Journal: Bioactive Materials

Article Title: Microfluidic intestinal organoid-on-a-chip uncovers therapeutic targets by recapitulating oxygen dynamics of intestinal IR injury

doi: 10.1016/j.bioactmat.2023.07.001

Figure Lengend Snippet: Bio-responsiveness and genetic signatures of microfluidic intestinal organoid-on-a-chip. (A) The schematic diagram of intestinal organoid damage caused by UV-induced artificial vessel injury. (B) The elevated ROS production of intestinal organoids resulted from UV-triggered artificial vessel injury. (C) The increase of cell apoptosis in intestinal organoids resulted from UV-triggered artificial vessel injury. The arrows label the apoptotic cells. (D) The increase of ROS production in HR-treated intestinal organoids. (E) The increase of cell apoptosis in HR-treated intestinal organoids. The arrows label the apoptotic cells. (F) Comparison of LDH release and caspase-3 activity in intestinal organoids treated with normoxia or HR, n = 4. (G) The heatmap of 1301 genes differentially expressed in intestinal organoids treated with normoxia vs HR (up-regulated: 741; down-regulated: 560). (H) The KEGG analysis of differentially-enriched pathways in HR-treated intestinal organoids compared to those treated with normoxia. (I) The volcano plot labeling differentially expressed genes, among which Olfm4 is the most significantly gene expressed at low levels. (J) Real-time quantitative PCR of the gene of interest, Olfm4 , to verify the findings of RNA-sequencing and compare the expression levels between two HR patterns by using the chip or the tri-gas incubator, n = 4. (K) Comparison of transcriptional levels of inflammatory cytokines (e.g., IL-6 , TNF-ɑ , and IL-10 ) in the intestinal organoids treated with HR or normoxia using the chip, n = 4. (L) Quantitative analysis of IL-6, TNF-ɑ, SOD, MDA in the intestinal organoids on chip treated with HR or normoxia, n = 3. **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: First, 10 mg 1,2-Distearoyl-sn-glycerol-3-phosphocholine (L130335, Aladdin) and 1 mg cholesterol (C104032, Aladdin) were dissolved in ethanol and added into a 25-mL eggplant-shaped flask to form a film by rotary evaporation in a vacuum dryer for 2 h. Then, 1 mL PBS with 12.5 μg OLFM4 recombinant protein (HY-P71179, MCE) was added to the flask filled with nitrogen and treated with 60 min of hydration by shaking and 5 min of ultrasonication in a water bath.

Techniques: Comparison, Activity Assay, Labeling, Real-time Polymerase Chain Reaction, RNA Sequencing, Expressing

Journal: Bioactive Materials

Article Title: Microfluidic intestinal organoid-on-a-chip uncovers therapeutic targets by recapitulating oxygen dynamics of intestinal IR injury

doi: 10.1016/j.bioactmat.2023.07.001

Figure Lengend Snippet: Regulation of OLFM4 on HR-induced intestinal inflammation in vitro . (A) The protocol for the lentivirus transduction of intestinal organoids. (B, C) Success on lentivirus infections on intestinal organoids confirmed by the emerging of GFP. (D) Transcriptional differences of pro-inflammatory cytokines in the HR-treated intestinal organoids with Olfm4 interference or Olfm4 overexpression compared with those infected with vector lentivirus. i and ii: on chip HR treatment; iii and iv: off chip HR treatment based on tri-gas incubator. n = 4. (E) The schematic diagram of intracellular delivery of OLFM4-loaded liposome. (H) The verification of intracellular delivery of OLFM4 by liposome and its inhibitory effects on the p65 phosphorylation of NF-kappa B signaling. (G) Quantitative analysis of genes coding pro-inflammatory cytokines in HR-treated FHC with different pre-interventions using blank liposome, OLFM4, or OLFM4-loaded liposome, n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: First, 10 mg 1,2-Distearoyl-sn-glycerol-3-phosphocholine (L130335, Aladdin) and 1 mg cholesterol (C104032, Aladdin) were dissolved in ethanol and added into a 25-mL eggplant-shaped flask to form a film by rotary evaporation in a vacuum dryer for 2 h. Then, 1 mL PBS with 12.5 μg OLFM4 recombinant protein (HY-P71179, MCE) was added to the flask filled with nitrogen and treated with 60 min of hydration by shaking and 5 min of ultrasonication in a water bath.

Techniques: In Vitro, Transduction, Over Expression, Infection, Plasmid Preparation, Phospho-proteomics

Journal: Bioactive Materials

Article Title: Microfluidic intestinal organoid-on-a-chip uncovers therapeutic targets by recapitulating oxygen dynamics of intestinal IR injury

doi: 10.1016/j.bioactmat.2023.07.001

Figure Lengend Snippet: Dynamic changes and functions of OLFM4 during intestinal IR injury in vivo . (A) The transcriptional changes of the pro-inflammatory cytokine ( IL-1β ) and Olfm4 at the different time points of intestinal IR injury, n = 6. IR-0, -1, or -2 means 30-min ischemia followed by 0, 1, or 2-h reperfusion. (B) The protein levels of the pro-inflammatory cytokine (TNF-ɑ) and OLFM4 at the different time points of intestinal IR injury, n = 6. (C) The change of Olfm4 RNA was observed by fluorescence in situ hybridization in intestinal tissues at the different time points of intestinal IR injury. The arrows label the positively stained cells. (D) The relation of OLFM4 with spatially different extents of intestinal damage in the patient with an ischemic incisional hernia. i, mild lesion; ii, moderate lesion; and iii, severe lesion. (E) The protocol on the lentivirus transduction of mice. i.p., intraperitoneal injection. (F) The lentivirus increased the expression of Olfm4 in the small intestine with Olfm4 transcript, n = 3. (G) The representative HE staining images and Chiu's score of small intestinal samples following IR-2 injury in mice treated with vector lentivirus or the lentivirus with olfm4 transcript, n = 6. (H) The representative in vivo imaging pictures of MPO production by an MPO inflammation probe following IR-2 injury in mice treated with vector lentivirus or the lentivirus with Olfm4 transcript. (I) The representative flow cytometry images and quantitative analysis on the measurement of small intestinal apoptotic cells following IR-2 injury in mice treated with vector lentivirus or the lentivirus with Olfm4 transcript, n = 3. (J) The comparison of pro-inflammatory cytokine production (e.g., IL-6 and TNF-ɑ) following IR-2 injury in mice treated with vector lentivirus or the lentivirus with Olfm4 transcript, n = 6. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: First, 10 mg 1,2-Distearoyl-sn-glycerol-3-phosphocholine (L130335, Aladdin) and 1 mg cholesterol (C104032, Aladdin) were dissolved in ethanol and added into a 25-mL eggplant-shaped flask to form a film by rotary evaporation in a vacuum dryer for 2 h. Then, 1 mL PBS with 12.5 μg OLFM4 recombinant protein (HY-P71179, MCE) was added to the flask filled with nitrogen and treated with 60 min of hydration by shaking and 5 min of ultrasonication in a water bath.

Techniques: In Vivo, Fluorescence, In Situ Hybridization, Staining, Transduction, Injection, Expressing, Plasmid Preparation, In Vivo Imaging, Flow Cytometry, Comparison

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

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: Cell Communication and Signaling : CCS

Article Title: Wnt5a suppresses colorectal cancer progression via TGF-β/NOTUM/OLFM4 axis in patient-derived organoids

doi: 10.1186/s12964-025-02364-z

Figure Lengend Snippet: Wnt5a represses OLFM4 and ATOH1 expression in the microwells array-based organoid. ( A-H ) Organoids that express doxycycline (Dox)-inducible Wnt5a were seeded in microwell array and cultured for 7 days. ( A ) Bright-field images showing the development of an organoid in microwells over a period of 7 days. Scale bars: 100 μm. ( B ) Immunofluorescence images of organoids grown in microwell, these cells were labeled with Ki67 (green), EpCAM (red) and DAPI (blue). Scale bars: 100 μm. ( C ) Representative images of organoids treated with DMSO (left) or Dox (right). The black scale bars represent 100 μm; the white scale bars represent 300 μm. ( D ) Quantification of organoid sizes in ( C ). (E ) Immunofluorescence images of organoids grown in microwell, these cells were labeled with phospho-Smad2 (green), Wnt5a-Flag (red) and DAPI (blue). Scale bars: 100 μm. ( F ) RT–qPCR analysis of the expression of LGR5 and OLFM4 . The data are presented as mean ± SEM ( n = 3). ( G ) Immunofluorescence staining of organoids in the presence of DMS or Dox for OLFM4 (green), Wnt5a-Flag (red) and DAPI (blue). Scale bars: 100 μm. ( H - I ) RT-qPCR analysis of secretory markers (H: MUC2 , DEFA6 ) and ATOH1 ( I ). The data are presented as mean ± SEM ( n = 3)

Article Snippet: Antibodies to p-Smad2 (18338 S) and NOTUM (92654 S) were purchased from Cell Signaling Technology; antibody to Flag (PM020) was purchased from MBL; Antibody to Wnt5a (MA5-15502) was purchased from Thermo Fisher Scientific; Antibody to OLFM4 (MAB9218-100) was purchased from R&D Systems; Antibody to MUC2 (MA5-12345) was purchased from Invitrogen; Goat Anti-Rabbit IgG H&L (HRP, ab205718) was purchased from Abcam; Goat Anti-Mouse IgG, Light-Chain Specific Antibody (HRP Conjugate, 91196 S) was purchased from Cell Signaling Technology.

Techniques: Expressing, Cell Culture, Immunofluorescence, Labeling, Quantitative RT-PCR, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Wnt5a suppresses colorectal cancer progression via TGF-β/NOTUM/OLFM4 axis in patient-derived organoids

doi: 10.1186/s12964-025-02364-z

Figure Lengend Snippet: Wnt5a-mediated inhibition of organoids growth requires NOTUM. ( A ) Organoids were treated with DMSO or Dox for 7days. RT–qPCR analysis of the expression of NOTUM and ATOH8 . The data are presented as mean ± SEM ( n = 3). ( B ) Protein association network for NOTUM using the STRING database. ( C ) Organoids were treated with DMSO or Dox for 7days. Western blot of NOTUM using anti-NOTUM. (D-E) Organoids were RT–qPCR ( D ) and Western blot ( E ) analysis of NOTUM expression in organoids treated with DMSO or Dox in the presence or absence of SB431542 for 7 days. The data are presented as mean ± SEM ( n = 3). ( F - G ) Organoids were treated with or without TGF-β (10 ng/mL) in the presence or absence of SB431542 for 7 days. Expression of NOTUM mRNA ( F ) and NOTUM levels were blotted with anti-NOTUM ( G ). The data are presented as mean ± SEM ( n = 3). ( H ) Representative images of organoids treated with DMSO or DOX in the presence or absence of ABC99 (500 nM) for 7 days. Scale bars: 100 μm. Quantification of organoid sizes in ( H ). ( I ) Organoids were treated with or without Dox in the presence or absence of ABC99 for 7 days. OLFM4 levels were blotted with anti-OLFM4

Article Snippet: Antibodies to p-Smad2 (18338 S) and NOTUM (92654 S) were purchased from Cell Signaling Technology; antibody to Flag (PM020) was purchased from MBL; Antibody to Wnt5a (MA5-15502) was purchased from Thermo Fisher Scientific; Antibody to OLFM4 (MAB9218-100) was purchased from R&D Systems; Antibody to MUC2 (MA5-12345) was purchased from Invitrogen; Goat Anti-Rabbit IgG H&L (HRP, ab205718) was purchased from Abcam; Goat Anti-Mouse IgG, Light-Chain Specific Antibody (HRP Conjugate, 91196 S) was purchased from Cell Signaling Technology.

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Western Blot

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: Endocrinology

Article Title: Olfactomedin 4 Deletion Improves Male Mouse Glucose Intolerance and Insulin Resistance Induced by a High-Fat Diet

doi: 10.1210/en.2018-00451

Figure Lengend Snippet: Olfactomedin 4 is expressed in human and mouse pancreatic islet β cells. (A) Immunofluorescence double staining of OLFM4 with insulin or glucagon in human pancreatic tissue sections. Scale bar, 50 µm. (B) Immunohistochemistry staining of Olfm4 and insulin in consecutive pancreatic tissue sections from WT and Olfm4−/− (knockout) mice. Scale bar, 50 µm. (C) Olfm4 mRNA expression in isolated islets from WT and Olfm4−/− mice by quantitative RT-PCR. Expression was normalized to β-actin expression. Data are expressed as mean ± SD (n = 3). (D) Western blot analysis of Olfm4 expression in isolated islets from WT and Olfm4−/− mice. β-Actin was used as a loading control.

Article Snippet: The sections were stained with anti-human OLFM4 antibody (RRID: AB_2732892 ; Sino Biological Inc., Beijing, China) ( 21 ), anti-mouse Olfm4 antibody (RRID: AB_2650511 ; Cell Signaling Technology, Danvers, MA) ( 22 ), glucagon antibody (RRID: AB_259852 ; Sigma-Aldrich) ( 23 ), and insulin antibody (RRID: AB_2532448 ; Thermo Fisher Scientific, Waltham, MA) ( 24 ).

Techniques: Immunofluorescence, Double Staining, Immunohistochemistry, Staining, Knock-Out, Expressing, Isolation, Quantitative RT-PCR, Western Blot

Journal: Endocrinology

Article Title: Olfactomedin 4 Deletion Improves Male Mouse Glucose Intolerance and Insulin Resistance Induced by a High-Fat Diet

doi: 10.1210/en.2018-00451

Figure Lengend Snippet: Olfm4 deletion improves glucose tolerance and increases plasma insulin levels after glucose challenge. (A) Body weight for WT (O+/+) (n = 12), Olfm4 heterozygous- (O+/−) (n = 10), and Olfm4 homozygous-deficient (O−/−) (n = 10) mice. (B) Blood glucose levels for WT and Olfm4-deficient mice after 16-h overnight fasting. (C) Plasma insulin levels for WT and Olfm4-deficient mice after 16-h overnight fasting. (D) Blood glucose levels during GTTs for WT and Olfm4-deficient mice. (E) Area under the curve (AUC) for (D). (F) Plasma insulin levels during GTTs for WT and Olfm4-deficient mice. (G) Blood glucose levels during ITTs for WT and Olfm4-deficient mice. Data are expressed as mean ± SD (n = 3). *P < 0.05 when compared with WT.

Article Snippet: The sections were stained with anti-human OLFM4 antibody (RRID: AB_2732892 ; Sino Biological Inc., Beijing, China) ( 21 ), anti-mouse Olfm4 antibody (RRID: AB_2650511 ; Cell Signaling Technology, Danvers, MA) ( 22 ), glucagon antibody (RRID: AB_259852 ; Sigma-Aldrich) ( 23 ), and insulin antibody (RRID: AB_2532448 ; Thermo Fisher Scientific, Waltham, MA) ( 24 ).

Techniques:

Journal: Endocrinology

Article Title: Olfactomedin 4 Deletion Improves Male Mouse Glucose Intolerance and Insulin Resistance Induced by a High-Fat Diet

doi: 10.1210/en.2018-00451

Figure Lengend Snippet: Olfm4 deletion increases insulin secretion, ATP production, and mitochondrial respiration in islets isolated from Olfm4-deficient mice. (A) Secreted insulin levels in islets isolated from WT (O+/+) and Olfm4 homozygous-deficient (O−/−) mice in the presence of basal glucose (3 mM) and high glucose (16.7 mM). (B) Insulin content in islets isolated from WT and Olfm4-deficient mice subjected to glucose stimulation. Secreted insulin levels in islets isolated from WT (O+/+) and Olfm4 homozygous-deficient (O−/−) mice in the presence of (C) 10 mM arginine or (D) 35 mM KCl for 20 min. (E) ATP production in islets isolated from WT and Olfm4-deficient mice in the presence of basal glucose (3 mM) and high glucose (16.7 mM). (F) Mitochondria respiration reflected by OCR levels was detected in islets from WT (O+/+) and Olfm4-deficient mice (O−/−) under basal conditions or following the addition of glucose (20 mM) or oligomycin (1 µM). The results presented as fold change compared with basal level. Data are expressed as mean ± SD (n = 3). *P < 0.05 when compared with WT. Glu, glucose.

Article Snippet: The sections were stained with anti-human OLFM4 antibody (RRID: AB_2732892 ; Sino Biological Inc., Beijing, China) ( 21 ), anti-mouse Olfm4 antibody (RRID: AB_2650511 ; Cell Signaling Technology, Danvers, MA) ( 22 ), glucagon antibody (RRID: AB_259852 ; Sigma-Aldrich) ( 23 ), and insulin antibody (RRID: AB_2532448 ; Thermo Fisher Scientific, Waltham, MA) ( 24 ).

Techniques: Isolation

Journal: Endocrinology

Article Title: Olfactomedin 4 Deletion Improves Male Mouse Glucose Intolerance and Insulin Resistance Induced by a High-Fat Diet

doi: 10.1210/en.2018-00451

Figure Lengend Snippet: Olfm4 overexpression decreases insulin secretion, ATP production, and mitochondria respiration. (A) Insulin secretion and (B) ATP production from Olfm4-overexpressing or empty vector–transfected pancreatic β Min6 cells in the presence of 3 mM or 16.7 mM glucose. Data are expressed as mean ± SD (n = 3). (C) OCR levels detected in vector or Olfm4-overexpressing Min6 cells under basal conditions or following the addition of oligomycin (1 µM), FCCP (1.5 µM), or antimycin A (1 µM) (n = 5). The rates of (D) basal respiration, (E) maximal respiration, and (F) ATP-linked respiration were quantified by normalization of OCR level to the total protein levels. *P < 0.05 when compared with vector control.

Article Snippet: The sections were stained with anti-human OLFM4 antibody (RRID: AB_2732892 ; Sino Biological Inc., Beijing, China) ( 21 ), anti-mouse Olfm4 antibody (RRID: AB_2650511 ; Cell Signaling Technology, Danvers, MA) ( 22 ), glucagon antibody (RRID: AB_259852 ; Sigma-Aldrich) ( 23 ), and insulin antibody (RRID: AB_2532448 ; Thermo Fisher Scientific, Waltham, MA) ( 24 ).

Techniques: Over Expression, Plasmid Preparation, Transfection

Journal: Endocrinology

Article Title: Olfactomedin 4 Deletion Improves Male Mouse Glucose Intolerance and Insulin Resistance Induced by a High-Fat Diet

doi: 10.1210/en.2018-00451

Figure Lengend Snippet: Olfm4 is localized in mitochondria and interacts with Grim-19. (A) Western blot analysis of Olfm4 and GRIM-19 expression in subcellular fractions of Olfm4-overexpressing Min6 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cytochrome c oxidase (COX) IV, and poly(ADP-ribose) polymerase-1 (PARP-1) were used as subcellular markers for cytoplasm, mitochondria, and nucleus, respectively. (B) Immunofluorescent staining of Olfm4 and Hsp60 or SDHA in Min6 cells. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. Scale bar, 10 μm. (C) Cell lysates from Olfm4-overexpressing Min6 cells were immunoprecipitated (IP) with normal IgG or Olfm4 antibody and subjected to Western blotting with GRIM-19 antibody (top panel). Cell lysates from Olfm4-overexpressing Min6 cells were immunoprecipitated with normal IgG or GRIM-19 antibody and subjected to Western blotting with Olfm4 antibody (bottom panel). The data are representative of three independent experiments.

Article Snippet: The sections were stained with anti-human OLFM4 antibody (RRID: AB_2732892 ; Sino Biological Inc., Beijing, China) ( 21 ), anti-mouse Olfm4 antibody (RRID: AB_2650511 ; Cell Signaling Technology, Danvers, MA) ( 22 ), glucagon antibody (RRID: AB_259852 ; Sigma-Aldrich) ( 23 ), and insulin antibody (RRID: AB_2532448 ; Thermo Fisher Scientific, Waltham, MA) ( 24 ).

Techniques: Western Blot, Expressing, Staining, Immunoprecipitation

Journal: Endocrinology

Article Title: Olfactomedin 4 Deletion Improves Male Mouse Glucose Intolerance and Insulin Resistance Induced by a High-Fat Diet

doi: 10.1210/en.2018-00451

Figure Lengend Snippet: Olfm4 deletion decreases insulin resistance and increases GSIS in HFD-fed mice. Male WT (n = 10) and Olfm4-deficient mice (n = 11) were fed an HFD for 2 months. (A) Body weight of WT and Olfm4-deficient mice. (B) Blood glucose levels in WT and Olfm4-deficient mice after 16-h overnight fasting. (C) Plasma insulin levels in WT and Olfm4-deficient mice after 16-h overnight fasting. (D) HOMA-IR index in WT and Olfm4-deficient mice. (E) Blood glucose levels during GTTs for WT and Olfm4-deficient mice. (F) AUC for (E). (G) Plasma insulin levels during GTTs for WT and Olfm4-deficient mice. (H) Fold increase (over baseline, 0 min) in insulin levels at 15 min in WT and Olfm4-deficient mice. (I) Blood glucose levels during ITTs for WT and Olfm4-deficient mice. Data are expressed as mean ± SD. *P < 0.05 when compared with WT.

Article Snippet: The sections were stained with anti-human OLFM4 antibody (RRID: AB_2732892 ; Sino Biological Inc., Beijing, China) ( 21 ), anti-mouse Olfm4 antibody (RRID: AB_2650511 ; Cell Signaling Technology, Danvers, MA) ( 22 ), glucagon antibody (RRID: AB_259852 ; Sigma-Aldrich) ( 23 ), and insulin antibody (RRID: AB_2532448 ; Thermo Fisher Scientific, Waltham, MA) ( 24 ).

Techniques:

Journal: Cellular and Molecular Life Sciences

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: 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 representative samples in each group are shown. Scale bar is 200 µm. C Relative quantification of OLFM4 expression by mean integrated density of the fluorescence signal. The bars depict the averages of samples from 10 patients ± standard deviation, *indicates a statistically 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, Quantitative Proteomics, Fluorescence, Standard Deviation

Journal: Cellular and Molecular Life Sciences

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: Upregulation of Olfm4 expression in regenerating mouse skin of cutaneous wounds. A Olfm4 expression was characterized by immunofluorescence microscopy in healthy skin (0 days) and at 2-, 4-, 6-, 8- and 12-day postwounding in a mouse model of full thickness splinted wound healing. 3 representative samples 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 quantification of Olfm4 expression by mean integrated density of the fluorescence signal. Bars show the average of 3 samples for each timepoint ± standard deviation, *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, Immunofluorescence, Microscopy, Quantitative Proteomics, Fluorescence, Standard Deviation

Journal: Cellular and Molecular Life Sciences

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: OLFM4 expression in healthy control skin and skin in psoriatic lesions. Three representative 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 quantification of OLFM4 expression by mean integrated density of the fluorescence signal n = 5. The plot depicts the distribution of 5 samples, *indicates a statistically 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, Quantitative Proteomics, Fluorescence

Journal: Cellular and Molecular Life Sciences

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: 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 quantification 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 ± standard deviation, *indicates a statistically 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, Standard Deviation, Control

Journal: Cellular and Molecular Life Sciences

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: OLFM4 supports keratinocyte migration. A Keratinocytes were untreated (control) or stimulated for 24 h with 1 µg/ml recombinant OLFM4 protein and subsequently allowed to migrate through a transwell chamber for 24 h. Representative images (left) and the quantification of the number of migrating cells in a field of view (right) are shown. B In vitro scratch migration assay with keratinocytes stimulated with recombinant OLFM4, representative images at indicated timepoints (left) and the quantification of the relative wound closure in time (right) is shown. Scale bar is 200 µm. The graphs depict the averages of at least 3 independent replicates ± standard deviation, *indicates a statistically significant ( p < 0.05) difference compared to unstimulated keratinocytes

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: Migration, Control, Recombinant, In Vitro, Standard Deviation

Journal: Cellular and Molecular Life Sciences

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: Proteomics analysis of OLFM4 stimulation in keratinocytes. Primary human keratinocytes were stimulated with 1 µg/ml recombinant OLFM4 protein for 24 h before being subjected to protein lysis and mass spectrometry analysis. The combined canonical pathway genes detected from proteomics analysis of OLFM4-stimulated keratinocytes are shown. Upper numbers indicate log2FC and lower p value for proteomics analysis, n = 3

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: Recombinant, Lysis, Mass Spectrometry

Journal: Cellular and Molecular Life Sciences

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: Immunofluorescence analysis of OCT4, PTEN, RND3 and MAP7D1 expression. Cultured primary keratinocytes were untreated (control) or stimulated with 1 µg/ml recombinant OLFM4 protein for 24 h. A Representative images of OCT4 and PTEN expression (left) and quantification of the percentage of cells expressing nuclear OCT4 and PTEN, respectively (right). B Representative images of RND3 and MAP7D1 expression (left) and quantification of the corrected total cell fluorescence levels (right). Scale bars are 200 µm. The graphs depict the averages of 3 replicates ± standard deviation; *indicates a statistically significant ( p < 0.05) difference compared to unstimulated keratinocytes

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: Immunofluorescence, Expressing, Cell Culture, Control, Recombinant, Fluorescence, Standard Deviation

Journal: Cellular and Molecular Life Sciences

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: Ingenuity Pathway Analysis of ESR1, SOX11, DIRAS3 and TFRC signalling. A RNAseq-based signalling pathways (left) and proteomics-based pathways (right) in keratinocyte transcriptional profile in response to OLFM4 stimulation. Single numbers indicate log2FC in proteomics and duplicate values indicate log2FC and p value for RNAseq, n = 3. B RT-qPCR measurements of SOX11 induction after 4 h stimulation with 1 µg/ml recombinant OLFM4 protein, fold changes in mRNA expression were calculated relative to control cells. *indicates a statistically significant ( p < 0.05) difference compared to unstimulated keratinocytes, n = 3

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: Quantitative RT-PCR, Recombinant, Expressing, Control

Journal: Cellular and Molecular Life Sciences

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: Downstream effect network of upregulated transcripts after keratinocyte stimulation with recombinant OLFM4 protein. String 11.5 network based on parameters: fold change > 2 and p < 0.01 in RNAseq and fold change > 1 and p < 0.01 in proteomics. Only upregulated DEGs (black) and DEPs (red), n = 81, are displayed

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: Recombinant

Journal: Cellular and Molecular Life Sciences

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: Transcriptomic analysis of OLFM4 stimulation in fibroblasts. Primary human skin fibroblasts were stimulated with 1 µg/ml recombinant OLFM4 protein for 4 h and separated RNA was analysed by RNAseq. Activated upstream signalling pathways (A) and inhibited upstream signalling pathways (B) are depicted. Upper numbers indicate the p value and lower log2FC for RNAseq, n = 2

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: Recombinant

Journal: Cellular and Molecular Life Sciences

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: OLFM4 supports wound healing in in vivo full-thickness cutaneous wound model. Either 1 µg of recombinant 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 measurements 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 statistically 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, Recombinant, Staining, Standard Deviation