monoclonal mouse antibodies against sox9  (Bio-Rad)

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 2. Immunohistochemical localization of SoxE family members in corneoscleral tissue sections. (A) Immunofluorescence microscopy demonstrates nuclear staining for Sox8 (clone 4E4.1) and Sox9 (clone 3C10) in suprabasal epithelial cells at the limbus (left column) and central cornea (right column), whereas Sox10 (clone BC34) is confined to few cells in the basal limbal epithelium. Higher magnification images of basal limbal regions (middle column), as indicated by boxed areas, show differential cytoplasmic (arrows) and nuclear localization of Sox8 and Sox9 in basal and suprabasal limbal epithelial cells. (B) High magnification images of individual channels show cytoplasmic localization of Sox9 in basal stem/progenitor cell clusters and nuclear localization in suprabasal limbal epithelial cells. (C) Double labeling experiments show nuclear co- localization of Sox8 (rabbit IgG) and Sox9 (clone 3C10) (left), distinct localization of Sox9 (clone 3C10) and Sox10 (rabbit IgG) (middle), and localization of Sox10 (clone BC34) to Melan A-positive cells (right). Nuclear counterstaining: DAPI. Individual channels of double labeling experiments are shown in Supplementary Fig. 1.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Immunohistochemical staining, Immunofluorescence, Microscopy, Staining, Labeling

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 3. Co-localisation of Sox9 with markers related to progenitor cell phenotype, differentiation and proliferation in the limbal epithelium. Double-labelling demonstrates co-localisation (arrows) of cytoplasmic Sox9 (red) with the stem/progenitor cell markers (green) N-cadherin, p75 nerve growth factor receptor (NGF-R), p63α, Oct4 and cytokeratin (CK) 15) in basal epithelial cells at the limbus. Suprabasal epithelial cells revealed co-localisation of nuclear Sox9 (red) with differentiation-related markers (green) CK3 and Pax6 as well as proliferation-related marker Ki-67. Sox9 monoclonal mouse antibody (clone 3C10) was used for double labelling experiments with polyclonal antibodies against Oct4, p63α, Pax6 and Ki-67, Sox9 polyclonal rabbit antibody (1) was used for double labelling experiments with monoclonal antibodies against p75 NGF- R, N-cadherin, CK3 and CK15. Nuclear counterstaining: DAPI. Individual channels of all double labelling experiments are shown in Supplementary Fig. 2.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Marker, Bioprocessing

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 4. Expression of Sox9 during limbal epithelial cell expansion and wound healing in vitro. (A) Relative expression levels of Sox9 in cultured limbal epithelial cells expanded as clones on a 3T3 feeder layer or as feeder- free monolayer in passage (P) 0 to P2. Expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) primer assays and normalized against GAPDH. Data are expressed as means (2−∆CT × 1,000) ± SD (n = 3) relative to clonal cultures; *p < 0.01, unpaired t-test. (B) Limbal epithelial cell (LEPC) clones (dashed lines) on 3T3 feeder cells (3T3) stain positively for Sox9 (clone 3C10, red), preferentially towards the proliferating border of the clones; Sox9-expressing cells partly co-localize with Ki-67 (green). Nuclear staining: DAPI. (C) Immunofluorescent staining shows increased levels of nuclear Sox9 (clone 3C10) in basal/suprabasal limbal epithelial cells and central corneal epithelial cells after epithelial debridement and regeneration compared to unwounded control corneas (the background fluorescence seen in the central corneal stroma may be attributed to the epithelial debridement allowing media and serum components to infiltrate the stroma during the wound healing process). Higher magnification images of limbal epithelial progenitor cell clusters show increased nuclear localization of Sox9 in basal epithelial cells in wound healing conditions compared to cytoplasmic retention of Sox9 in control tissues (inserts). Nuclear staining: DAPI. (D) Relative expression of SOX9 in limbal epithelial cells of wounded and unwounded corneas as determined by quantitative real-time polymerase chain reaction (qRT-PCR) primer assays. Normalized data are expressed as means (2−∆CT × 1,000) ± SD (n = 5) relative to unwounded controls; p = 0.08, unpaired t-test.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, In Vitro, Cell Culture, Clone Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Control, Fluorescence

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 5. Changes in mRNA expression following knockdown of SOX9 in cultured limbal epithelial cells by RNAi. (A) Results of quantitative real-time polymerase chain reaction (qRT-PCR) showing reduction of SOX9 mRNA transcripts in cultured limbal epithelial cells 24–96 hours after transfection with siRNA to SOX9 relative to control cells transfected with scramble siRNA (Control) (n = 6; Mean ± SD). (B) Transcriptional changes following knockdown of SOX9 in cultured limbal epithelial cells as determined by qRT-PCR. Significant or no relevant changes were seen in the expression levels of stemness-related genes ABCG2 (ATP Binding Cassette Subfamily G Member 2), TP63 (ΔNp63α) and CEBPD (CCAAT/enhancer-binding protein delta); progenitor cell marker genes KRT15 (keratin 15), KRT14 and CDH2 (N-cadherin); differentiation marker genes KRT3, KRT12 and IVL (involucrin); and proliferation-related genes PCNA (proliferating cell nuclear antigen),

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, Knockdown, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Control, Binding Assay, Marker

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 6. Changes in protein expression and proliferation following knockdown of SOX9 in cultured limbal epithelial cells by RNAi. (A) Western blot analysis of limbal epithelial cells transfected with either siRNA that targets SOX9 or non-targeting, scrambled siRNA as a control. Protein expression of Sox9, cytokeratin (CK) 15, CK3 and PCNA was detected with monoclonal antibodies, normalized to the house-keeping gene ß-actin, and expressed as percent of the expression in control cells (Ctrl); (n = 3; mean ± SD); *p < 0.05, **p < 0.005, ***p < 0.0001, unpaired t-test. Uncropped versions of Western blots are shown in Supplementary Fig. 4. (B) BrdU incorporation (i.e., cell proliferation) was determined by measuring absorbance at 450 nm. Statistically significant differences were observed at 72 (*p = 0.005) and 96 hours (**p = 0.009) between cells transfected with siRNA that targets SOX9 (Si-Sox9) and control cells transfected with scramble siRNA (Scr-Crtl) (n = 3; mean ± SD).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, Knockdown, Cell Culture, Western Blot, Transfection, Control, Bioprocessing, BrdU Incorporation Assay

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 7. Interactions between Sox9 and cell signaling pathways. (A) Changes in mRNA expression of genes centrally involved in Wnt/ß-catenin signaling, i.e., WNT4 (Wnt-4), CTNNB1 (ß-catenin) and GSK3B (glycogen synthase kinase 3 beta), following knockdown of SOX9 in cultured limbal epithelial cells by RNAi relative to mock-transfected control cells. Normalized data are expressed as means (2−∆CT × 1,000) ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test. (B) Changes in SOX9 mRNA expression following exposure of cultured limbal epithelial cells with signaling activators lithium chloride (LiCl), IM- 12, BMP-2, JAG-1 and SAG as well as signaling inhibitors C-59, DMH1, DAPT and Cyclopamine (Cyclo) for 24 hours relative to vehicle-treated control cells (Ctrl). Normalized data are expressed as means (2−∆CT × 1,000) ± SD (n = 3); *p < 0.05, **p < 0.01, unpaired t-test. (C) Changes in Sox9 protein expression following exposure of cultured limbal epithelial cells to Hedgehog signaling activators Sonic hedgehog (SHH), SAG and Purmorphamine (Purmo); BMP-2; Wnt signaling activators Wnt-3a, lithium chloride (LiCl) and IM-12; and Notch signaling ligand JAG-1 for 48 hours relative to vehicle-treated control cells (Ctrl). Sox9 protein expression was detected with the monoclonal antibody (clone 3C10), normalized to the house-keeping gene ß-actin, and expressed as percent of the expression in control cells (Ctrl); (n = 3; mean ± SD). Uncropped versions of Western blots are shown in Supplementary Fig. 1.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Protein-Protein interactions, Expressing, Knockdown, Cell Culture, Transfection, Control, Western Blot

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 8. Model illustrating the mutually repressive interaction of Sox9 and Wnt/ß-catenin signaling as well as potentially involved regulatory signaling pathways in the limbal stem cell niche (mod. after Xu, Z. et al., Elife 4, e10567 (2015) (https://creativecommons.org/licenses/by/4.0).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Protein-Protein interactions

Journal: Oncology reports

Article Title: MiR-124 inhibits cell proliferation, migration and invasion by directly targeting SOX9 in lung adenocarcinoma.

doi: 10.3892/or.2016.4648

Figure Lengend Snippet: Figure 1. Expression of miR-124 and SOX9 in lung ADC tissues. (A) miR‑124 expression was significantly lower in the lung ADC tissues than that in the corresponding non-cancerous tissues; *P<0.05. (B) Expression of SOX9 protein in lung ADC tissues and corresponding non-cancerous tissues. SOX9 protein levels were measured by western blot analysis and GAPDH was used as an internal control. (C) Semi‑quantitative analyses of western blot analyses for SOX9 protein; *P<0.05.

Article Snippet: The membranes were blocked with 5% fat-free milk at room temperature for 2 h, followed by incubation with the mouse anti-human primary monoclonal antibody against SOX9 (1:500; ab76997; Abcam, Cambridge, MA, USA) or GAPDH (1:5,000; D190090; Sangon Biotech, Shanghai, China) at 4 ̊C overnight.

Techniques: Expressing, Western Blot, Control

Journal: Oncology reports

Article Title: MiR-124 inhibits cell proliferation, migration and invasion by directly targeting SOX9 in lung adenocarcinoma.

doi: 10.3892/or.2016.4648

Figure Lengend Snippet: Figure 3. SOX9 is a direct target of miR-124. (A) The putative miR‑124‑binding sites in the 3'UTR of SOX9. (B) pEXZ-SOX9 vector was cotransfected into the A549 cells with the miR-124 mimics, mutant miR-124 or miR-124 mimic control. The relative luciferase activity was measured 48 h after transfection and firefly activities were normalized to Renilla luminescence; *P<0.05.

Article Snippet: The membranes were blocked with 5% fat-free milk at room temperature for 2 h, followed by incubation with the mouse anti-human primary monoclonal antibody against SOX9 (1:500; ab76997; Abcam, Cambridge, MA, USA) or GAPDH (1:5,000; D190090; Sangon Biotech, Shanghai, China) at 4 ̊C overnight.

Techniques: Plasmid Preparation, Mutagenesis, Control, Luciferase, Activity Assay, Transfection

Journal: Oncology reports

Article Title: MiR-124 inhibits cell proliferation, migration and invasion by directly targeting SOX9 in lung adenocarcinoma.

doi: 10.3892/or.2016.4648

Figure Lengend Snippet: Figure 4. miR‑124 regulates SOX9 protein in A549 cells. (A) Expression of miR-124 was significantly upregulated in the A549 cells transfected with the miR‑124 mimics. When A549 cells were transfected with the miR-124 inhibitors, the level of miR-124 was markedly decreased; *P<0.05. (B) Expression of SOX9 protein was significantly downregulated in the A549 cells transfected with the miR-124 mimics. Inhibition of miR-124 resulted in a significant increase in SOX9 protein levels in the A549 cells; *P<0.05. (C) Semi‑quantitative analyses of western blot analyses for SOX9 protein; *P<0.05.

Article Snippet: The membranes were blocked with 5% fat-free milk at room temperature for 2 h, followed by incubation with the mouse anti-human primary monoclonal antibody against SOX9 (1:500; ab76997; Abcam, Cambridge, MA, USA) or GAPDH (1:5,000; D190090; Sangon Biotech, Shanghai, China) at 4 ̊C overnight.

Techniques: Expressing, Transfection, Inhibition, Western Blot

Journal: Oncology reports

Article Title: MiR-124 inhibits cell proliferation, migration and invasion by directly targeting SOX9 in lung adenocarcinoma.

doi: 10.3892/or.2016.4648

Figure Lengend Snippet: Figure 5. SOX9 is an important functional mediator of miR-124 in A549 cells. (A) miR-124 mimics were cotransfected into the A549 cells with the SOX9 expression plasmid or control plasmid. Western blot analysis showed that the level of SOX9 protein was recovered after treatment with the SOX9 plasmid. (B) MTT assay showed that recovered expression of SOX9 rescued the proliferation capacity induced by overexpression of miR-124 in the A549 cells; *P<0.05. (C) Transwell migration assay showed that exogenous expression of SOX9 rescued the migration capacity induced by overexpression of miR-124 in the A549 cells; *P<0.05. (D) Transwell invasion assay showed that exogenous expression of SOX9 rescued the invasion capacity induced by overexpression of miR-124 in the A549 cells; *P<0.05.

Article Snippet: The membranes were blocked with 5% fat-free milk at room temperature for 2 h, followed by incubation with the mouse anti-human primary monoclonal antibody against SOX9 (1:500; ab76997; Abcam, Cambridge, MA, USA) or GAPDH (1:5,000; D190090; Sangon Biotech, Shanghai, China) at 4 ̊C overnight.

Techniques: Functional Assay, Expressing, Plasmid Preparation, Control, Western Blot, MTT Assay, Over Expression, Transwell Migration Assay, Migration, Transwell Invasion Assay

Journal: Oncotarget

Article Title: Immunohistochemical staining of ERG and SOX9 as potential biomarkers of docetaxel response in patients with metastatic castration-resistant prostate cancer

doi: 10.18632/oncotarget.13407

Figure Lengend Snippet: Correlation between IHC-measured ERG and SOX9 expression in 71 mCRPC patients (P, positive; N, negative)

Article Snippet: The sections were incubated overnight at 4°C with primary mouse monoclonal antibodies against SOX9 (Abnova, Taipei, Taiwan), followed by a 1-hour incubation at room temperature (RT) with ChemMate TM DAKO EnVision TM /HRP (DAKO, Denmark) and an additional hour at RT with DakoCytomation TechMateTM (DAKO, Denmark).

Techniques: Expressing

Journal: Oncotarget

Article Title: Immunohistochemical staining of ERG and SOX9 as potential biomarkers of docetaxel response in patients with metastatic castration-resistant prostate cancer

doi: 10.18632/oncotarget.13407

Figure Lengend Snippet: Waterfall plot of PSA levels in response to docetaxel treatment according to A. ERG and B. SOX9 expression.

Article Snippet: The sections were incubated overnight at 4°C with primary mouse monoclonal antibodies against SOX9 (Abnova, Taipei, Taiwan), followed by a 1-hour incubation at room temperature (RT) with ChemMate TM DAKO EnVision TM /HRP (DAKO, Denmark) and an additional hour at RT with DakoCytomation TechMateTM (DAKO, Denmark).

Techniques: Expressing

Journal: Oncotarget

Article Title: Immunohistochemical staining of ERG and SOX9 as potential biomarkers of docetaxel response in patients with metastatic castration-resistant prostate cancer

doi: 10.18632/oncotarget.13407

Figure Lengend Snippet: Kaplan-Meier analysis depicting PSA progression-free survival, clinical/radiologic progression-free survival and overall survival according to A. ERG, B. SOX9 and C. ERG and SOX9 expression.

Article Snippet: The sections were incubated overnight at 4°C with primary mouse monoclonal antibodies against SOX9 (Abnova, Taipei, Taiwan), followed by a 1-hour incubation at room temperature (RT) with ChemMate TM DAKO EnVision TM /HRP (DAKO, Denmark) and an additional hour at RT with DakoCytomation TechMateTM (DAKO, Denmark).

Techniques: Expressing

Journal: Oncotarget

Article Title: Immunohistochemical staining of ERG and SOX9 as potential biomarkers of docetaxel response in patients with metastatic castration-resistant prostate cancer

doi: 10.18632/oncotarget.13407

Figure Lengend Snippet: Multivariate Cox proportional hazard regression analyses in the prediction of PSA-progression free survival (PFS), C/R-PFS and overall survival (OS) in mCRPC patients

Article Snippet: The sections were incubated overnight at 4°C with primary mouse monoclonal antibodies against SOX9 (Abnova, Taipei, Taiwan), followed by a 1-hour incubation at room temperature (RT) with ChemMate TM DAKO EnVision TM /HRP (DAKO, Denmark) and an additional hour at RT with DakoCytomation TechMateTM (DAKO, Denmark).

Techniques:

Journal: Oncotarget

Article Title: Immunohistochemical staining of ERG and SOX9 as potential biomarkers of docetaxel response in patients with metastatic castration-resistant prostate cancer

doi: 10.18632/oncotarget.13407

Figure Lengend Snippet: Representative images of ERG and SOX9 detection by IHC in mCRPC patients according to intensity (Magnification x 200)

Article Snippet: The sections were incubated overnight at 4°C with primary mouse monoclonal antibodies against SOX9 (Abnova, Taipei, Taiwan), followed by a 1-hour incubation at room temperature (RT) with ChemMate TM DAKO EnVision TM /HRP (DAKO, Denmark) and an additional hour at RT with DakoCytomation TechMateTM (DAKO, Denmark).

Techniques: