Journal: International Journal of Cancer

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

doi: 10.1002/ijc.32535

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

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

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

Journal: International Journal of Cancer

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

doi: 10.1002/ijc.32535

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

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

Techniques: Expressing, Translocation Assay

Journal: International Journal of Cancer

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

doi: 10.1002/ijc.32535

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

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

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

Journal: International Journal of Cancer

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

doi: 10.1002/ijc.32535

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

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

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

Journal: International Journal of Cancer

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

doi: 10.1002/ijc.32535

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

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

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

Journal: International Journal of Cancer

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

doi: 10.1002/ijc.32535

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

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

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

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: bioRxiv

Article Title: REEP6 deficiency impairs ER and Golgi morphologies and causes retinal degeneration by attenuating the expression of phototransduction proteins

doi: 10.1101/2025.03.02.641069

Figure Lengend Snippet: ( A ) Immunoblotting analysis of the expression of phototransduction proteins, including RHO, GC1, GC2, GRK1, and PDE6B in retinas of P30 WT and KO mice. GAPDH was used as a loading control. ( B ) Quantification of immunoblotting signals shown in ( A ). Data are expressed as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; unpaired t -test. ( C, D ) Immunostaining of P30 mouse retinal sections to label RHO, GC1, GC2, PDE6B, GRK1, and GCAP2 (green). Nuclei were label with DAPI (blue). OS, outer segment; IS, inner segment; ONL, outer nuclear layer. Scale bar, 20 μm. ( E ) COS-7 cells transfected to co-express recombinant RHO-mCherry with EGFP (upper panel) or REEP6-EGFP (lower panel). Nuclei were stained with DAPI (blue). Scale bar, 10 μm.

Article Snippet: The sections were immunostained as previously described using primary antibodies against the following proteins: rhodopsin (1:1,000, custom-made ( )), GC1 (1:200, Proteintech, China), GC2 (1:500, a gift from Dr. Wolfgang Baehr at the University of Utah), PDE6B (1:500, Proteintech, China), GRK1 (1:200, Proteintech, China), and GCAP2 (1:500, a gift from Dr. Wolfgang Baehr at the University of Utah).

Techniques: Western Blot, Expressing, Control, Immunostaining, Transfection, Recombinant, Staining

Journal: Advanced Science

Article Title: Deciphering the Metabolic Impact and Clinical Relevance of N‐Glycosylation in Colorectal Cancer through Comprehensive Glycoproteomic Profiling

doi: 10.1002/advs.202415645

Figure Lengend Snippet: Screening for Potential CRC Prognostic Signature. (A) Point plot showing the AUC values for each IGP. Differential colors represent differential N‐Glycoform. Differential size represents the number of values detected in 90 samples. (B) Boxplot illustrating the AUC for each glycoform. Differential colors represent differential N‐Glycoform. (C) Comparison of AUC values for glycoproteins versus non‐glycoproteins. (D) Top 10 glycoproteins with the highest number of IGPs. The color represents the average number of N‐glycan across all sites in each protein, with higher saturation representing a higher average. (E) ROC curve displaying the performance of multiple glycoproteins logistic regression modules in identifying CRC. Each red dot represents an independent ROC value for each protein. (F) ROC curve showing the results of constructing a random forest model using a combination of CLCA1 and OLFM4. Each red dot represents an independent ROC value for each protein. (G) ROC curve presenting the training results of a random forest model for CLCA1 and OLFM4 using public data. Each red dot represents an independent ROC value for each protein. (H) ROC curve illustrating the testing results of a random forest model for CLCA1 and OLFM4 using public data. Each red dot represents an independent ROC value for each protein. (I) Multivariate survival analyses of IHC patients involved Cox regression analysis. The red line segments represent high risk and the blue color represents low risk.

Article Snippet: IHC staining of OLFM4 (1:1000, 28432‐1‐AP, Proteintech) and CLCA1 (1:100, A15041, Abcam) were performed on paraffin‐embedded human colorectal cancer tissue microarray (HColA180Su21, Shanghai Outdo Biotech Co. Ltd., Shanghai, China), as previously described.

Techniques: Comparison, Glycoproteomics