Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation

doi: 10.1016/j.xpro.2024.103297

Figure Lengend Snippet: Reagents and small molecules used in the β-like cell differentiation

Article Snippet: Sobetirome (GC-1) , Tocris , Cat# 4554.

Techniques: Concentration Assay, Solvent, Recombinant

Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation

doi: 10.1016/j.xpro.2024.103297

Figure Lengend Snippet:

Article Snippet: Sobetirome (GC-1) , Tocris , Cat# 4554.

Techniques: Recombinant, Membrane, Knock-Out, Electroporation, Passaging, Transfection, Control, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Pore Size

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: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 1. (a) Photo of the implantable medical grade polyether ether ketone (PEEK) capsules with a 250 μl drug reservoir and nanochan- nel membranes loaded with GC-1. (b) Scanning electron micro- scopic image of the nanofluidic membrane.

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques: Capsules, Membrane

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 2. Body weight (a), lean mass (b) and fat mass (c) (n = 10) for NMD–PBS- and NMD–GC-1-treated DIO mice (*Po0.05, **Po0.01 and ***Po0.001).

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques:

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 4. (a) TSH and (b) total T4 for (n = 10) NMD–PBS- and NMD–GC-1-treated DIO mice (***Po0.001).

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques:

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 3. Cholesterol (a), triglycerides (b), insulin (c) and glucose (n = 10) (d) for NMD–PBS- and NMD–GC-1-treated DIO mice (*Po0.05, **Po0.01 and ***Po0.001).

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques:

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 5. Thermogenic gene expression (n = 4) from white adipose tissue collected from (a) inguinal deposits (Wing), (b) WAT deposits near the NMD implant (Wimp) and from (c) BAT for NMD–GC-1-treated DIO mice normalized to PBS treatment (*Po0.10 and **Po0.05). Histology (hematoxylin–eosin (H&E)) for (d, g) WATing, (e, h) WATimp and (f, i) BAT for NMD–PBS- (d–f) and NMD–GC-1- (g–i) treated DIO mice. All images taken at original magnification × 10; scale bar is 100 μm.

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques: Gene Expression

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 6. Representative images of pancreatic islets from DIO mice treated with (a–c) NMD–PBS and (d–f) NMD–GC-1. Islets were double stained for insulin (green) and glucagon (red). All images taken at original magnification × 4; scale bar is 200 μm. Histomorphometric comparisons of (g) islet number and (h) β-cell area for DIO mice treated with NMD–PBS and NMD-GC-1 (*Po0.10). Four mice per group were analyzed.

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques: Staining

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