Journal: Molecular and Cellular Biology

Article Title: Distinct Functions of Sox2 Control Self-Renewal and Differentiation in the Osteoblast Lineage

doi: 10.1128/mcb.05798-11

Figure Lengend Snippet: FIG. 1. Identification of Sox2 domains required for self-renewal and the inhibition of Wnt signaling. (A) Schematic representation of Sox2 constructs and summary of their activity in rescue of colony formation and Wnt inhibition. , complete effect; and , partial effect; /, negligible effect; , no effect; HMG, DNA binding domain of Sox2; LEXA, DNA binding domain of LexA; RI, R2, and R3, transactivation domains of Sox2; VP16, activation domain of VP16; Eng, repressor domain of Engrailed. The results of a colony assay and a Wnt reporter assay

Article Snippet: For detection of Sox2 deletion mutants, specific Sox2 antibodies from Novus Biologicals (catalog no. NB110-79875) and Activemotif (catalog no. 39823) were used.

Techniques: Inhibition, Construct, Activity Assay, Binding Assay, Activation Assay, Colony Assay, Reporter Assay

Journal: Molecular and Cellular Biology

Article Title: Distinct Functions of Sox2 Control Self-Renewal and Differentiation in the Osteoblast Lineage

doi: 10.1128/mcb.05798-11

Figure Lengend Snippet: FIG. 2. Independent domains of Sox2 inhibit the Wnt–-catenin pathway. (A and B) Wnt-responsive luciferase activity in osteoblasts expressing Sox2 mutants. OB1-TOP cells (osteoprogenitor cells containing a stably integrated Wnt-responsive luciferase reporter construct) were infected with empty vector or the indicated constructs for 36 h and plated in triplicate. The next day, cells were treated with Wnt3A (100 ng/ml) for 10 h (A) or with CHIR 99021 (4.5 M) for 18 h (B), and luciferase activity was measured. *, P 0.05. (C and D) Wnt-responsive luciferase activity in osteoblasts expressing Sox2-chimeric fusion proteins. OB1-TOP cells were infected with empty vector, Sox2, HMG-VP16, or HMG-Eng and treated with Wnt3A (100 ng/ml) for 10 h (C) or with CHIR 99021 (4.5 M) for 18 h (D), and luciferase activity was measured as described in Materials and Methods. *, P 0.05. Error bars, SD.

Article Snippet: For detection of Sox2 deletion mutants, specific Sox2 antibodies from Novus Biologicals (catalog no. NB110-79875) and Activemotif (catalog no. 39823) were used.

Techniques: Luciferase, Activity Assay, Expressing, Stable Transfection, Construct, Infection, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Distinct Functions of Sox2 Control Self-Renewal and Differentiation in the Osteoblast Lineage

doi: 10.1128/mcb.05798-11

Figure Lengend Snippet: FIG. 3. Sox2 inhibits Wnt signaling by induction of APC and GSK3 mRNA and by binding to -catenin. (A) Gene expression analysis of APC and GSK3 in osteoblasts expressing Sox2 and chimeric proteins. OB1 cells were infected with vector, Sox2, LexA-121-319, or HMG-VP16 for 48 h. Expression levels of APC and GSK3 were analyzed using qRT-PCR and specific primers. All values are normalized to actin and are expressed as severalfold increases compared to vector-infected control results. *, P 0.05. (B) Western analysis of phosphorylated -catenin. Empty vector, Sox2, LexA 121-319, and HMG-VP16 viral vectors were expressed in OB1 cells, and phospho--catenin (serine 33 and threonine 45) was detected after 48 h. (C) Interaction of Sox2 (R1) with -catenin. OB1 cells were infected with empty vector, Sox2, Sox2-129-254 (containing R1 only), LexA 121-319, or Sox2 1-255 for 36 h. Cells were treated with control media or Wnt3A-conditioned media for 24 h. Immunoprecipitation (IP) was performed with anti-Sox2 antibody against the C terminus of Sox2 or against the HMG domain, and blot analysis was performed with anti--catenin antibody, anti-Sox2 HMG antibody, or anti-Sox2 C-terminal antibody. The lower panel shows expression levels of proteins in the whole-cell lysate used in IP. Error bars, SD.

Article Snippet: For detection of Sox2 deletion mutants, specific Sox2 antibodies from Novus Biologicals (catalog no. NB110-79875) and Activemotif (catalog no. 39823) were used.

Techniques: Binding Assay, Gene Expression, Expressing, Infection, Plasmid Preparation, Quantitative RT-PCR, Control, Western Blot, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Distinct Functions of Sox2 Control Self-Renewal and Differentiation in the Osteoblast Lineage

doi: 10.1128/mcb.05798-11

Figure Lengend Snippet: FIG. 4. Gene expression changes following Sox2 deletion. (A) Expression profiles of GFP- and CRE-infected Sox2flox/flox cells, showing entities upregulated 1.66-fold at 72 h. Normalized expression values plotted on a log2 scale were determined relative to expression in GFP-infected samples. Expression changes upregulated more than 2-fold are shown in red, and those downregulated over 2-fold are shown in blue. The right panel shows Sox2 and CRE protein expression as determined by Western analysis of GFP- and CRE-infected Sox2flox/flox cells at the indicated time points. Tubulin was used as a loading control. (B) Cell cycle genes are downregulated upon Sox2 deletion. A GSEA enrichment plot showing expression enrichment of a set of cell cycle-related genes is presented. A negative enrichment score indicates that expression of the majority of these genes in the GFP-infected cells was enriched. Bars represent individual genes in a ranked data set list. A heat map of the genes in the leading edge (most highly ranked) with the greatest significant differences revealed in experiments performed using triplicate samples is shown.

Article Snippet: For detection of Sox2 deletion mutants, specific Sox2 antibodies from Novus Biologicals (catalog no. NB110-79875) and Activemotif (catalog no. 39823) were used.

Techniques: Gene Expression, Expressing, Infection, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Distinct Functions of Sox2 Control Self-Renewal and Differentiation in the Osteoblast Lineage

doi: 10.1128/mcb.05798-11

Figure Lengend Snippet: FIG. 5. Stem cell genes are downregulated upon Sox2 deletion. (A) A GSEA enrichment plot showing expression enrichment of a set of common stem cell-related genes at 48 h is presented. Bars represent individual genes in a ranked data set list. Expression of the majority of these genes was enriched in the GFP-infected cells. A heat map of the genes in the leading edge showing the strongest downregulation in CRE-infected

Article Snippet: For detection of Sox2 deletion mutants, specific Sox2 antibodies from Novus Biologicals (catalog no. NB110-79875) and Activemotif (catalog no. 39823) were used.

Techniques: Expressing, Infection

Journal: Molecular and Cellular Biology

Article Title: Distinct Functions of Sox2 Control Self-Renewal and Differentiation in the Osteoblast Lineage

doi: 10.1128/mcb.05798-11

Figure Lengend Snippet: FIG. 7. BMI-1 is a critical regulator of Sox2-dependent self-renewal. (A) Western analysis of Sox2, Foxp1, and BMI-1 expression. Sox2flox/

Article Snippet: For detection of Sox2 deletion mutants, specific Sox2 antibodies from Novus Biologicals (catalog no. NB110-79875) and Activemotif (catalog no. 39823) were used.

Techniques: Western Blot, Expressing

Journal: Molecular and Cellular Biology

Article Title: Distinct Functions of Sox2 Control Self-Renewal and Differentiation in the Osteoblast Lineage

doi: 10.1128/mcb.05798-11

Figure Lengend Snippet: FIG. 9. Model for the multiple mechanisms by which Sox2 regu- lates the osteoblast lineage. Sox2 maintains stemness and self-renewal by transcriptional mechanisms, fostering expression of genes such as Bmi-1 that are necessary for the self-renewal of osteoprogenitor cells. Sox2 also regulates transcription of genes in the Wnt pathway (e.g., APC, GSK3, and Fzd receptor genes) whose upregulation or down- regulation would inhibit Wnt signaling. Additionally, Sox2 downregu- lates canonical Wnt signaling by interacting through its C-terminal activation domain (R1) with -catenin, thus inhibiting the prodiffer- entiation Wnt pathway. FIG. 8. Expression of Sox2-regulated genes in the bones of mice with osteoblast-specific conditional knockout of Sox2 (6). Total RNA was extracted from femurs of 8-week old mice (A) and calvaria of P1 pups (B). Expression of indicated genes was analyzed by qRT-PCR using specific primers. All values are normalized to 18S rRNA and are expressed as fold change compared to wild-type (WT) results. Sox2 CKO, Sox2 conditional knockout. *, P 0.05. Error bars, SD.

Article Snippet: For detection of Sox2 deletion mutants, specific Sox2 antibodies from Novus Biologicals (catalog no. NB110-79875) and Activemotif (catalog no. 39823) were used.

Techniques: Expressing, Activation Assay, Knock-Out, Quantitative RT-PCR

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 3. Knockdown of WPMY-1 suppresses LIF-driven neuroendocrine differentiation (NED) and malignant progression in prostate cancer (PCa) cells (A) Relative protein levels of LIF, LIFR, WISP1, SOCS3, and PDL1 were measured in AR-positive PCa cell lines (LNCaP, C4-2, and 22Rv1), an AR-negative PCa cell line (PC3), and an NEPC cell line (LASCPC-01). (B) Relative protein levels of WISP1, phosphorylated (p)-STAT3, STAT3, PDL1, and SOCS3 were measured in LNCaP cells treated with PBS or 100 ng/mL of the LIF recombinant protein for a duration ranging 0 to 96 h. (C) Relative mRNA levels of WISP1, neuroendocrine (CHGA, SYP, and ENO2), stem cell (SOX2 and NANOG), and anti-inflammatory (SOCS3 and PDL1) markers in LNCaP cells treated with PBS or 100 ng/mL of the LIF recombinant protein or combined treatment with DMSO or 35 nM EC330 for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (D) Relative protein levels of WISP1, p-STAT3, STAT3, PDL1, and SOCS3 in LNCaP and C4-2 cells cultured in PBS or 100 ng/mL of the LIF recombinant protein, followed by DMSO or 35 nM EC330 treatment for 48 h. (E) Relative mRNA expression levels of WISP1, neuroendocrine markers, stem cell markers, and anti-inflammatory markers (SOCS3 and PDL1) were measured in LNCaP cells expressing the non-target control (NC) or WISP1 siRNA, followed by treatment with PBS or 100 ng/mL of the LIF recombinant protein for 48 h * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. (F and G) Relative cell proliferation (F) and sphere formation (G) were measured in LNCaP cells expressing either NC or WISP1 siRNA, followed by treatment with either PBS or 100 ng/mL of LIF recombinant protein for 5 days (F) or 1 week (G). Scale bars represent 100 mm (G). * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. (H and I) Relative cell migration (H) and invasion through Matrigel (I) were measured in LNCaP cells expressing the NC or WISP1 siRNA, followed by treatment with PBS or 100 ng/mL of LIF recombinant protein for 12 h. Scale bars representing 20 mm are shown. * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. Quantification of relative mRNA levels, cell proliferation, sphere formation, and cell invasion through Matrigel is presented as the mean G SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.005. (J) A GSEA of the TCGA PCa dataset revealed significant associations between high WISP1 expression in prostate tissues and a gene signature representing NEPC-responsive signaling. NES, normalized enrichment score; FDR, false discovery rate.

Article Snippet: Antibodies CXCL5 ThermoFisher 710010 CXCR2 Abcam ab217314 CHGA Cell signaling Cat#85798 SOX2 NOVUS NB110-37235 LIF Cloud PAA085Hu01 p-STAT3 Cell signaling Cat#9145 LIFR Proteintech 22779-1-AP STAT3 Cell signaling Cat#9139 PDL1 Proteintech 66248-1-Ig WISP1 ThermoFisher PA5-106451 ENO2 Abcam ab218388 b-actin Merck MAB1501

Techniques: Knockdown, Recombinant, Cell Culture, Expressing, Control, Migration

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 4. Crosstalk between prostate cancer (PCa) cells and prostate stromal cells promotes neuroendocrine differentiation (NED) and expressions of immunosuppressive cytokines in the tumor microenvironment (TME) through the activation of LIF/LIFR signaling (A) Relative mRNA expression levels of WISP1, neuroendocrine markers (CHGA, SYP, and ENO2), stem cell markers (SOX2 and NANOG), anti-inflammatory markers (SOCS3 and PDL1), LIF, CXCR2, and CXCL5 in LNCaP cells. These cells were cultured with conditioned medium (CM) collected from human WPMY-1 stromal cells, at concentrations of 0%, 15%, or 50%, for a duration of 48 h * vs. 0%, as determined by a one-way ANOVA. (B) Relative mRNA expression levels of WISP1, neuroendocrine markers, stem cell markers, anti-inflammatory markers, LIF, CXCR2, and CXCL5 were measured in LNCaP cells. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMSO or 35 nM EC330 for 48 h * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO, as determined by a one-way ANOVA. (C) Relative mRNA expression levels of WISP1, neuroendocrine, stem cell, anti-inflammatory markers, LIF, CXCR2, and CXCL5 in LNCaP cells cultured with CM collected from the non-target control (Luc) or LIFR shRNA-expressing WPMY-1 cells for 48 h * vs. Veh; # vs. WPMY-1/shLuc CM, as determined by a one-way ANOVA. (D) Relative mRNA expression levels of LIFR, LIF, CXCL5, WISP1, and anti-inflammatory markers (IL10, IL4, IL1RN, TGFB1, VEGFA, IFNA17, and SOCS3) in WPMY-1 cells stably expressing the shLuc or LIFR shRNA. * vs. shLuc, as determined by a one-way ANOVA. (E) Relative mRNA expression levels of LIFR, LIF, CXCL5, WISP1, and anti-inflammatory markers in WPMY-1 cells treated with PBS or 100 ng/mL of the LIF recombinant protein, followed by DMSO or 35 nM EC330 treatment for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (F and G) Cell proliferation (F) and sphere formation (G) of LNCaP cells were evaluated. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMOS or 35 nM EC330 for 48 h. Scale bars in (G) represent 100 mm. Statistical comparisons were performed using a one-way ANOVA. * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO. (H and I) Relative cell migration (H) and invasion through Matrigel (I) were measured in LNCaP cells. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMSO or 35 nM EC330 for 12 h. Scale bars representing 20 mm are shown. * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO, as determined by a one-way ANOVA. Quantification of relative mRNA levels, cell proliferation, sphere formation, and cell invasion through Matrigel is presented as the mean G SEM, based on three biological replicates. Significance levels are denoted as *p < 0.05, **p < 0.01, and ***p < 0.005.

Article Snippet: Antibodies CXCL5 ThermoFisher 710010 CXCR2 Abcam ab217314 CHGA Cell signaling Cat#85798 SOX2 NOVUS NB110-37235 LIF Cloud PAA085Hu01 p-STAT3 Cell signaling Cat#9145 LIFR Proteintech 22779-1-AP STAT3 Cell signaling Cat#9139 PDL1 Proteintech 66248-1-Ig WISP1 ThermoFisher PA5-106451 ENO2 Abcam ab218388 b-actin Merck MAB1501

Techniques: Activation Assay, Expressing, Cell Culture, Control, shRNA, Stable Transfection, Recombinant, Migration

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 6. WISP1 abundance in serum relative to prostate cancer (PCa) progression (A) Relative mRNA levels of CXCL5, CXCR2, LIF, WISP1, neuroendocrine (CHGA, SYP, and ENO2), and stem cell (SOX2 and NANOG) markers in C4-2 cells expressing the empty vector (EV) or CXCL5-expressing vector, followed by treatment with DMSO or 35 nM EC330 for 48 h * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a one-way ANOVA. (B and C) Tumor growth analysis was conducted by subcutaneously inoculating male nude mice with C4-2 cells expressing either the EV or a CXCL5-expressing vector. The mice were then treated bi-daily with either DMSO or 2.5 mg/kg EC330 via intraperitoneal injection and allowed to grow for 8 weeks. Tumor sizes were measured weekly (B). Tumor weights were measured upon tumor collection (C). n = 5 per group. * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a one- way ANOVA and t-test. (D and E) Immunohistochemical (IHC) staining and intensity analyses were performed to assess protein levels of CXCL5, CXCR2, LIF, WISP1, and ENO2 in subcutaneous tumors derived from (B). * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a two-tailed Student’s t-test. Scale bars, 100 mm. (F) WISP1 concentrations were measured in patient sera derived from samples of benign prostatic hyperplasia (BPH; n = 10), hormone-sensitive PCa (HSPC, n = 10), and metastatic castration-resistant PCa (mCRPC; n = 8). * vs. BPH; # vs. HSPC, analyzed by a one-way ANOVA.

Article Snippet: Antibodies CXCL5 ThermoFisher 710010 CXCR2 Abcam ab217314 CHGA Cell signaling Cat#85798 SOX2 NOVUS NB110-37235 LIF Cloud PAA085Hu01 p-STAT3 Cell signaling Cat#9145 LIFR Proteintech 22779-1-AP STAT3 Cell signaling Cat#9139 PDL1 Proteintech 66248-1-Ig WISP1 ThermoFisher PA5-106451 ENO2 Abcam ab218388 b-actin Merck MAB1501

Techniques: Expressing, Plasmid Preparation, Injection, Immunohistochemical staining, Immunohistochemistry, Derivative Assay, Two Tailed Test

Journal: iScience

Article Title: N6-methyladenosine modification of B7-H3 mRNA promotes the development and progression of colorectal cancer

doi: 10.1016/j.isci.2024.108956

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SOX2 , Novus , Cat#NB110-79875; RRID: AB_1110593.

Techniques: Recombinant, Software

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: Difference in Tissue Expression Scores of Studied Markers in Examined Lesions

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: Difference in Markers Expression Scores in Different Ggrades of Hepatitis Activity and Stages of Fibrosis

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing, Activity Assay

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: Relation between the Expression of Tissue Markers and the Grades of HCC

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: The Correlation between Serum TTF-1, RAGE, GLUT-1 and SOX-2 Genes Expression in Relation to Tumor Grade and Stage of Liver Fibrosis

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: The Validation of SOX-2, GLUT-1, RAGE and TTF-1 Genes as a Diagnostic Biomarker in the Serum

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Biomarker Discovery, Diagnostic Assay