Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) Immunohistochemical staining of Sox2 demonstrating representative nuclear basal epithelial staining (dark red) in normal glands (Region #3), and two distinct tumor regions that are either uniformly Sox2-positve (Region #2) or Sox2-negative (Region #1). B ) Expression of Sox2 in representative castration-resistant metastatic lesions. The box in the 8× magnification corresponds to the region shown in the 40× image. C ) Percentage and distribution of Sox2 expression among prostate disease states. In normal, BPH, and HGPIN tissues, Sox2 is expressed in basal-epithelial cells (Grey Bars). Positive expression is defined by more than 50% of the cancer cells positive with a relative intensity of 1 or more on a scale of 0–3. In cancer tissues and metastases, Sox2 is either uniformly expressed or absent, and the percentage of Sox2-positive tumors increases with Gleason Score (Black Bars). BPH: Benign Prostatic Hyperplasia; HGPIN: high-grade prostatic intraepithelial neoplasia; GS: Gleason Score (N = number of individual patient specimens analyzed).

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) Immunofluorescent co-staining of Sox2 (green) with the basal-specific marker p63 (red), showing that 75% of normal basal-epithelial cells are positive for both Sox2 and p63 (yellow) while the remaining 25% are Sox2-negative (red). DAPI staining highlights nuclei (representative images of normal prostate epithelium acquired from three different individual patient specimens). B ) Western blotting of a series of patient-derived Prostate (PrEC) and Seminal Vesicle (SVEC) epithelial sell (PrEC) cultures demonstrates that a portion of these cultures express detectable Sox2, while other PrEC and all SVEC cultures do not. C ) Immunocytochemical staining of Sox2 showing uniform nuclear Sox2 expression in Sox2-positive PrECs and lack of Sox2-positive cells within Sox2-negative PrECs (representative images of three independent experiments).

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Staining, Marker, Western Blot, Derivative Assay, Expressing

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) To test whether AR signaling impacted Sox2 expression, we ectopically expressed AR (LV-AR) and control lentivirus (LV-Control) in two different Sox2-positive PrEC lines (#1 and #2). Western blotting shows that expression and further ligand activation of AR using 1 nM R1881 results in decreased Sox2 protein expression and decreased p63 expression. GAPDH was used as a loading control. B ) To test whether AR-mediated inhibition of Sox2 expression was specific to PrECs or could also occur in Sox2-positive human ES cells, we treated the WA01(H1) human ES cell line with androgen (1 nM R1881). Western blotting documents detectable endogenous AR expression in hESCs, which increases in response to androgen. β-Actin was used as a loading control. C ) AR activation in hESCs results in a significant decrease in Sox2 mRNA expression as measured by qPCR (* indicates p<0.05). This was compared to untreated and vehicle treated cells (EtOH).

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Expressing, Control, Western Blot, Activation Assay, Inhibition

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) Western blot of a panel of non-malignant and prostate cancer cell lines for Sox2, Nanog, Oct4, and AR. β-Actin was used as a loading control. Expression of Sox2 in castration-resistant CWR cells is not accompanied by co-expression of Nanog or Oct4. LNCaP, C4-2B, LAPC-4, and MDA-PCa2B cells expressed detectable Nanog, which is presumably the NanogP8 retrogene (Jeter et al., 2011). The human embryonal carcinoma cell line NCCIT was used as a positive control for Sox2, Nanog, and Oct4 at a 1∶10 dilution. B ) Decreased expression of Sox2 upon AR stimulation with physiologic levels of androgen (1 nM R1881) in castration-resistant CWR-R1 prostate cancer cells. Protein lysates from cells treated at defined intervals (3–48 hours) after androgen treatment were subjected to western blotting, and accumulation of secreted PSA expressed in the media validates increased AR signaling. C ) Rapid decrease of Sox2 mRNA in CWR-R1 cells upon AR stimulation as measured by qPCR. Levels at 0.5 hrs and beyond represent a statistically significant decrease in Sox2 mRNA (p<0.05). D ) AR Chromatin Immunoprecipitation (ChIP) documents direct binding of ligand-activated AR to the Sox2 enhancer region in response to AR stimulation by R1881. CWR-R1 cells were treated with vehicle control or 1 nM R1881, and enrichment of the Sox2 promoter after AR-ChIP was normalized as a percentage of total chromatin input. IgG and Histone H3 served as negative and positive controls, respectively. When compared to total input, both the positive control Histone H3 and ligand-activated AR significantly enriched for the Sox2 enhancer (p<0.05). Data represents three independent experiments.

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Western Blot, Control, Expressing, Positive Control, Chromatin Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: Levels of Sox2 mRNA.

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques:

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) To verify that the decrease of Sox2 protein is specific to AR activation and can be reversed by an AR antagonist, CWR-R1 cells were grown in 1 nm R1881 or vehicle for 24 hrs, and then either 10 µM MDV3100 or vehicle was added to the culture medium for an additional 48 hrs. Western blots show a decrease in Sox2 protein with R1881 that can be returned to basal levels with addition of MDV3100, without any change in AR protein. β-Actin was used as a loading control. A schematic outlines the time frame of these experiments. B ) A decrease of Sox2 mRNA with R1881 treatment was measured using qPCR (*p<0.05), which was brought back to control levels upon treatment with MDV3100. C ) A time course of treatment with a known inhibitor of transcription, 10 µM Actinomycin D, yielded a similar rapid decrease of Sox2 mRNA in CWR-R1 cells upon AR stimulation with R1881 as measured by qPCR. Levels at 0.25 hrs and beyond represent a statistically significant decrease in Sox2 mRNA (p<0.05), showing similar kinetics of transcriptional repression with both R1881 and Actinomycin D drug treatments.

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Activation Assay, Western Blot, Control

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) To test whether Sox2 expression is associated with resistance to AR pathway inhibition, we developed a series of prostate cancer cell lines that were resistant to MDV3100. After 30 days of continuous treatment, MDV3100-resistant lines expressed significantly higher Sox2 as measured using qPCR (*p<0.05). AR-mediated PSA expression was also significantly reduced in these lines. B ) Comparison between the castration-sensitive LNCaP and isogenic castration-resistant C4-2 cell lines documents a significant increase in Sox2 expression in castration-resistant C4-2 cells (*p<0.05). C ) Increased Sox2 expression in castration-resistant xenografts of LAPC-4 and CWR-R1 tumors. Tumors were allowed to establish and then host mice were castrated; 30 days later castration-resistant tumors were harvested and total mRNA analyzed for Sox2 and PSA. These data show increased Sox2 within castration-resistant LAPC-4 and CWR-R1 tumors (*p<0.05; data represents quantitation from multiple tumor specimens).

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Expressing, Inhibition, Comparison, Quantitation Assay

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) Ectopic expression of Sox2 in castrate-sensitive LAPC-4 (LV-Sox2, n = 10 mice) cells is sufficient to promote tumor take in castrated male nude hosts compared to control mice (LV-Control, n = 10) (p = 0.019). Inset: western blot documenting ectopic lentiviral Sox2 expression in LAPC-4 cells. GAPDH was used as a loading control. NCCIT cells are used as a Sox2-positive control. B ) Castrated LAPC-4-Sox2 versus LAPC-4-Control tumors do not have differences in serum PSA density. These data support that Sox2 expression does not confer a less-differentiated tumor phenotype. PSA density is the ng/mL total PSA per gram tumor. C ) Expression of Sox2 in LAPC-4 does not significantly increase the percentage of rare CD133-positive cells, which are thought to be putative cancer stem/initiating cells.

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Expressing, Control, Western Blot, Positive Control

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) Depletion of Sox2 protein expression using four different lentiviral shRNA constructs. To test the impact of inhibiting Sox2 expression in castration-resistant CWR-R1 cells, we used a series of shRNAs that targeted different regions of the Sox2 mRNA transcript. These data show that each shRNA sequence results in decreased Sox2 protein expression after 72 hours, and combinations of three were used to obtain levels below detection while controlling for potential off-target (i.e. non-Sox2 specific) effects. A non-silencing shRNA (NSC) control was used in comparison. GAPDH was used as a loading control. B ) Decreased expression of Sox2 results in significant growth inhibition. Cell growth was measured using MTT reduction after five days in complete media. In all four instances of Sox2 knockdown, cell growth was significantly diminished compared to the non-silencing control.

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Expressing, shRNA, Construct, Sequencing, Control, Comparison, Inhibition, Knockdown

Journal: PLoS ONE

Article Title: Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

doi: 10.1371/journal.pone.0053701

Figure Lengend Snippet: A ) The expression of 83 embryonic stem cell-associated genes was analyzed via quantitative real-time PCR analyses to identify Sox2-associated gene changes in LAPC-4-Sox2 cells (compared to LAPC-4-Control cells). Open bars represent genes that are previously identified Sox2-target genes in hESCs (Boyer et al. 2005). No changes in any of these 18 genes were associated with Sox2 expression in LAPC-4 cells. Rather, significant changes in expression were detected for FGF5, Kit, NR5A2, PDX1, and RUNX2 (black bars). B ) Elevated expression of FGF5 in castration-resistant LAPC-4 and CWR-R1 tumors (*p<0.05; same mRNA as described in ).

Article Snippet: Knockdown of Sox2 expression using shRNA expression was achieved using the anti-human Sox2 shRNA gene set, which consists of 4 different lentiviral targeting sequences against Sox2 and a non-silencing control [HSH017628-HIVH1(Sox2) and CSHCTR001-HIVH1 (control) vectors containing a GFP and puromycin-resistance components; Genecopoeia].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SnRNA-seq reveals cellular diversity and dynamic differentiation in NOTCH-driven CP tumors characterized by a glial progenitor-like signature. (A) Major cell types of a NOTCH-driven CPP from an adult Lcre;NICD1 mouse. Uniform Manifold Approximation and Projection (UMAP) of 6428 single-nucleus profiles, colored by post hoc annotated cell type; also see . (B) UMAP showing mesenchymal, epithelial, and NOTCH-activated profiles from snRNA-seq in NOTCH-driven CPP, colored by expression of Wnt5a (mesenchymal marker, left ), Hes1 (NOTCH pathway target, middle ), and Otx2 (CP epithelial marker, right ); also see . (C) GO analysis of differentially expressed genes in the epithelial-like tumor cell compartment in NOTCH-driven CPP. (D) The expression of markers for glial progenitors in the rhombic lip in NOTCH-driven CPP. UMAP shows 6428 single-nucleus profiles from snRNA-seq in NOTCH-driven CPP, colored by expression of genes associated with glial progenitors in the rhombic lip in the hindbrain ( Rspo1 , Zfp423 , Zic3 , Msx1 , Sox2 , and Slc1a3 ). (E) Western blot analysis of MSX1 expression in the CP of wild-type mice, and NOTCH-driven CPP in Lcre;NICD1 animals ( n = 3 per genotype, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Data were generated from 2 independent experiments. (F) RT-qPCR analysis of Rspo1 and Gdf7 mRNA levels in NOTCH-driven CPP and wild-type CP ( n = 3 per time point per genotype, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Three independent experiments were conducted. (G) UMAP showing single-nucleus profiles in the epithelial-like compartment in (A) colored by subgroups; also see . (H) Cell trajectory analysis of single-nucleus profiles of subgroups of the epithelial-like compartment, and the glia-like compartment in NOTCH-driven CPP. (I) Violin plots for the expression of Rspo1 , Zfp423 , Msx1 , and Zic4 in subgroups of the epithelial-like compartment in NOTCH-driven CPP.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Marker, Western Blot, Generated, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: Increased SOX2 expression in CP tumors in humans and mice. (A) Immunohistochemistry of SOX2 is shown in the upper rhombic lip/roof plate (red dotted lines) and hindbrain CP (arrow) in wild-type mice, NOTCH-driven CPP and CPC (arrowheads) in Lcre;NICD1 and Lcre;Ptch cko ;NICD1 animals at embryonic (E) day 14.5 (E14.5). Scale bar, 50 µm. Black dotted line marks the border of the ventricle with SOX2-expressing ependymal cells. (B) Immunofluorescence of SOX2 and OTX2 is shown in Rb1/Trp53 -deficient CPC in adult Lcre;p53 cko ;Rb cko mice. Nuclei are labeled with DAPI. Scale bar, 50 µm. (C) RT-qPCR analysis of Sox2 expression in wild-type CP, NOTCH-driven CPP, and Rb1/Trp53 -deficient CPC ( n = 5 per tissue type, mean ± SEM, 1-way ANOVA, **** P < 0.0001). (D) RNAscope of Sox2 and Myc expression in hindbrain CP (black arrow) in adult wild-type mice, and NOTCH-driven CPP (arrowhead) in adult Lcre;NICD1 animals. The dotted line marks ventricular walls with ependymal cells (red arrow). Scale bar, 50 µm. (E) Immunofluorescence of SOX2 and ARL13B is shown in NOTCH-driven CPP in adult Lcre;NICD1 animals. DAPI labels nuclei. Scale bar, 20 µm. (F) Immunofluorescence of SOX2 is shown in NOTCH-driven CP tumor cells treated with DMSO or RIN-1 (10 µM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of fluorescence intensity is shown ( n = 333 [DMSO]; n = 101 [RIN-1], mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). (G) Immunohistochemistry of SOX2 in human CP tumors are shown. Scale bar, 50 µm. (CPC: n = 14; CPP and atypical CPP: n = 26). All results were obtained from 3 independent experiments. (H) RT-qPCR analysis of gene expression in human CP organoids and CP tumors ( n = 6 [CP organoid], n = 9 [CPP], n = 13 [CPC], mean ± SEM, 1-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.001; NS, nonsignificant). (I) CosMx analysis of the expression of NOTCH1 , NOTCH2 , NOTCH3 , and SOX2 in a human CPC sample. Boxed region is shown in higher magnification on the right. (J) RNAscope studies of SOX2 and HES5 expression in a human CPP. Scale bar, 200 µm.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Labeling, Quantitative RT-PCR, RNAscope, Fluorescence, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 is essential for the glial progenitor-like signature and NOTCH-driven CP tumor development. (A) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP cells treated with control scrambled siRNAs or siRNAs against Sox2 at different concentrations. DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, **** P < 0.0001). Data were obtained from 3 independent experiments; see also . (B) Bisected brain hemispheres and hematoxylin and eosin (H&E) staining of the hindbrain CP in adult wild-type mice, and CPP in adult Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals. Red arrows point to wild-type CP, arrowheads point to CPP, black arrows point to SOX2-deficient CPP. Scale bar, 50 µm. (C) Immunofluorescence of Ki-67 and GFP is shown in CPP in Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals at day E13.5. Nuclei are labeled with DAPI. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Results were obtained from 3 independent experiments; also see . (D) Principal component analysis (PCA) of CP in wild-type mice, CPP in Lcre;NICD1 mice, and Sox2 -deficient CPP in Lcre;NICD1;Sox2 cko animals; also see . (E) Gene set enrichment analysis (GSEA) of the effect of Sox2 loss on NOTCH-driven CP tumors. Pathways regulating pluripotency of the stem cells are shown as an example; also see . NES, normalized enrichment score. FDR, false discovery rate. (F) RT-qPCR analysis of gene expression in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, nonsignificant); also see .

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Immunofluorescence, Control, Expressing, Staining, Labeling, Quantitative RT-PCR, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 transcriptional targets in CP tumors are enriched in markers and transcriptional regulators of progenitors in the rhombic lip. (A) Pie chart illustrating the distribution of SOX2-binding sites in relation to genes in NOTCH-driven CPP in Lcre;NICD1 animals; also see . (B) Heatmap of tag densities of SOX2 (left) or H3K23Ac (right) ChIP-seq signals at all of the binding regions identified in ChIP-seq experiments. In each heat map the tag density is plotted for 10 Kb at either side of its binding peak summit. (C) Comparison of SOX2 and H3K27Ac signals generated from ChIP-seq fragment counts in the 20 Kb genomic regions surrounding SOX2 peaks in NOTCH-driven CPP. (D) Logos for the motif enriched in SOX2-binding sequences identified by motif analysis in NOTCH-driven CPP; also see . TF: transcription factor; FDR: false discovery rate. (E) Venn diagram shows the overlap of SOX2-associated genes and differentially expressed genes in NOTCH-driven CPPs at days P0 and P21, respectively. (F) GO analysis of candidate SOX2 transcriptional targets in NOTCH-driven CPP. (G) Venn diagram shows the overlap of SOX2 candidate transcriptional targets in (E) and significantly downregulated genes in Sox2 -deficient tumors. (H) Hierarchical clustering of the expression of 52 candidate SOX2 transcriptional targets identified in (G, FDR < 0.05) in wild-type CP, and Sox2 -wild-type or Sox2 -deficient NOTCH-driven CPP. Lmx1b and Hes5 on the heatmap are marked by arrows. (I) The peak density plot of fragment counts is shown in genomic regions that encompass Lmx1b , Lmx1a , Hes5 , and Zic4 , and bound by SOX2 and H3K27Ac, respectively. Genes are labeled in black with sequence in a single exon as a rectangle; also see . (J) RT-qPCR analysis of gene expression in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, *** P < 0.001; **** P < 0.0001; NS, nonsignificant).

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Binding Assay, ChIP-sequencing, Comparison, Generated, Expressing, Labeling, Sequencing, Quantitative RT-PCR, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 regulates transcription factors LMX1A and LMX1B in NOTCH-driven CP tumors. (A) UMAP of 6428 single-nucleus profiles from a NOTCH-driven CPP colored by Lmx1a and Lmx1b expression, respectively. (B) t-distributed stochastic neighbor embedding (t-SNE) plot shows the annotated scATAC-seq profiles of different cell populations in NOTCH-driven CPP. Different subclusters of cells are marked by different colors. (C) Violin plots show the activity of different genes in each subcluster of cells. (D) Western blot analysis of the expression of SOX2, LMX1A, and LMX1B in CP in wild-type mice, and NOTCH-driven CPP in Lcre;NICD1 animals ( n = 3 per group, mean ± SEM, 2-tailed unpaired t test, ** P < 0.01; *** P < 0.001). Three independent experiments were conducted; also see . (E, F) Immunofluorescence of LMX1A (E) and LMX1B (F) is shown in Rb1/Trp53 -deficient CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bars, 50 µm. Three independent experiments were conducted. (G) Immunofluorescence of LMX1A is shown in TP53 -deficient human CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bar, 50 µm. Three independent experiments were conducted. (H, I) Immunofluorescence of LMX1A (H) LMX1B (I) is shown in CPP in Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals at postnatal (P) day 7 (P7). Arrowheads point to tumor cells, arrows point to SOX2-deficient tumor cells. DAPI labels nuclei. Scale bars, 50 µm. Fluorescence intensity is quantified ( n = 1101 [LMX1A], n = 754 [LMX1B] for NOTCH-driven CPP cells; n = 457 [LMX1A], n = 563 [LMX1B] for Sox2 -deficient tumor cells; mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). The experiments were repeated 3 times independently; also see and . (J) RT-qPCR analysis of the expression of Lmx1a and Lmx1b in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, *** P < 0.001; **** P < 0.0001; NS, nonsignificant). Results were obtained from 1 experiment. (K) t-SNE plots show that motifs of LMX1A and LMX1B are enriched in tumor cell subclusters in NOTCH-driven CPP. Colors represent average gene activity score of cells in each subcluster. Dark red means high gene activity score, blue means low gene activity score.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Activity Assay, Western Blot, Immunofluorescence, Infection, Fluorescence, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: LMX1A and LMX1B mediate SOX2 functions to support tumor cell proliferation. (A) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP treated with control scrambled siRNAs or siRNAs against Lmx1a and/or Lmx1b (40 nM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, ** P < 0.01). The experiments were repeated 3 times independently; also see . (B) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP treated with control scrambled siRNAs or siRNAs against Sox2 (40 nM), and infected with viruses expressing HA-tagged Lmx1a , Lmx1b , or control viruses. DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, **** P < 0.0001; NS, nonsignificant). Three independent experiments were conducted; also see . (C) Immunofluorescence of LMX1B is shown in NOTCH-driven CPP cells treated with DMSO or RIN-1 (10 µM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of fluorescence intensity is shown ( n = 233 [DMSO]; n = 201 [RIN-1], mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). (D) RT-qPCR analysis of the expression of SOX2 , LMX1A , and LMX1B in human CP organoids and CP tumors ( n = 6 [CP organoid], n = 9 [CPP], n = 13 [CPC]; mean ± SEM, NS, nonsignificant). The experiments were repeated 1 time independently. (E) RNAscope studies of LMX1A and SOX2 expression in human CP tumors. Scale bar, 200 µm. (F) Immunofluorescence of LMX1A in human CP tumor samples is shown. DAPI labels nuclei. Scale bar, 50 µm. Three independent experiments were conducted. (G) RNAscope studies of LMX1A and HES5 expression in a human CPP. Scale bar, 200 µm.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Immunofluorescence, Control, Expressing, Infection, Fluorescence, Quantitative RT-PCR, RNAscope

Journal: Prostate cancer and prostatic diseases

Article Title: SOX2 expression in the developing, adult, as well as diseased prostate

doi: 10.1038/pcan.2014.29

Figure Lengend Snippet: The level of SOX2 was knocked down in PC3 cells and protein lysate was prepared from parental PC3 cells and PC3 cells that have stably integrated with control shRNA, or with variant shSOX2 construct. Beta-Actin was used as loading control.

Article Snippet: The shSOX2 constructs were purchased from Origene (Rockville, MD).

Techniques: Stable Transfection, Control, shRNA, Variant Assay, Construct

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: Expression levels of SOX2 and LPCAT1 in osteosarcoma. (A,B) qRT-PCR analysis of SOX2 and LPCAT1 expression in tumor tissues (n = 20) and adjacent normal tissues (n = 20) from osteosarcoma patients. (C,D) Western blot analysis of SOX2 and LPCAT1 protein levels in osteosarcoma tissues and paired adjacent normal tissues. (E–H) qRT-PCR and Western blot detection of SOX2 and LPCAT1 expression in normal osteoblast cell line hFOB1.19 and osteosarcoma cell lines (MG63, 143B, U2OS, and MNNG/HOS). Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001 vs. PT or hFOB1.19.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: SOX2 mediated transcriptional regulation of LPCAT1. (A) JASPAR prediction of SOX2 binding sites in the LPCAT1 promoter region. (B,C) Dual-luciferase reporter assay validating the interaction between SOX2 and LPCAT1 promoter. (D) ChIP assay demonstrating the binding of SOX2 to LPCAT1 promoter region. (E,F) qRT-PCR confirmation of SOX2 overexpression and knockdown efficiency. (G–I) Western blot and qRT-PCR analysis of LPCAT1 expression following SOX2 modulation. Data are representative of three independent experiments (mean ± SD). ** P < 0.01, *** P < 0.001 vs. NC, anti-IgG or shNC.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Over Expression, Knockdown, Western Blot, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: In vitro validation of SOX2/LPCAT1 axis in regulating osteosarcoma cell biological behaviors and cholesterol metabolism. MG63 cells were co-transfected with oe-LPCAT1 and/or shSOX2, while U2OS cells were co-transfected with shLPCAT1 and/or SOX2. (A,B) Cell viability was determined by CCK-8 assay. (C,D) Migration ability was assessed by wound healing assay. (E,F) Invasion capacity was evaluated by Transwell assay. (G,H) Intracellular cholesterol level measurement. (I,J) Western blot and qRT-PCR analysis of metastasis- and cholesterol metabolism-related proteins. Data are representative of three independent experiments (mean ± SD). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. NC+shNC, shLPCAT1+NC or LPCAT1+shNC, SOX2+shNC or shSOX2+NC.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: In Vitro, Biomarker Discovery, Transfection, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Assay, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: In vivo validation of SOX2/LPCAT1 axis in osteosarcoma progression and cholesterol metabolism. For (A–D) , MG63 cells were transfected with oe-LPCAT1 and/or shSOX2, and then subcutaneously injected into nude mice to construct a xenograft model. (A) Representative images of xenograft tumors. (B) Tumor volume. (C) Cholesterol content measurement in tumor tissues. (D,E) Western blot analysis of metastasis- and cholesterol metabolism-related proteins in xenograft tumors. (F) MG63 cells transfected with oe-LPCAT1 and/or shSOX2 were injected into the tail vein of nude mice to construct a lung metastasis model. The number of pulmonary nodules was counted. Data are representative of three independent experiments (mean ± SD). ** P < 0.01, *** P < 0.001 vs. NC+shNC, LPCAT1+shNC, and shSOX2+NC.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: In Vivo, Biomarker Discovery, Transfection, Injection, Construct, Western Blot

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: PAX6-induced cancer stemness properties via SOX2. ( A ) Re-expression of the PAX6 gene after treatment of LUAD cells with 5-Aza-dC ± Trichostatin A (TSA), as determined by Q-RT-PCR. The relative expression levels were calculated as a ratio of the values of 5-Aza-dC ± TSA treatment relative to the value of mock treated cells considered as 1.0. Plus (+) and minus (-) marks represent cell lines with and without methylation of the PAX6 gene, respectively. ( B ) The PM values of the PAX6 gene in isogeneic parental and spheroid LUAD cells, as measured by Q-MSP. ( C ) Western blotting analysis of PAX6 in isogeneic parental and spheroid cells. ( D ) Sphere formation and self-renewal assays through the second (P2) passage from the first passage (P1) in stable PAX6 knockdown (PAX6-sh) NCI-H1650 and overexpressed (PAX6-LV) NCI-H1975 cells compared with control (PAX6-Ctrl) cells. Left, representative images of sphere formation (scale bars, 200 μm); Right, number of the spheres over 100 μm. ( E ) Western blotting analysis of PAX6 and SOX2 expression in stable PAX6-sh NCI-H1650 and PAX6-LV NCI-H1975 cells. ( F ) The in vivo tumorigenesis after xenotransplantation of stable PAX6-sh NCI-H1650 (left) and PAX6-LV NCI-H1975 (right) cells (four mice per group). ( G ) Limiting dilution xenograft assays in stable PAX6-sh NCI-H1650 spheroid (upper) and PAX6-LV NCI-H1975 (lower) cells. Tumor-initiating capacity is shown as the numbers of tumors / the number of injections. ( H ) Sphere formation assay (upper left) and western blotting analysis (lower left) in PAX6-sh or PAX6-Ctrl NCI-H1650 cells transduced with SOX2-LV or SOX2-Ctrl (PAX6-Ctrl/SOX2-Ctrl, PAX6-sh/SOX2-Ctrl, and PAX6-sh/SOX2-LV). Right, representative images of sphere formation (scale bars, 200 μm). The spheres over 100 μm were counted. ( I ) Sphere formation assay (upper left) and western blotting analysis (lower left) of PAX6-LV or PAX6-Ctrl NCI-H1975 cells transduced with SOX2-sh or SOX2-Ctrl (PAX6-Ctrl/SOX2-Ctrl, PAX6-LV/SOX2-Ctrl, and PAX6-LV/SOX2-sh). Right, representative images of sphere formation (scale bars, 200 μm). Each error bar indicates mean ± SEM. *, P <0.05; **, P <0.01 (Wilcoxon–Mann–Whitney test [ D and F ] Chi-squared test [ G ], and Kruskal–Wallis with post-hoc test [ H and I ]). See also .

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Methylation, Western Blot, Knockdown, Control, In Vivo, Tube Formation Assay, Transduction, MANN-WHITNEY

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: The PAX6-GLI-SOX2 signaling axis in LUAD CSCs. ( A ) Gene set enrichment analysis (GSEA) related to the oncogenic signatures and hallmarks in the groups with high and low expression of PAX6 in the TCGA LUAD cohort. Left, the enhanced oncogenic pathways in group with high PAX6 expression, as determined by a normalized enrichment score (NES); Right, the enrichment of Hedgehog pathway in group with high PAX6 expression. The GSEAs from both oncogenic signatures gene set (left and middle panels) and hallmarks gene set (right panel) showed the enriched Hedgehog pathway. SHH; Sonic hedgehog. ( B ) Western blotting analysis of GLI in stable PAX6-sh NCI-H1650 or PAX6-LV NCI-H1975 cells. ( C ) ChIP assays conducted on the proximal promoter region of the SOX2 gene using the indicated antibodies in NCI-H1650 or PAX6-LV NCI-H1975 cells. Histone H3 and normal IgG were used as the positive and negative control, respectively. ( D ) Sphere formation assay (upper left) and western blotting analysis of indicated molecules (lower left) of PAX6-LV NCI-H1975 cells transfected with GLI siRNA ± SOX2-LV. Right, representative images of sphere formation (scale bars, 200 μm). The spheres over 100 μm were counted. ( E ) Sphere formation assay (upper left) and western blotting analysis (lower left) of PAX6-sh NCI-H1650 cells stimulated with recombinant human Sonic Hedgehog ligand (Shh; 1μg /ml) after transfection with SOX2-sh or SOX2-Ctrl. Right, representative images of sphere formation (scale bars, 200 μm). ( F ) The in vivo tumorigenesis of stable PAX6-LV or PAX6-Ctrl NCI-H1975 cells in the presence or absence of the GLI antagonist GANT61 (50 mg/kg) treatment for 21 days (five mice per group). Each error bar indicates mean ± SEM. **, P <0.01 (Kruskal–Wallis with post-hoc test [ D, E, and F ]). See also

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Expressing, Western Blot, Negative Control, Tube Formation Assay, Transfection, Recombinant, In Vivo

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: PAX6-regulated differentiation lineage factors (HOPX and NKX2-1) and stemness-related factors (SOX2 and GLI) in LUAD. ( A ) The enrichment scores of molecular subtype-related genes from GSEA in the groups with high and low expression of PAX6 in the TCGA LUAD cohort. The enrichment score of each gene was summarized in . The alveolar-related genes, especially HOPX and NKX2-1, were enriched in group with low PAX6 expression compared with high group. DASC, distal airway stem cell. ( B ) The relative expression levels of HOPX and NKX2-1 in PAX6-sh (NCI-H1650 and NCI-H1299) and PAX6-LV (NCI-H1975 and NCI-H23) cells compared with PAX6-Ctrl cells, as measured by Q-RT-PCR. ( C ) The promoter methylation levels of the PAX6 gene and expression levels of HOPX and NKX2-1 in re-differentiated cells compared with spheroid cells. When spheroid cells were cultured under standard conditions containing the fetal bovine serum (FBS), the floating spheroid cells could adhere and acquire epithelial morphology similar to parental cells. The cells showed higher expression of HOPX and NKX2-1 along with re-methylation of the PAX6 gene compared with corresponding spheroid cells, as measured by Q-RT-PCR and Q-MSP. ( D ) A linear correlation analysis of PAX6 with SOX2, GLI1, GLI2, HOPX, and NKX2-1 in 75 human lung cancer tissues. The relative mRNA expression levels were calculated as the Q-RT-PCR values of each molecules vs. β-actin (The values of PAX6, GLI1, and HOPX were multiplied by 1000 for easy tabulation). The extent of the correlation is indicated by the R coefficient. ( E ) The association between PAX6 expression and the status of histological differentiation (well/moderate vs. poor differentiation). Scatter plots show the distribution of relative mRNA expression values of PAX6 according to the differentiation status. ( F ) The association of PAX6 promoter methylation with the expression levels of HOPX and NKX2-1. Each error bar indicates mean ± SEM. *, P <0.05; **, P <0.01 (Wilcoxon–Mann–Whitney test [ A, B, E, and F ]). See also .

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Methylation, Cell Culture, MANN-WHITNEY

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: CSC expansion following chemotherapy abrogated by targeting the PAX6-GLI-SOX2 axis in xenograft models. ( A ) Sphere formation assay of cancer cells isolated from xenografted tumor tissues after treatment with pemetrexed (75 mg/kg, once daily, 5 days on and 2 days off) plus cisplatin (4 mg/kg, once every 7 days; Pem-Cis) chemotherapy for 14 days in PAX6-sh or PAX6-Ctrl xenograft models (NCI-H1650 and NCI-H1299 cells). Upper, representative images of sphere formation (scale bars, 200 μm); Lower, number of spheres over 100 μm. ( B ) The expression levels of GLI, PAX6, and SOX2 of xenografted tumor tissues after treatment with Pem-Cis chemotherapy for 14 days in PAX6-sh or PAX6-Ctrl NCI-H1650 xenograft models, as measured by western blotting analysis for GLI and PAX6 (left) and flow cytometry for SOX2 (right). ( C ) Sphere formation assay of cancer cells isolated from xenografted tumor tissues after treatment with Pem-Cis chemotherapy in the presence or absence of the GLI antagonist GANT61 (50 mg/kg) treatment for 14 days in PAX6-LV or PAX6-Ctrl NCI-H1975 xenograft models. ( D ) The promoter methylation levels of the PAX6 gene of xenografted tumor tissues after treatment with Pem-Cis chemotherapy for 14 days in xenograft models, as measured by Q-MSP. ( E ) The in vivo therapeutic efficacy of the combination of Pem-Cis chemotherapy for 14 days with GANT61 (50 mg/kg) for 21 days in NCI-H1650 and NCI-H1975 xenograft models (five mice per group). Left, tumor growth curve; Right, tumor weight from both flanks of five mice per group. Growth curves were calculated by comparing the tumor size before any treatment with the size at different time points of therapy. ( F ) The in vivo therapeutic efficacy of the combination of Pem-Cis chemotherapy with GANT61 (50 mg/kg) in patient derived xenograft (PDX) models (five mice per group). Each error bar indicates mean ± SEM. *, P <0.05; **, P <0.01 (Wilcoxon–Mann–Whitney test [ A and C ] and Kruskal–Wallis with post-hoc test [ E and F ]). See also .

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Tube Formation Assay, Isolation, Expressing, Western Blot, Flow Cytometry, Methylation, In Vivo, Drug discovery, Derivative Assay, MANN-WHITNEY

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Increased SOX2 gene amplification and protein overexpression in LUSC (A) Analysis conducted on the C-Bio portal Pan-Cancer Analysis revealed SOX2 gene amplification in 39.4% of LUSC. (B) The GEPIA interactive software was used to determine the expression of stem cell markers SOX2, EPCAM, CD133, CD44, and ITGB4 in the LUSC TCGA dataset. The significant changes in expression between normal and tumor tissue were determined (∗p < 0.01). (C and D) The gene expression analysis of SOX2 and ITGB4 was performed on different sub-histologies of LUSC within the TCGA dataset, utilizing the GEPIA interactive software. The analysis demonstrated a statistically significant association (∗p < 0.01). (E) The overall survival of LUSC subtypes expressing median high or low levels of ITGB4 normalized to SOX2 expression was investigated. Except for the basal subtype, the overall survival was poor for all other subtypes. (F) Immunofluorescence analysis was performed on a LUSC tumor microarray, demonstrating variations in the expression and spatial distribution of ITGB4 and SOX2. The SOX2 protein was represented by red fluorescence, ITGB4 by yellow fluorescence, and DAPI staining was used for blue visualization.

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Amplification, Over Expression, Software, Expressing, Gene Expression, Immunofluorescence, Microarray, Fluorescence, Staining

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Patient-derived cells exhibited the stem cell-like phenotype (A) Schematic representation illustrating the process of tumor tissue processing, isolation, and characterization of patient-derived primary cell lines. (B) Flow cytometry analysis demonstrating the expression of cancer stem cell surface markers EPCAM, CD133, and CD44. (C) Quantitative PCR analysis revealing the expression levels of stem cell markers, including SOX2, CD44, ALDH1A1, EPCAM, ITGB4, and PXN, in two primary patient-derived cell lines. Statistical significance was determined using ordinary one-way ANOVA (∗p < 0.05, ∗∗p < 0.001, ∗∗∗p = 0.0001, ∗∗∗∗p < 0.0001). (D) Immunoblotting analysis showcasing variations in the expression of stem cell markers among the BEAS2B, COH1, and COH2 cell lines. (E) Spheroid formation assay depicting the ability of COH2 cells to form spheroids in specialized media, while BEAS2B cells failed to form spheroids, indicating a stemness phenotype in COH2 cells. (F and G) Cell viability assay was conducted on COH2 primary cells in both attached and spheroid conditions after a 3-day treatment with cisplatin. The results are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (∗∗p < 0.01). (H) Western blot analysis was performed on COH2 cells following treatment with increasing concentrations of cisplatin for 3 days, revealing no significant changes in the expression of SOX2 or ITGB4.

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Derivative Assay, Isolation, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Tube Formation Assay, Viability Assay

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Subclones retain cisplatin-resistant phenotype, and ITGB4 knockdown sensitizes the primary cells to cisplatin (A) Schematic representation illustrating the isolation of single clones from the primary patient-derived cell line. (B and C) Representative images demonstrating the differential growth patterns of subclones derived from single cells. (D and E) Immunoblotting analysis depicting the differences in the expression of SOX2 and ITGB4 in the subclones compared to the mixed parental cells. (F and G) Cell viability assay (CCK8 assay) was conducted on subclones in both attached and spheroid conditions after a 3-day treatment with cisplatin. The subclones exhibited a resistant phenotype similar to parental cells. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns, not significant). (H) Western blot analysis was performed after 3 days of cisplatin treatment on subclone 1, revealing no significant changes in the expression of SOX2 or ITGB4. (I) Immunoblotting analysis conducted after 72 h of transfection with siRNA targeting SOX2, siRNA targeting ITGB4, or both, in COH2 cells. ITGB4 knockdown resulted in suppressed SOX2 expression. The numbers below each immunoblot represent the percentage difference in intensity compared to the control. The band intensity was normalized to actin and then compared to the control. (J) The knockdown of ITGB4 or SOX2 in COH2 cells sensitized the cells to cisplatin. Statistical significance was determined using ordinary one-way ANOVA. (K) Stable cell lines expressing ITGB4 shRNA were generated using COH2 cells. Immunoblotting confirmed ITGB4 knockdown and a reduction in SOX2 expression. The numbers below each immunoblot represent the percentage difference in intensity compared to the control. (L) These stable cell lines expressing ITGB4 shRNA exhibited high sensitivity to cisplatin. The bar graph represents the mean ± SD. Statistical significance was determined using one-way ANOVA (∗∗∗p = 0.0001, ∗∗∗∗p < 0.0001).

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Knockdown, Isolation, Clone Assay, Derivative Assay, Western Blot, Expressing, Viability Assay, CCK-8 Assay, Transfection, Control, Stable Transfection, shRNA, Generated

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Inhibition of SOX2 expression by ITGB4 knockdown (A) Western blot analysis revealing the expression levels of SOX2 and ITGB4 in different cell lines of LUSC and SCLC. H520 and SBC5 cell lines exhibit higher expression of SOX2 and lower expression of ITGB4. (B) Cell viability assays were performed on H520 and SBC5 cells after 3 days of cisplatin treatment. The IC50 value indicates that SBC5 is resistant to cisplatin. (C) Immunoblotting data showed no significant reduction in the expression patterns of SOX2 and ITGB4 after 3 days of cisplatin treatment. (D and E) The knockdown of SOX2 sensitizes H520 and SBC5 cells to lower concentrations (2μM) of cisplatin. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns, not significant, ∗∗p < 0.001, ∗∗∗∗p < 0.0001). (F) Immunoblots confirmed reduced expression of SOX2 in the H520 and SBC5 cells with no significant change in ITGB4 expression. (G and H) H520 and SBC5 cells with ITGB4 knockdown exhibited sensitivity to cisplatin treatment at a lower concentration of 2μM. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns, not significant, ∗∗p < 0.001, ∗∗∗∗p < 0.0001). (I) Immunoblots confirmed ITGB4 knockdown in both H520 and SBC5 cell lines in addition to reduced expression of SOX2 and YAP1. The percentage reduction compared to the control is quantified and mentioned below each immunoblot. (J) Chromatin immunoprecipitation was performed using H3K27Ac and H3Kme4 antibodies on H520 control and ITGB4 knockdown cell lines. A significant reduction in histone acetylation was observed at the SOX2 promoter site. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns-not significant, ∗∗∗p = 0.0001).

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Inhibition, Expressing, Knockdown, Western Blot, Concentration Assay, Control, Chromatin Immunoprecipitation

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Sensitivity of primary cell lines to proteasome inhibitors (A) CCK8 assay performed on COH2 cells after 3 days of treatment with increasing concentrations of carfilzomib (CFZ) or ixazomib (IXA) in 2D culture. (B) CCK8 assay conducted on COH2 cells treated with increasing concentrations of CFZ or IXA in 3D culture. Data are presented as mean ± SD (n = 3), and IC50 values were calculated using GraphPad Prism 9.0. (C) Immunoblotting analysis of SOX2, ITGB4, ALDH1A1, EPCAM, and cytokeratin in COH2 cells after 3 days of CFZ and IXA treatment at their respective IC50 doses. The normalized changes in protein expression are indicated below each immunoblot. Treatment with the drugs resulted in reduced expression of SOX2 and ITGB4. (D) CCK8 assay showing the significant inhibition of Clone 1 cell viability after 3 days of CFZ treatment. Mean ± SD shown, with statistical significance determined by one-way ANOVA (ns, not significant, ∗∗∗∗p < 0.0001). (E) Immunoblot and densitometry analysis revealed reduced SOX2 expression following CFZ treatment at IC50 dose for 3 days. (F) Evaluation of the inhibitory effect of the cisplatin and CFZ combination using synergy experiments. The average synergy score was calculated using Bliss analysis. A synergy score ≥10 indicates synergism. (G) COH2 cells treated with the cisplatin and CFZ combination exhibited inhibition of CD44, EPCAM, SOX2, and ITGB4 expression, as shown by immunoblotting. (H and I) CCK8 assay was performed on H520 and SBC5 cells after 3 days of CFZ or IXA treatment in 2D culture. Data are presented as mean ± SD (n = 3), and IC50 values were calculated for both inhibitors. CFZ demonstrated efficacy at lower doses. Analysis was performed using GraphPad Prism 9.0.

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: CCK-8 Assay, Western Blot, Expressing, Inhibition

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: CFZ reduced SOX2 expression by inhibiting promoter activity (A) Immunofluorescent images of COH2 cells treated with 40 nM and 80 nM CFZ for 3 days, showing differential expression of SOX2 (Green) among cells. High SOX2-expressing cells are indicated by white arrows, and low SOX2-expressing cells are indicated by red arrows. Blue represents DAPI staining. Scale bar: 100 μm. Quantification of high SOX2 expression cell numbers using QuPath. Data presented as mean ± SD (n = 10), ∗∗p < 0.01 one-way ANOVA. (B–D) Time course measurement of SOX2 mRNA expression in COH2, H520, and SBC5 cells after 6 h of CFZ treatment up to 24 h using qPCR. (E) qPCR assay measuring changes in SOX2 mRNA expression in COH2, H520, and SBC5 cells after 6 h of CFZ or Act D treatment. Both drugs significantly reduced SOX2 expression. (F and G) qPCR measurement of ALDH1A1, CD44, and EPCAM mRNA expression in COH2 and H520 cells after 6 h of CFZ or Act D treatment. No significant reduction was observed in these markers. (H–J) COH2, H520, and SBC5 were transfected with p-GL3-SOX2 promoter expressing plasmid. A significant reduction in promoter activity was observed after treatment with ActD and CFZ (IC50 dose, 12 h, and 24 h), but not with cisplatin. Data represented as mean ± SD (n = 3). (K) CFZ or IXA treatment significantly inhibits the binding of acetylated H3K27 at the SOX2 promoter site in COH2 cells, correlating with the reduction in its transcription. (L) SOX2 chromatin immunoprecipitation (ChIP) reveals the binding of SOX2 at the promoter region of PXN and ITGB4. Statistical significance was determined using one-way or two-way ANOVA, with p values as follows: ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and NS (not significant).

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Expressing, Activity Assay, Quantitative Proteomics, Staining, Transfection, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Differential regulation of gene expression by CFZ or SOX2 (A) Heatmap illustrating the differential gene expression changes in response to Act D or CFZ treatment. (B) Volcano plot depicting the differentially expressed genes with a 2-fold change in expression and <0.05 false discovery rate (FDR). Significant changes were observed primarily in downregulated genes compared to upregulated genes. (C) Venn diagram representing the overlap and uniqueness between the upregulated or downregulated genes within the cell line in response to Act D or CFZ treatment. 461 downregulated genes overlap between the treatment for the primary cells. (D) Venn diagram representing the overlap and uniqueness between the down-regulated genes across all the cells in response to Act D or CFZ treatment. About 911 genes were found to overlap across all the cell lines in response to Act D treatment, and 171 genes overlapped for the CFZ treatment. (E) The heatmap displays the significant downregulation of histone expression following CFZ treatment.

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Gene Expression, Expressing

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Identifying differential expression of SOX2 regulated genes and validating cisplatin/CFZ synergy in vivo (A–C) The downregulated gene list resulting from CFZ treatment was analyzed to identify enriched molecular functions (MF) in COH2, H520, and SBC5 cells. COH2 cells exhibited enriched MF associated with transcriptional regulation (red), DNA binding (green), and enzymatic activity (blue). (D–F) The downregulated genes from differential gene expression analysis, along with chromatin immunoprecipitation data, were utilized to predict the functional status of transcription factors (TFs). The stemness-associated factors KLF4 and SOX2 were enriched in all three cell lines and predicted to be inactive after CFZ treatment. Additionally, COH2 cells displayed enrichment of multiple TFs associated with stemness, which were also predicted to be inactive. (G) Screening 775 known targets across all cell lines revealed 16 overlapping genes in the three cell lines (Venn diagram). Heatmap analysis indicated significant downregulation of these genes upon CFZ treatment. (H and I) Athymic nude (nu/nu) mice were implanted with H520 NSCLC cells and subsequently treated with cisplatin (5 mg/kg.b.w), CFZ (4 mg/kg.b.w), or their combination. Changes in tumor area (mm 2 ) and weight (g) were measured. The combination treatment significantly affected tumor size and weight. Statistical significance was determined using one-way ANOVA (∗∗∗∗p < 0.0001). (J and K) The data represent changes in tumor area and weight of SBC5-generated xenografts after treatment with CFZ and cisplatin. Statistical significance was determined using one-way ANOVA (∗∗∗∗p < 0.0001).

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Quantitative Proteomics, In Vivo, Binding Assay, Activity Assay, Gene Expression, Chromatin Immunoprecipitation, Functional Assay, Generated

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet:

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Control, Virus, Recombinant, Live Cell Imaging, Chromatin Immunoprecipitation, Luciferase, CCK-8 Assay, Small Interfering RNA, shRNA, Software

Journal: Scientific reports

Article Title: Efficient extravasation of tumor-repopulating cells depends on cell deformability.

doi: 10.1038/srep19304

Figure Lengend Snippet: Figure 4. Silencing Sox2 inhibits metastasis of TRCs. Sox2 shRNA (a) and scrambled shRNA (b) treated TRCs were injected into the yolk of 2 dpf Tg(kdrl:mCherry) embryos respectively; cell proliferation, survival, and metastasis were quantified every 24 hr from 0 dpi to 6 dpi respectively. Representative images show metastatic tumor foci at 6 dpi zebrafish by comparing the scrambled shRNA group with the Sox2 shRNA group. Arrowheads indicate disseminated tumor foci or tumor foci aggregates. Color code: Sox2 shRNA or scrambled shRNA treated TRCs are green; zebrafish blood vessels are red. Scale bars, 500 μ m.

Article Snippet: Sox2 shRNAs were obtained from Origene (TG515613).

Techniques: shRNA, Injection

Journal: Scientific reports

Article Title: Efficient extravasation of tumor-repopulating cells depends on cell deformability.

doi: 10.1038/srep19304

Figure Lengend Snippet: Figure 5. Silencing Sox2 downregulates proliferation and metastases of TRCs in zebrafish. (a) Quantification of total tumor sizes from 0 dpi to 6 dpi relative to the injected tumor size at the yolk (n >10 fish per group; ≥3 independent experiments). (b–d) Quantification of tumor cell metastasis from 0 dpi to 6 dpi at Head (b), Trunk (c), and Tail (d) (n >10 fish per group, ≥3 independent experiments). Mean + s.e.m.; *p < 0.05, **p < 0.01.

Article Snippet: Sox2 shRNAs were obtained from Origene (TG515613).

Techniques: Injection

Journal: The Journal of urology

Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

doi: 10.1016/j.juro.2009.12.092

Figure Lengend Snippet: Representative pluripotent stem cell gene expression results in 3 independent experiments in prostate cancer tissue. A to E, semiquantitative RT-PCR using TissueScan prostate tissue panels shows normal prostate (N), PSs and ESs as controls. Band intensity was calculated using AlphaEase. Transcript levels were normalized to β-actin and are shown as relative units standardized to normal tissue pool. Statistical significance was considered at p <0.05. Asterisk indicates statistically different from normal. Double dagger indicates statistically different from ES. F, relative SOX2 mRNA levels vs relative OCT3/4 mRNA levels in tissue samples (Spearman correlation coefficient 0.4730, p <0.0001). Other possible transcription level combinations did not attain significance.

Article Snippet: Mice were sacrificed when moribund or when subcutaneous tumors were 15 mm in diameter. shRNA OCT3/4 and SOX2 Knockdown Plasmid vectors (OriGene) encoding OCT3/4 or SOX2 were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of urology

Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

doi: 10.1016/j.juro.2009.12.092

Figure Lengend Snippet: Immunohistochemical staining for OCT3/4 and SOX2 in human prostate cancer tissues. A, representative results of at least 2 independent experiments reveal immunostaining for OCT3/4 and SOX2 using prostate tissue arrays. Neg., negative. Int., intermediate. Brown areas indicate positive nuclear staining. Reduced from ×20. Scale bar represents 70 µm. Insets, reduced from ×40. B and C, classification of different GS samples based on staining intensity. GS 5–6 and 7–8 tissues were significantly different than normal (N) and BPH tissue (2-tailed Mann-Whitney rank test p <0.05). Red lines indicate mean.

Article Snippet: Mice were sacrificed when moribund or when subcutaneous tumors were 15 mm in diameter. shRNA OCT3/4 and SOX2 Knockdown Plasmid vectors (OriGene) encoding OCT3/4 or SOX2 were used.

Techniques: Immunohistochemical staining, Staining, Immunostaining, MANN-WHITNEY

Journal: The Journal of urology

Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

doi: 10.1016/j.juro.2009.12.092

Figure Lengend Snippet: Representative results show identification of stem cell-like tumor cells with pluripotent stem cell reprogramming factors in 3 independent experiments in prostate cancer cell lines. A and B, RT-PCR or Western blot detected OCT3/4, SOX2, Nanog, c-Myc and Klf4 expression in DU145 and PC3 cell lines with human embryonic stem cells (hESC) as control. Data were normalized to β-actin. C, DU145 and PC3 cells were immunostained for OCT3/4 (red areas), SOX2 (green areas) and DAPI (blue areas). Arrow indicates merged OCT3/4 and SOX2 staining (Merge). Scale bar represents 20 µm. Reduced from ×40.

Article Snippet: Mice were sacrificed when moribund or when subcutaneous tumors were 15 mm in diameter. shRNA OCT3/4 and SOX2 Knockdown Plasmid vectors (OriGene) encoding OCT3/4 or SOX2 were used.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Staining

Journal: The Journal of urology

Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

doi: 10.1016/j.juro.2009.12.092

Figure Lengend Snippet: Isolation of stem cell-like prostate tumor cells. A, to identify surface markers for isolating stem cell-like cells from prostate cell lines DU145 and PC3 cells were immunostained for OCT3/4 (red areas), E-cadherin (Ecad) (green areas) and DAPI (blue areas). OCT3/4 and E-cadherin staining was also merged (arrow). Reduced from ×40. Scale bar represents 20 µm. B and C, for phenotypic analysis of DU145 and PC3 cells using double staining with E-cadherin and CD44 or integrin-α2β1 cells were gated on E-cadherin+ (green curves) or E-cadherin− (blue curves) population. FITC, fluorescein isothiocyanate. D and E, flow cytometry (FCS) analysis of DU145 and PC3 cells reveals E-cadherin (E-cad) expression. Isotype matched controls were used to set analysis gates for E-cadherin cell sorting. F, representative results of 3 independent experiments show that RT-PCR identified OCT3/4, SOX2, Nanog, c-Myc and Klf4 expression in E-cadherin+ and E-cadherin− cells isolated from DU145 and PC3 cells. Data were normalized to β-actin.

Article Snippet: Mice were sacrificed when moribund or when subcutaneous tumors were 15 mm in diameter. shRNA OCT3/4 and SOX2 Knockdown Plasmid vectors (OriGene) encoding OCT3/4 or SOX2 were used.

Techniques: Isolation, Staining, Double Staining, Flow Cytometry, Expressing, FACS, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of urology

Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

doi: 10.1016/j.juro.2009.12.092

Figure Lengend Snippet: Functional analysis of stem cell-like prostate tumor cells. A, colony forming assay using sorted E-cadherin+ and E-cadherin− cells shows mean ± SD of 2 independent experiments. Asterisks indicate p <0.01. B, spheroid culture assay in E-cadherin (Ecad) sorted cells. Western blot of unsorted parental line (P) and E-cadherin+ spheroids (S) demonstrates OCT3/4, SOX2 and E-cadherin protein levels. Data were normalized to β-actin. Reduced from ×5. C, tumorigenic assay of sorted E-cadherin+ and E-cadherin− cells reveals representative mean ± SD tumor volume in 5 SCID mice per group in 2 independent experiments. D, immunostaining of E-cadherin− and E-cadherin+ DU145 cells with E-cadherin or β-catenin antibodies during 3-day differentiation. Phase contrast microscopy reduced from ×5 (scale bar indicates 100 µm). Fluorescence microscopy reduced from ×40 (scale bar indicates 20 µm).

Article Snippet: Mice were sacrificed when moribund or when subcutaneous tumors were 15 mm in diameter. shRNA OCT3/4 and SOX2 Knockdown Plasmid vectors (OriGene) encoding OCT3/4 or SOX2 were used.

Techniques: Functional Assay, Western Blot, Immunostaining, Microscopy, Fluorescence

Journal: The Journal of urology

Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

doi: 10.1016/j.juro.2009.12.092

Figure Lengend Snippet: OCT3/4 or SOX2 knockdown tumorigenicity in DU145 and PC3 cells. A and B, Western blot shows decreased OCT3/4 or SOX2 protein levels in human DU145 and PC3 prostate cancer cells transfected with shRNA. Unsorted DU145 (1 × 105) or PC3 (3 × 105) cells were subcutaneously injected in SCID mice. C and D, mean ± SD tumor volume after OCT 3/4, SOX2 or control shRNA treatment.

Article Snippet: Mice were sacrificed when moribund or when subcutaneous tumors were 15 mm in diameter. shRNA OCT3/4 and SOX2 Knockdown Plasmid vectors (OriGene) encoding OCT3/4 or SOX2 were used.

Techniques: Western Blot, Transfection, shRNA, Injection