC-GFP tag-vector and pCMV-6- OLFM4 -GFP tag plasmids were purchased from Origene.

Techniques: Staining, Transfection, Clone Assay, Plasmid Preparation, Control, Stable Transfection, Expressing, FLAG-tag, Immunoprecipitation, Western Blot, Negative Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway

doi: 10.1038/srep16974

Figure Lengend Snippet: Effects of the OLFM4 gene on GLI-reporter activity in PC-3 cells ( a ) and 22RV1 cells ( b ). Bar graph represents the relative GLI-reporter activity that was normalized by using cotransfection with Renilla luciferase and detected using the dual-luciferase reporter assay system. The mean percent was obtained by comparing activity in triplicate transfections for each experimental condition to the activity for the SHH-N–treated sample (number 2; value set at 100%). SHH-N protein (100 nM) was added 48 h after transfection, and GLI-reporter activity was measured 24 h later. Carry plasmid indicates plasmid carried empty vector. Data represent the mean ± SD of triplicate experiments. * P < 0.05. The significance of differences between experimental groups was determined by ANOVA. Shadow triangle indicates dose of OLFM4 cDNA plasmid.

Article Snippet: The pCMV-6-AC-GFP tag-vector and pCMV-6- OLFM4 -GFP tag plasmids were purchased from Origene.

Techniques: Activity Assay, Cotransfection, Luciferase, Reporter Assay, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway

doi: 10.1038/srep16974

Figure Lengend Snippet: ( a ) Gene-expression levels in published human prostate tissue GSE35988 microarray data. Scott plot graphs represent the relative expression of OLFM4 and SHH in normal prostate, primary prostate tumors, and metastatic prostate tumors. NS, not significant. CRPC, castrate-resistant prostate cancer. The significance of differences between any 2 stages was determined by Mann-Whitney U tests. ( b) Representative images of immunohistochemistry analysis of SHH and OLFM4 expression in human prostate-cancer tissue-array specimens with different Gleason scores. Scale bar, 100 μm. ( c ) Bar graph represents quantitation of immunohistochemistry staining results from ( b ). ( d ) A model illustrating the function of OLFM4 in regulating hedgehog signaling-pathway activities. OLFM4 protein binds to the SHH protein and blocks its binding to the PTCH1 receptor, therefore inhibiting autocrine and paracrine signaling-pathway activities that regulate cellular proliferation and EMT.

Article Snippet: The pCMV-6-AC-GFP tag-vector and pCMV-6- OLFM4 -GFP tag plasmids were purchased from Origene.

Techniques: Gene Expression, Microarray, Expressing, MANN-WHITNEY, Immunohistochemistry, Quantitation Assay, Staining, Binding Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: Olfactomedin-4 improves cutaneous wound healing by promoting skin cell proliferation and migration through POU5F1/OCT4 and ESR1 signalling cascades.

doi: 10.1007/s00018-022-04202-8

Figure Lengend Snippet: Fig. 1 OLFM4 expression in healthy human skin and in regenerating skin after a burn injury. (A) Healthy control skin; (B) biopsy samples from the skin of burn injury patients collected at indicated timepoints after the wound excision. E— epidermis; D—dermis; OLFM4 expression in small blood vessels in dermis is indicated by red arrows. Three representa- tive samples in each group are shown. Scale bar is 200 µm. C Relative quantification of OLFM4 expression by mean integrated density of the fluores- cence signal. The bars depict the averages of samples from 10 patients ± standard deviation, *indicates a statistically signifi- cant (p < 0.05) difference

Article Snippet: For testing the effect of OLFM4 protein, 1 μg of purified recombinant human OLFM4 protein (R&D Systems, Minneapolis, MN, USA, product code 10261-OL-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily to the wounds.

Techniques: Expressing, Control, Quantitative Proteomics, Standard Deviation

Journal: Cellular and molecular life sciences : CMLS

Article Title: Olfactomedin-4 improves cutaneous wound healing by promoting skin cell proliferation and migration through POU5F1/OCT4 and ESR1 signalling cascades.

doi: 10.1007/s00018-022-04202-8

Figure Lengend Snippet: Fig. 2 Upregulation of Olfm4 expression in regenerating mouse skin of cutaneous wounds. A Olfm4 expression was characterized by immu- nofluorescence microscopy in healthy skin (0 days) and at 2-, 4-, 6-, 8- and 12-day post- wounding in a mouse model of full thickness splinted wound healing. 3 representative sam- ples in each group are shown. Ki67 marks proliferating cells, yellow arrows indicate Olfm4 expression in hair follicles. W— wound area; S—scab. Scale bar is 200 µm. B Relative quantifi- cation of Olfm4 expression by mean integrated density of the fluorescence signal. Bars show the average of 3 samples for each timepoint ± standard devia- tion, *indicates a statistically significant (p < 0.05) difference from the values at day 0

Article Snippet: For testing the effect of OLFM4 protein, 1 μg of purified recombinant human OLFM4 protein (R&D Systems, Minneapolis, MN, USA, product code 10261-OL-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily to the wounds.

Techniques: Expressing, Microscopy, Fluorescence

Journal: Cellular and molecular life sciences : CMLS

Article Title: Olfactomedin-4 improves cutaneous wound healing by promoting skin cell proliferation and migration through POU5F1/OCT4 and ESR1 signalling cascades.

doi: 10.1007/s00018-022-04202-8

Figure Lengend Snippet: Fig. 3 OLFM4 expression in healthy control skin and skin in psoriatic lesions. Three repre- sentative samples in each group are shown (A) and a higher magnification view of the OLFM4 expression in psoriatic skin lesions (B). Scale bar is 200 µm on image A and 50 µm on image B. C Relative quantifi- cation of OLFM4 expression by mean integrated density of the fluorescence signal n = 5. The plot depicts the distribution of 5 samples, *indicates a statisti- cally significant (p < 0.05) difference

Article Snippet: For testing the effect of OLFM4 protein, 1 μg of purified recombinant human OLFM4 protein (R&D Systems, Minneapolis, MN, USA, product code 10261-OL-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily to the wounds.

Techniques: Expressing, Control, Fluorescence

Journal: Cellular and molecular life sciences : CMLS

Article Title: Olfactomedin-4 improves cutaneous wound healing by promoting skin cell proliferation and migration through POU5F1/OCT4 and ESR1 signalling cascades.

doi: 10.1007/s00018-022-04202-8

Figure Lengend Snippet: Fig. 4 OLFM4 supports keratinocyte but not fibroblast proliferation in vitro. A Human primary keratinocytes were cultured in the presence of 1 µg/ ml recombinant OLFM4 protein for 24 h, representative images (left) and the quantification of Ki67+ positive cells (right) are shown. The cytoskeleton of the cells was visualised by keratin-5 staining. B Human primary fibroblasts were cultured in the presence of recombinant OLFM4 protein, representative images (left) and the quan- tification of Ki67+ positive cells (right) are shown. The cytoskeleton of the cells was visualised by vimentin staining. Scale bar is 200 µm. The graphs depict the averages of at least 3 independent replicates ± stand- ard deviation, *indicates a sta- tistically significant (p < 0.05) difference compared to control cells

Article Snippet: For testing the effect of OLFM4 protein, 1 μg of purified recombinant human OLFM4 protein (R&D Systems, Minneapolis, MN, USA, product code 10261-OL-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily to the wounds.

Techniques: In Vitro, Cell Culture, Recombinant, Staining, Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: Olfactomedin-4 improves cutaneous wound healing by promoting skin cell proliferation and migration through POU5F1/OCT4 and ESR1 signalling cascades.

doi: 10.1007/s00018-022-04202-8

Figure Lengend Snippet: Fig. 11 OLFM4 supports wound healing in in vivo full- thickness cutaneous wound model. Either 1 µg of recom- binant OLFM4 or vehicle (PBS) was applied on inflicted dorsal wounds daily for up to 12 days. Representative images of the wounds from indicated timepoints (A) and measure- ments of the wound area (B). C Representative histology at the time of sacrifice, hematoxylin and eosin staining. Epithelium thickness at wound edge is indicated by green arrows. H—healthy skin; W—wound; E—epidermis; D—dermis. The graphs depict the averages of 10 biological replicates ± standard deviation, *indicates a statisti- cally significant (p < 0.05) difference

Article Snippet: For testing the effect of OLFM4 protein, 1 μg of purified recombinant human OLFM4 protein (R&D Systems, Minneapolis, MN, USA, product code 10261-OL-050) dissolved in 10 μl of phosphate buffered saline (PBS) was applied daily to the wounds.

Techniques: In Vivo, Staining, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

doi: 10.1016/j.jcmgh.2019.12.005

Figure Lengend Snippet: List of Antibodies

Article Snippet: Olfm4 , Novus Biologicals (Centennial, CO)/NBP2-24535 , 1:800 , WB.

Techniques: Transduction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

doi: 10.1016/j.jcmgh.2019.12.005

Figure Lengend Snippet: Deletion of Cldn7 resulted in the loss of active crypt stem cells and an increase in epithelial cell proliferation in gKO SIs. ( A ) The qRT-PCR analysis of IESC marker genes in PN3 WT and gKO SIs. Lgr5 , Olfm4 , and Sox9 are active intestinal stem cell marker genes. Hopx is a quiescent stem cell marker gene and Prom1 is a transit-amplifying cell marker gene. n = 4–5; 2-tailed, paired t test; * P < .05, ** P < .01, *** P < .001. ( B ) The protein levels of Olfm4 and Hopx were detected by immunoblotting ( left ) and quantified ( right ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. n = 4; 2-tailed, unpaired t test; * P < .05, ** P < .01. ( C ) The IESC maker Olfm4 gene was detected by FISH in PN3 ( left ) and PN0 ( right ) WT and gKO SIs. Arrowheads indicate the Olfm4+ cells. ( D ) Quantification of Olfm4+ cells in panel C (PN3). n = 5; 2-tailed, unpaired t test; **** P < .0001. ( E ) Double-immunofluorescent labeling of Sox9 (red) and claudin-7 (green). Arrowheads indicate the Sox9+ cells. ( F ) Co-immunolabeling of proliferating cell marker PCNA (green) with enterocyte marker FABP-1 (red) ( left ). The quantification of PCNA-positive cells is shown ( right ). n = 5; 2-tailed, unpaired t test; * P < .05. Each point represents the calculation from 50 crypt–villus in each mouse. Data are represented as means ± SEM. Images are representative of more than 3 independent experiments. ( C , E , and F ) Scale bars : 100 μm.

Article Snippet: Olfm4 , Novus Biologicals (Centennial, CO)/NBP2-24535 , 1:800 , WB.

Techniques: Quantitative RT-PCR, Marker, Western Blot, Control, Labeling, Immunolabeling

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

doi: 10.1016/j.jcmgh.2019.12.005

Figure Lengend Snippet: Cldn7 deletion in adult cKO SIs led to the loss of active crypt stem cells, increased epithelial cell proliferation, and aberrant positioning of Paneth cells. ( A ) The qRT-PCR analysis of Cldn7 , Lgr5 , Olfm4 , and Hopx mRNA in 3-month-old control and cKO SIs. n = 4–5; 2-tailed, paired t test; * P < .05, ** P < .01. ( B ) Active IESCs were labeled using FISH ( left ) and quantified ( right ) in 3-month-old control and cKO SIs. Each point represents the calculation from 50 crypts in each mouse. n = 5; 2-tailed, unpaired t test, **** P < .0001. ( C ) The control and cKO SIs were triple-labeled with lysozyme/PCNA/Hoechst to show the positions of Paneth and proliferating cells ( left ). Arrowheads and circles highlight the epithelial cells with positive PCNA and lysozyme signals. The statistical analysis of PCNA-positive cells is shown ( right ). Each point represents the calculation from 50 crypt–villus in each mouse. n = 5; 2-tailed, unpaired t test, **** P < .0001. ( D ) Paneth cells were labeled using antilysozyme antibody by immunohistochemistry to compare the position of the Paneth cells between control and cKO SIs ( left , images in the square were enlarged as indicated by corresponding lines). The statistical analysis of Paneth cells is shown ( right ). Each point represents the calculation from 50 crypt–villus in each mouse. n = 5; 2-tailed, unpaired t test; **** P < .0001. Data are represented as means ± SEM. ( E ) Parallel labeling of lysozyme and mucin 2 (muc2) on the neighboring control and cKO SI sections. Arrowheads highlight the epithelial cells with positive lysozyme and muc2 signals. The images in the square were enlarged as indicated by corresponding lines . ( F ) Co-labeling of Alcian blue (blue) and antilysozyme antibody by immunohistochemistry (brown). Arrowheads indicate the epithelial cells with positive Alcian blue and lysozyme signals (n = 4). Scale bars : 100 μm.

Article Snippet: Olfm4 , Novus Biologicals (Centennial, CO)/NBP2-24535 , 1:800 , WB.

Techniques: Quantitative RT-PCR, Control, Labeling, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

doi: 10.1016/j.jcmgh.2019.12.005

Figure Lengend Snippet: Cldn7 is essential for in vitro organoid survival and growth of adult cKO SIs. ( A ) Representative bright-field images of control and cKO organoids isolated from 3-month-old control and cKO SI crypts treated with tamoxifen in vivo. Images were captured on days 2 and 6 after plating. ( B ) Quantification of organoid numbers. The percentage of organoids were compared by 1-way analysis of variance, followed by the Dunnett multiple comparison test; n = 3; **** P < .0001, P > .05. Scale bars : 100 μm. ( C ) Representative bright-field images of organoids isolated from 3-month-old cCldn7 fl/fl-W and cCldn7 fl/fl-T SI crypts. Then these organoids were treated with either DMSO or 4OH-TAM (3 μmol/L) in vitro. All images were captured on day 6 after plating. ( D ) Quantification of organoid numbers. The percentage of organoids were compared by 1-way analysis of variance, followed by the Dunnett multiple comparison test; n = 3; ** P < .01, P > .05. ( E ) Representative images of bright-field ( top ), whole-mount cleaved caspase-3 and TUNEL staining of cCldn7 fl/fl-T organoids treated with either DMSO or 4OH-TAM after a 6-day culture. Images are representative of 3 independent experiments. ( F ) Detection of Cldn7 (green) and proliferating cells (red) by immunofluorescent staining in DMSO- or 4OH-TAM–treated cCldn7 fl/fl-T organoids after a 6-day culture (n = 3). ( G ) Detection of Olfm4+ stem cells by immunohistochemistry in DMSO- or 4OH-TAM–treated cCldn7 fl/fl-T organoids after a 6-day culture (n = 3).

Article Snippet: Olfm4 , Novus Biologicals (Centennial, CO)/NBP2-24535 , 1:800 , WB.

Techniques: In Vitro, Control, Isolation, In Vivo, Comparison, TUNEL Assay, Staining, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

doi: 10.1016/j.jcmgh.2019.12.005

Figure Lengend Snippet: Oligonucleotide Primers

Article Snippet: Olfm4 , Novus Biologicals (Centennial, CO)/NBP2-24535 , 1:800 , WB.

Techniques: Sequencing

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: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: OLFM4 expression is enhanced by Notch activation. Cells were stimulated with dH 2 O (control) or doxycycline (DOX, 100 ng/ml) for 24 h unless otherwise stated. (A) LS174T and DLD1 parent cells (Parent), and their respective tet-on NICD cells (NICD) were treated with DOX and collected for immunoblot analysis of NICD1 and Hes1. (B) Cells were treated with DOX and collected for qRT-PCR analysis of OLFM4 expression. data were normalized to β-actin levels. ** P < 0.01; **** P < 0.0001. n.s. not significant. (C) LS174T tet-on NICD cells were treated with DOX and collected for immunoblot analysis of intracellular OLFM4 protein. Two different forms of OLFM4 protein (B1 and B2) were observed.

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Activation Assay, Control, Western Blot, Quantitative RT-PCR

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: OLFM4 expression is enhanced by the synergy of TNF-α and Notch activation. (A) Cells were treated with DOX, TNF-α (50 ng/ml), IL-1β (25 ng/ml), IFN-γ (50 ng/ml), IL-6 (50 ng/ml), or LPS (100 ng/ml) for 24 h and collected for qRT-PCR analysis of OLFM4 expression. (B) After pre-treatment with DOX (100 ng/ml) for 24 h, LS174T cells were treated with TNF-α (50 ng/ml) for the indicated time-period. (C) After pre-treatment by DOX (100 ng/ml) for 24 h, LS174T cells were treated with TNF-α at the indicated concentration for 24 h. (D) Colonic organoids established from non-inflamed human colonic tissue (3 cases) were treated with TNF-α (50 ng/ml) for 24 h and collected for qRT-PCR analysis of OLFM4 expression. Data were normalized to β-actin levels. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Concentration Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: Increased expression of OLFM4 is regulated at the transcriptional level by TNF-α and Notch activation in human IECs.Cells were stimulated with dH 2 O (control) or DOX (100 ng/mL) for 24 h, unless otherwise indicated. (A) Luciferase reporter analysis using OLFM4-Luc. The transcriptional activity of the human OLFM4 gene was quantified in LS174T tet-on NICD and DLD1 tet-on NICD cells using a luciferase reporter plasmid containing the -2000 to +10 region of the human OLFM4 gene. (B) Luciferase reporter analysis using OLFM4-Luc with the addition of cytokines to LS174T tet-on NICD cells. (C) A ChIP assay for the human OLFM4 promoter region was performed in LS174T tet-on NICD cells. Cells were stimulated with DOX and TNF-α (50 ng/mL) for 24 h and subjected to ChIP analysis. Immunoprecipitation was performed using either rabbit IgG or anti-NICD1 antibodies. Primer sets were designed to amplify the proximal region of the human OLFM4 promoter, including an area with putative binding sites for RBP-Jκ and NF-κB (Site A). Data were normalized to the initial chromatin input. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, n.s. not significant.

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Activation Assay, Control, Luciferase, Activity Assay, Plasmid Preparation, Immunoprecipitation, Binding Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: Synergy between TNF-α and Notch activation promotes cytoplasmic accumulation of OLFM4 protein in human IECs. (A) LS174T tet-on NICD cells were treated with DOX and TNF-α (50 ng/ml) for 24 h, before the supernatants were collected for ELISA. The secretion levels of the OLFM4 protein are indicated. (B) LS174T tet-on NICD Cells were treated with DOX and TNF-α (50 ng/ml), IL-1β (25 ng/ml), or IFN-γ (50 ng/ml) for 24 h before immunoblot analysis. Protein levels of OLFM4, NICD1, and Hes1 are shown. (C) LS174T tet-on NICD Cells were treated with DOX and TNF-α (50 ng/ml) for 24 h before immunostaining for OLFM4 (green). Scale bar, 10 μm. (D) Apoptotic response under transient overexpression of cytoplasmic OLFM4 in LS174T cells. Cells were treated with TNF-α (50 ng/ml) for 24 h before collection for immunoblot analysis. Levels of PARP and OLFM4 are shown. * P < 0.05; n.s. not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining, Over Expression

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: OLFM4 expression is enhanced and accumulates intracellularly in IECs of IBD patients. (A) Colonic organoids from patients were treated with TNF-α (50 ng/ml) for 24 h, before immunostaining for OLFM4 (green). (B) Inflamed and non-inflamed tissues from the small intestine and colon of patients were immunostained for OLFM4 expression (green). Small intestinal tissue of a patient with Crohn's disease and colon tissue of a patient with ulcerative colitis (UC) were used to show representative inflammatory patterns of OLFM4 expression. An enlarged view of an area of the left-side panel (white dotted square) is shown in the right panel. (C) Non-inflamed and inflamed colon tissues of a patient with UC were stained for OLFM4 (green), p65 (red), and NICD1 (red) using serial sections. Scale bar, 100 μm. All tissues or organoids were counterstained using DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Immunostaining, Staining