Journal: Oncotarget

Article Title: Loss of zfp36 expression in colorectal cancer correlates to wnt/ β-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of zeb1, sox9 and macc1

doi: 10.18632/oncotarget.10828

Figure Lengend Snippet: (Panel A ) Boxplot of Log 2 expression values of MACC1, SOX9 and ZEB1 in 23 normal colon mucosa (Normal), 30 primary colon carcinoma (CRC) and 27 liver metastases (Mts) samples. The thick line indicates the median value, the coloured box indicates the interquartile range and the whiskers the minimum and maximum values excluded outliers. Open circles represent data points outside the whiskers. (Panel B ) Schematic representation of the 3′UTRs sequences of MACC1, SOX9 and ZEB1. A-U rich sequences (ARE) are highlighted in bold. (Panel C) HCT116 and SW480 cells were transfected with an empty vector (pCDNA3.1) or a ZFP36-overexpressing vector (pCDNA3.1-ZFP36). RNA was extracted after 48 hours and MACC1, SOX9, ZEB1 mRNA levels were analysed through qRT-PCR analysis. Results are represented as means of three experiments (+/−SEM) and GAPDH was used as endogenous control. * p < 0.05. (Panel D ) HCT116 and SW480 were infected with an empty vector (pRRL) or a ZFP36-overexpressing vector (ZFP36) and corresponding total protein lysates were analysed through Western blotting techniques with antibodies against ZEB1, MACC1, SOX9 and ZFP36. Actin was used as loading control. (Panel E ) A fragment of the 3′UTRs of MACC1, SOX9 and ZEB1 was cloned in a pGL3 vector, downstream of the Luciferase gene. These constructs where co-transfected with a Δ-gal reporter plasmid and with an empty vector (pCDNA3.1) or ZFP36 overexpressing vector (pCDNA3.1-ZFP36) in HEK293T cells. Cells were harvested after 48 hours, luciferase activity was measured and normalized over Δ-gal signals. Results are represented as means of three independent experiments +/−SEM. * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The membranes were then blocked with 5% non-fat milk in TBST 0.1% and immunoblotted overnight at 4°C with different primary antibodies listed below together with their working dilution: N-cadherin (910920 BD Transduction Laboratories); Vimentin (Dako MO725); E-Cadherin (610181 BD); ZO-1 (Zymed 61-7300); MACC1 (Bioss bs-4293R); SOX9 (Bioss bs-4177R); ZEB1 (Sigma HPA027524); ZFP36 (S. Cruz Biotechnology sc-14030).

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Infection, Western Blot, Clone Assay, Luciferase, Construct, Activity Assay

Journal: BMC Veterinary Research

Article Title: Neural, adipocyte and hepatic differentiation potential of primary and secondary hair follicle stem cells isolated from Arbas Cashmere goats

doi: 10.1186/s12917-022-03420-3

Figure Lengend Snippet: Expression of pluripotency-related genes in HFSCs. a Immunofluorescence was used to check the expression of SOX9, OCT4, and SOX2 in PHFSCs; nuclei (blue) and target proteins (green) (Scale bar, 100 µm). b Detection of SOX9, OCT4, and SOX2 expression in SHFSCs by the immunofluorescence (Scale bar, 100 µm). c qRT-PCR was used to evaluate the transcript-level expression of SOX9 , OCT4 , and SOX2 in PHFSCs. PDPCs were used as a control. d qRT-PCR was used to evaluate the transcript-level expression of SOX9 , OCT4 , and SOX2 in SHFSCs. SDPCs were used as a control. e Western blotting was used to check the expression of SOX9, OCT4, and SOX2 in PHFSCs and PDPCs. Full-length blots are presented in Supplementary Fig. . f Western blotting was used to check the expression of SOX9, OCT4, and SOX2 was assessed in SHFSCs and SDPCs. Full-length blots are presented in Supplementary Fig. . g Grey-value quantitative analysis of SOX9, OCT4, and SOX2 expression in PHFSCs. PDPCs were used as a control. h Grey-value quantitative analysis of SOX9, OCT4, and SOX2 expression in SHFSCs. SDPCs were used as a control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: All primary antibodies used in this step and their dilution were as follows: Rabbit Anti-CD34 antibody (Bioss, China) [1:500], Rabbit Anti-CK14 antibody (Bioss, China) [1:1,000], Rabbit Anti-LGR5 antibody (Bioss, China) [1:1,000], Rabbit Anti-K15 antibody (Bioss, China) [1:500], Rabbit Anti-K19 antibody (Bioss, China)[1:1,000], Rabbit Anti-SOX9 antibody (Bioss, China) [1:500], OCT4 Polyclonal Antibody (Proteintech, USA) [1:500], SOX2 Polyclonal Antibody (Proteintech, USA), [1:500], GAPDH Polyclonal Antibody (Proteintech, USA) [1:10000].

Techniques: Expressing, Immunofluorescence, Quantitative RT-PCR, Western Blot

Journal: BMC Veterinary Research

Article Title: Neural, adipocyte and hepatic differentiation potential of primary and secondary hair follicle stem cells isolated from Arbas Cashmere goats

doi: 10.1186/s12917-022-03420-3

Figure Lengend Snippet: The primers of HFSCs markers

Article Snippet: All primary antibodies used in this step and their dilution were as follows: Rabbit Anti-CD34 antibody (Bioss, China) [1:500], Rabbit Anti-CK14 antibody (Bioss, China) [1:1,000], Rabbit Anti-LGR5 antibody (Bioss, China) [1:1,000], Rabbit Anti-K15 antibody (Bioss, China) [1:500], Rabbit Anti-K19 antibody (Bioss, China)[1:1,000], Rabbit Anti-SOX9 antibody (Bioss, China) [1:500], OCT4 Polyclonal Antibody (Proteintech, USA) [1:500], SOX2 Polyclonal Antibody (Proteintech, USA), [1:500], GAPDH Polyclonal Antibody (Proteintech, USA) [1:10000].

Techniques:

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Western blotting of total SOX9 protein levels in the cytoplasmic and nuclear fractions of HCT116‐FBW7 WT versus KO cells. The β‐actin was used as a loading control. General evolutionary conservation for SOX9 amino acid sequence surrounding the human CPD motif (highlighted in red) of threonine 236–240 across species. Western blotting of FLAG‐FBW7α eluted from the immunoprecipitated HA‐SOX9 wild‐type (WT) or CPD mutants (‐T236/240A and ‐T240A). The HA‐SOX9‐WT and the CPD mutant constructs were transiently co‐expressed for 24 h with FLAG‐FBW7α in HEK293 prior to immunoprecipitation with anti‐HA antibody. Equal protein expression of FBW7α across the HEK293 cells transfected with different SOX9 constructs was assessed by immunoblotting of the whole‐cell extract. Co‐expression of FBW7α with HA‐SOX9 WT or various other CPD mutant constructs (‐T236/240A, ‐T236A, or ‐T240A) in HEK293 cells. Whole‐cell lysates were collected 24 h following transfection for Western blotting of the total exogenous and the phosphorylated SOX9 proteins using anti‐HA and our pT236‐SOX9 antibody, respectively. Immunoblot of GAPDH protein was used to indicate protein loading in each lane. Detection of both exogenous and endogenous phosphorylated SOX9 protein from SOX9 immunoprecipitates. HA‐SOX9‐transfected or non‐transfected HEK293 cells were used as sources for exogenous and endogenous SOX9 protein, respectively. Following SOX9 immunoprecipitation with either anti‐HA (for exogenous) or anti‐SOX9 (for endogenous) antibody, the resulting immunoprecipitates were divided and either treated with λ‐phosphatase or left untreated prior to gel electrophoresis and immunoblotting with pT236‐SOX9 antibody. The SOX9 protein blot shows the total protein level present in each sample. Treatment of HEK293 with proteasome inhibitor MG132 (10 μM) increased the level of phosphorylated SOX9. Immunoblots of endogenous pT236 and total SOX9 protein 24 h following transfection of D324MED medulloblastoma cell line with either non‐targeting scramble RNA (siScr) or increasing concentrations of siRNA against SOX9. GAPDH protein was used to indicated protein loading for each sample Representative immunofluorescence staining depicting high intensity of pT236‐SOX9 (Alexa Fluor 488; green) staining in the nucleus (counterstained with DAPI; blue) in Daoy medulloblastoma cells. Transfection of Daoy cells with 20 nM siSOX9 depleted the nuclear staining of pT236‐SOX9. Images were taken using a 40× objective. Scale bar: 20 μm. Bead‐immobilized IVT HA‐SOX9 WT were subjected to in vitro kinase reaction with 1 unit of recombinant active GSK3α, GSK3β, or their combination (i.e., 0.5 unit for each isoform) for 90 min at 37°C prior to elution and gel electrophoresis. The SOX9 blot shows total SOX9 protein eluted from the beads from each in vitro kinase reaction.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Western Blot, Control, Sequencing, Immunoprecipitation, Mutagenesis, Construct, Expressing, Transfection, Nucleic Acid Electrophoresis, Immunofluorescence, Staining, In Vitro, Recombinant

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: General sequence alignment of SOX9 Cdc4 phosphodegron (CPD) motif against other high‐affinity motifs present in previously established FBW7α substrates including cyclin E, cMYC, and c‐Jun. Immunoblot analysis of the immunoprecipitated HA‐SOX9 or FLAG‐FBW7α and their pull‐down products. Western blots from whole‐cell extract (WCE) of the transfected HEK293 show the levels of exogenous HA‐SOX9 or FLAG‐FBW7α proteins. Cells were treated with 10 μM MG132 for 4 h prior to harvesting and immunoprecipitation. Blots are representative of three independent experiments. FBW7α in vitro binding assay. FBW7α was eluted from the agarose bead‐bound SOX9 peptide (encompassing amino acids 231‐245), which had been incubated with the recombinant SCF FBW7α for 1 h at 37°C. The agarose bead‐bound peptide contains either the non‐phosphorylated SOX9 amino acid sequence (SOX9 peptide) or the threonine phosphorylated amino acids (pSOX9 peptide). The input (10%) show the level of the supplemented recombinant SCF FBW7α in the in vitro binding reaction. Blot is representative of two independent experiments. Representative Western blots from three independent repeats of pT236‐SOX9 from lysates of HEK293 transfected with either HA‐EV or HA‐SOX9. Each lysate was divided and left untreated or subjected to lambda phosphatase (λ‐PPase) treatment for 1 h at 37°C prior to gel electrophoresis. SOX9 blot show the presence of SOX9 protein in both the untreated and the phosphatase‐treated SOX9‐transfected cell lysates. Bead‐immobilized in vitro ‐translated (IVT) HA‐SOX9 wild‐type (WT) or T236/240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 min at 37°C prior to elution and gel electrophoresis. SOX9 immunoblots are representative of three independent experiments. GSK3‐mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF FBW7α in vitro . IVT HA‐SOX9 immobilized on beads were either untreated or phosphorylated with GSK3 as described in (E) prior to further incubation with recombinant SCF FBW7α for in vitro binding assay. Immunoblot show FBW7α protein in the in vitro binding reaction (input) and FBW7α eluted from the untreated and GSK3‐phosphorylated SOX9. Blots are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Sequencing, Western Blot, Immunoprecipitation, Transfection, In Vitro, Binding Assay, Incubation, Recombinant, Nucleic Acid Electrophoresis, Mutagenesis, Phospho-proteomics

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α, FBW7β, or FBW7γ. HEK293 cells were transfected with 20 nM of scrambled siRNA or siRNAs specifically targeting FBW7α, FBW7β , or FBW7γ for 72 h prior to chase with the addition of 100 ng/ml cycloheximide. Immunoblot of cyclin E, an established SCF FBW7 substrate, was used to assess the efficacy of RNAi‐mediated depletion of FBW7 protein. GAPDH served as a protein loading control. Immunofluorescence staining of D324MED medulloblastoma cells showing nuclear co‐localization of HA‐SOX9 WT (Alexa Fluor 568; red) and FLAG‐FBW7α (Alexa Fluor 488; green). The cell nuclei were counterstained with DAPI (blue). Images are representative of multiple fields taken at 40× objective magnification. Scale bar indicates 20 μm. Endogenous SOX9 protein levels following transfection (24 h) of 100, 250, 500, and 1,000 ng of plasmid expressing FLAG‐FBW7α. Changes in SOX9 protein levels were analyzed relative to GAPDH levels using ImageJ. The blots shown are representative of three independent experiments. Expression of FLAG‐FBW7α enhance HA‐SOX9‐WT protein turnover but not HA‐SOX9‐T236A or HA‐SOX9‐T236A/T240A protein turnover over a 4‐h cycloheximide (100 ng/ml) chase in HEK293 cells. β‐actin protein served as a loading control. The blots shown are representative of four independent experiments. Treatment of HEK293 cells with increasing concentration of the GSK3α/β inhibitor BIO, increase HA‐SOX9‐WT protein level in a dose‐dependent manner. HA‐SOX9‐WT and FLAG‐FBW7α were co‐expressed in HEK293 cells prior to treatment with different concentrations of BIO for 4 h. Whole‐cell lysates were collected for Western blotting with anti‐HA (SOX9) and anti‐FLAG (FBW7) antibodies. GAPDH served as a protein loading control. The blots shown are representative of two independent experiments. RNAi depletion of GSK3β attenuate FBW7α‐induced HA‐SOX9‐WT turnover in HEK293 cells. HA‐SOX9‐WT protein turnover was examined following 48 h depletion of siGSK3b (20 nM) in the presence of cycloheximide (100 ng/ml). Immunoblots with GSK3β antibodies demonstrated depletion of GSK3β protein with the siRNA. Changes in SOX9 protein levels were analyzed relative to GAPDH levels using ImageJ. The blots shown are representative of three independent experiments. FLAG‐FBW7α‐WT expression promote poly‐ubiquitylation of endogenous SOX9 in HEK293 cells. Expression of FBW7α lacking the F‐box domain (ΔF), or containing R465A mutation did not induce SOX9 poly‐ubiquitylation in vivo . Ubiquitylation assay was performed under denaturing condition to disrupt non‐covalently linked ubiquitin as described in the Materials and Methods. Expression of different FLAG‐FBW7α constructs and endogenous SOX9 protein were examined in the whole‐cell lysates. GAPDH protein was used as a loading control. The blots shown are representative of four independent experiments. Reconstitution of SOX9 poly‐ubiquitylation by FLAG‐FBW7α in vitro . Immobilized IVT HA‐SOX9 WT was incubated with FLAG‐FBW7α‐WT or the ΔF mutant for 60 min at 37°C. Reaction mixture lacking the UbE1, an E1 ubiquitin‐activating enzyme, or UbcH3, an E2 ubiquitin‐conjugating enzyme, served as a negative control for the in vitro reaction. SOX9 poly‐ubiquitylation was assessed following elution of the protein from the immobilized beads under denaturing condition as described in . The blots shown are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Transfection, Western Blot, Control, Immunofluorescence, Staining, Plasmid Preparation, Expressing, Concentration Assay, Mutagenesis, In Vivo, Ubiquitin Assay, Ubiquitin Proteomics, Construct, In Vitro, Incubation, Negative Control

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Endogenous SOX9 protein turnover in HCT116‐FBW7 WT and KO cells over the course of 8 h following the addition of 100 ng/ml cycloheximide. β‐Actin indicates total protein loading for each sample. Quantitative PCR analysis of FBW7 and SOX9 mRNA transcripts in Daoy at 8 and 24 h following transfection with 20 nM siFBW7. The FBW7 and SOX9 mRNAs were adjusted to the B2M mRNA prior to being expressed relative to the siScr control. Data are expressed as mean + standard deviation from two independent experiments, with statistical significance determined by multiple‐comparison two‐way ANOVA with Bonferroni's post‐test. Immunoblots of endogenous SOX9 protein in medulloblastoma cell line Daoy and breast cancer cell line HCC1143 following depletion of FBW7 by RNAi (20 nM) for 48 h. Accumulation of cyclin E is used to assess the efficiency of FBW7 knockdown. GAPDH immunoblot is shown as a loading control. Cycloheximide chase of endogenous SOX9 protein over the course of 6 h in glioma cell line U343MG. The cells were transfected with either non‐targeting (siScr) or FBW7‐specific siRNA for 72 h prior to experiments. Immunoblots of cyclin E, established SCF FBW7 substrate, indicated the efficacy of siFBW, while GAPDH protein was used as total protein loading control for each sample. Western blotting of endogenous SOX9 protein level upon treatment with 10 μM MG132. HEK293 cells were transfected with 1 μg FLAG‐FBW7α for 24 h prior to treatment with the proteasome inhibitor. Whole‐cell lysates were collected 4 h following MG132 treatment for gel electrophoresis and immunoblotting. GAPDH protein was immunoblotted to indicate total protein loading for each sample. RNAi depletion of FBW7α decreased endogenous SOX9 ubiquitylation in HEK293. The cells were transfected with either scramble (siScr) or siFBW7α for 72 h prior to assessment of endogenous SOX9 ubiquitylation in the absence or presence of MG132. Endogenous SOX9 protein was immunoprecipitated under denaturing condition (1% SDS) from the whole‐cell lysate using the pT236‐SOX9 antibody and eluted as described in . Total SOX9, cyclin E, and GAPDH proteins in the whole‐cell lysate were immunoblotted. Reconstitution of ubiquitylation reaction in vitro using bead‐immobilized IVT HA‐SOX9‐WT and recombinant, active human SCF FBW7α . Reaction mixture lacking the UbcH3, SCF FBW7α , and IVT HA‐SOX9‐WT served as control for the experiments. Ubiquitylation was assessed following elution of IVT HA‐SOX9‐WT from the bead. Immunoblots of SOX9 and FBW7 proteins present in the eluted fraction are shown.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Control, Standard Deviation, Comparison, Western Blot, Knockdown, Nucleic Acid Electrophoresis, Immunoprecipitation, In Vitro, Recombinant

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: FBW7 mutations across medulloblastoma molecular subgroup as identified by exome sequencing. In the SHH medulloblastoma subgroup, FBW7 mutations occurred approximately 11% in a cohort containing 133 SHH medulloblastoma cases (Kool et al , ) and can be categorized as stop‐gain ( n = 3) and non‐synonymous ( n = 7). One case of splicing error mutation is detected in the Group 3 medulloblastoma. Immunoblotting of HA‐SOX9‐WT protein following expression of FLAG‐FBW7α constructs including wild‐type (WT), R465A or R479A mutation in Daoy cells. The cells were transfected with equal amounts of HA‐SOX9‐WT and FLAG‐FBW7α, and the whole‐cell lysates were collected 24 h following the transfection. GAPDH expression was used as a loading control. The blots shown are representative of two independent experiments. Co‐immunoprecipitation of HA‐SOX9 with either wild‐type (WT) or R465A‐FLAG‐FBW7α. Unlike the wild‐type protein, FLAG‐FBW7α containing arginine 465 mutation did not significantly co‐immunoprecipitate with HA‐SOX9 (IP with anti‐HA antibody) and vice versa (IP with anti‐FLAG antibody). HA‐SOX9 and FLAG‐FBW7α‐WT or R465A mutant were assessed in the whole‐cell extract (WCE) of the transfected HEK293 cells with GAPDH protein used to indicate total protein loading. Comparison of FBW7 mRNA expression across the medulloblastoma subgroups (WNT, SHH, Group 3, and Group 4) in a cohort containing 423 medulloblastoma cases. Expression of FBW7 in both normal fetal ( n = 5) and adult cerebellum ( n = 13) was used for comparison against the medulloblastoma. Overall survival analysis of medulloblastoma patients based upon FBW7 expression level. Analysis was performed on 383 out of the 423 cases from which the survival data were available. Cohort was divided into high (blue line)‐ and low‐FBW7 (red line)‐expressing subgroup using FBW7 median expression as group classifier. A log‐rank test was used to show differences between groups. Correlation analysis between SOX9 protein/mRNA and FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples. SOX9 protein level was scored following immunohistochemistry staining, while SOX9 and FBW7 mRNA expression was analyzed by the RNAscope as described in . A negative correlation was determined by Kendall's correlation test (τ = −0.223, P < 0.001) between SOX9 protein/mRNA and FBW7 mRNA expression. Affiliation of samples with medulloblastoma subgroups is indicated by different colored circle (blue = WNT, red = SHH, yellow = Group 3, green = Group 4, gray = undetermined). Source data are available online for this figure.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Sequencing, Mutagenesis, Western Blot, Expressing, Construct, Transfection, Control, Immunoprecipitation, Comparison, Microarray, Immunohistochemistry, Staining, RNAscope

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Overall survival in the different MB molecular subgroups of patients with low vs. high FBW7 expression. After Bonferroni correction, the results were not significant in any of the MB subgroups (Bonferroni P = 1.000). SOX9 expression in the four molecular subgroups of MB (423 tumors). Adult ( n = 13) and fetal ( n = 5) cerebellums were used as controls. ANOVA: P = 2.7e‐05. FBW7 and SOX9 mRNA levels do not correlate with each other in a cohort of 423 SHH patients ( R = −0.030; P = 0.54). Examples of FBW7 (green circles) and SOX9 RNAscope (red circles) images and corresponding SOX9 IHC protein staining (red arrows). Overall tumor expression analyses were performed based upon the “percentage of positive cells” and the staining “intensity” from at least three different image fields by two independent, blinded subjects for each tumor as described in detail in Materials and Methods. The overall score was subsequently converted to “Rank” for correlation analysis of SOX9 protein:RNA ratios against FBW7 expression.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Expressing, RNAscope, Staining

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Immunoblots of WCE from SOX9‐expressing Daoy cells with dox‐inducible FBW7α expression. FBW7α expression was induced with different levels of dox for 24 h prior to cell harvesting for Western blotting. GAPDH immunoblot is shown as a loading control. Assessment of changes in Daoy cell viability upon constitutive expression of SOX9 and doxycycline induction of FBW7α. Changes in cell viability are expressed relative to day 0, at the time of seeding. Cell‐doubling time compared to parental cells (Daoy T d = 31.47 h vs. SOX9 T d = 35.67 h), and FBW7α induction reversed this effect (SOX9 + FBW7α T d = 30.20 h). Time‐course Western blotting profiling of Daoy cells constitutively expressing SOX9 upon doxycycline induction of FBW7α. Quantification of Ki67‐positive cells in SOX9‐expressing Daoy cells with dox‐inducible FBW7α tumors (+ and − dox). Representative IHC stainings used for quantification are shown. Scale bars: 50 μm. Box‐and‐whisker plots displaying the quantification of Ki67‐positive cells. The horizontal line inside each box represents the median value, lower and upper box borders display the 25 th and 75 th percentiles, respectively, and the interval between the two whiskers include non‐outlier values (min to max). IHC staining targeting human‐specific epitope (STEM121) identifying transplanted human medulloblastoma cells in SOX9‐expressing Daoy cells without FBW7α expression. Between the primary tumor and the metastatic compartment human tumor cell “droplets” are evident, as seen in the marked region 1. IHC targeting human‐specific epitope (STEM121), identifying transplanted human medulloblastoma cells in SOX9‐expressing Daoy cells with dox‐induced FBW7α expression. Black arrow indicates the center part of the tumor (similar to the region indicated by the black arrow in H). Antibody targeting HA epitope identifying HA‐SOX9 levels in SOX9‐expressing Daoy cells without FBW7α expression. Between the primary tumor and the metastatic compartment human SOX9‐positive tumor cell “droplets” are evident, as seen in the marked region 2. Antibody targeting HA epitope identifying HA‐SOX9 levels in SOX9‐expressing Daoy cells with dox‐induced FBW7α expression. Black arrow indicates the center part of the tumor (similar to F) with reduced SOX9 expression to be compared to center regions of the tumor in (G).

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Western Blot, Expressing, Cell Harvesting, Control, Whisker Assay, Immunohistochemistry

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Overall survival analysis of mice bearing Daoy cells expressing SOX9 with doxycycline (dox)‐inducible FBW7α (Daoy‐SOX9/T‐FBW7α). The dox‐preconditioned or the control cells (10 5 cells) were orthotopically xenografted to the cerebellum of 6‐week‐old athymic nude Foxn1 nu mice, which were continuously fed with either dox‐containing chow (red line) or regular chow food (green line), respectively. Mice xenografted with Daoy‐SOX9/T‐FBW7α cells and fed continuously with dox‐containing chow (red line) survived longer ( P = 0.0285) than the control group (green line). Representative hematoxylin and eosin (H&E) staining of medulloblastoma tumors arising from the orthotopic xenograft experiment described in (A). Both the control (non‐dox‐treated) and the dox‐treated mice developed primary intracerebellar tumors (green and red arrows, respectively). SOX9‐expressing Daoy tumors disseminated to the spinal cord (3/4; green asterisks), while the group continuously receiving dox‐containing chow showed no metastatic spread (0/4). Representative bright‐field images of migrating Daoy‐SOX9/T‐FBW7α cells in the absence or presence of dox. Daoy‐SOX9/T‐FBW7α cells were either maintained in regular culture or preconditioned with dox prior to Transwell experiments. The cells (2 × 10 5 cells/insert) were seeded and allowed to migrate across 5‐μm filter pore toward the laminin‐coated Transwell for 4 h prior to fixation and staining with crystal violet. Histogram quantification of cell migration was performed by measuring the crystal violet absorbance of the stained migrating cells and presented as mean + standard deviation from two independent experiments, each containing two technical replicates. Representative H&E staining of primary (arrow) and disseminated tumors (asterisk) arising from orthotopic xenograft of parental and SOX9‐expressing medulloblastoma‐initiating cells (MIC and MIC‐Sox9, respectively). Quantification of primary and metastatic tumors arising from both of MIC ( n = 16) and MIC‐SOX9 ( n = 8) is shown in the table ( P = 0.0000136). Quantitative comparison of MIC and MIC‐SOX9 cells migrating across 5‐μm filter pore toward the laminin‐coated Transwell for 4 h. Quantification of cell migration was performed as described in (C) and presented as mean + standard error of the mean from six replicates. Transwell migration of SOX9‐expressing MICs with dox‐inducible FBW7α or dominant negative ΔF FBW7α. The cells were either maintained in regular cell culture media or preconditioned dox‐media prior to assessment of in vitro Transwell migration as described in (C). Data are mean + standard deviation from two independent experiments, each containing two technical replicates. Statistical analysis comparing the effect of dox‐induction of WT or ΔF FBW7 was carried out using unpaired, two‐tailed Student's t ‐test. Representative bright‐field images of (F). Correlation between clinical metastatic staging and SOX9 protein across the TMA of 142 cases of human medulloblastoma. The SOX9 protein levels were determined by IHC in Fig F. Tumors with high SOX9 protein level frequently presented with the M3 metastatic staging (* P = 0.038, Fisher's exact test) and spread to the spinal cord (red bars). Analysis was carried out by comparing patients with high SOX9 protein rank (rank 5–7) versus patients with low SOX9 protein rank (rank 1–3).

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Expressing, Control, Staining, Migration, Standard Deviation, Comparison, Dominant Negative Mutation, Cell Culture, In Vitro, Two Tailed Test

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Scatter plot depicting the relationship between transcriptional changes measured as log2 FC between EV to SOX9‐WT ( x ‐axis) and EV to SOX9‐T236/T240A ( y ‐axis). Each dot represents one transcript, with colored dots indicating transcripts upregulated (log2 FC > 0.585; P < 0.05; red dots) or downregulated (log2 FC < −0.585; P < 0.05; blue dots) in both comparisons. The transcript corresponding to the SOX9 gene is highlighted as a red filled dot. Indicated R ‐value indicates Spearman's rank correlation coefficient between the two FC profiles. Box‐and‐whisker plot displaying the sample specific expression of three FBW7 isoforms: FBW7 α (NM_033632), FBW7 β (NM_018315), and FBW7 γ (NM_001013415'). Each box represents the expression distribution over three replicates. The horizontal line inside each box represents the median value, lower and upper box borders display the 25 th and 75 th percentiles, respectively, and the interval between the two whiskers includes all non‐outlier values. Venn diagram illustrating the overlap of differentially expressed (FC > 1.5, P < 0.05) transcripts between any of the three comparisons: EV to SOX9‐WT (upregulated: 145 + 308 + 21; downregulated 146 + 333 + 44), EV to SOX9‐T236/T240A (upregulated: 294 + 308 + 95 + 21; downregulated 345 + 333 + 95 + 44), or SOX9‐WT to SOX9‐T236/T240A (upregulated: 112 + 95 + 21; downregulated 114 + 95 + 44). Each oval represents the number of transcripts either upregulated or downregulated in the respective comparison, where the direction of differential expression is indicated by the inequality sign in the label. Numbers in overlapping regions signify the number of transcripts commonly differentially expressed in all contributing comparisons. Heat map of the row‐wise z‐scores of 16 EMT hallmark genes in SOX9‐WT and SOX9‐T236/T240A samples. Heat map was generated using the GenePattern software (Reich et al , ). Quantitative PCR (qPCR) of S NAI2 (SLUG), VIM , and POU3F1 ( OCT6 ) (normalized to GAPDH levels) in MB002 cells stably transduced with EV (set to 1), SOX9‐WT, and SOX9‐T236/240A. Cells were induced for 8 or 24 h with doxycycline. The data shown represents mean expression values and error bars indicate the standard error of the mean. Western blot of SOX9, HA‐SOX9, pSOX9‐T236, SNAI2 (SLUG), VIM, and POU3F1 (OCT6) in MB002 cells stably transduced with EV, SOX9‐WT, and SOX9‐T236/240A. Cells were induced for 24 and 48 h with doxycycline, and GAPDH was used as a loading control. Heat map of the row‐wise z‐scores of 14 pro‐metastasis genes differentially expressed between SOX9‐WT and SOX9‐T236/T240A samples and previously identified as SOX9 target genes (Kadaja et al , ; Oh et al , ; Larsimont et al , ). Heat map was generated using the GenePattern software (Reich et al , ).

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Whisker Assay, Expressing, Comparison, Quantitative Proteomics, Generated, Software, Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, Western Blot, Control

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Hierarchical clustering of 103 human medulloblastoma samples with known molecular subgroups (Northcott et al , ) together with triplicates each of MB002 cells transduced with EV (blue circles), SOX9‐WT (orange circles), or SOX9‐T236/240A (purple circles). The clustering was performed on the 3,167 signature genes most variably expressed between the four MB subgroups. Extraction of signature genes, hierarchical clustering, and plotting of the dendrogram were performed using the metagene code for cross‐platform, cross‐species projection of transcription profiles (Tamayo et al , ). Gene sets with significant enrichment in SOX9‐expressing MB002 cells. A GSEA on a list of 19 gene sets related to metastasis, migration, or EMT ( Table EV5 ) was performed for each pairwise comparison of MB002 EV, SOX9‐WT, and SOX9‐T236/240A. The figure shows the five gene sets (rows) with significant positive enrichment in either SOX9‐WT against EV (left column), SOX9‐T236/240A against EV (middle column), or SOX9‐T236/240A against SOX9‐WT (right column).

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Transduction, Extraction, Expressing, Migration, Comparison

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Cisplatin dose–response curves of MB002 cells (left), Daoy cells (middle), and MICs (right) in the absence (EV) or presence of SOX9 as analyzed by alamarBlue assay. Cells were preconditioned with doxycycline to induce expression of SOX9 (or EV) prior to treatment with increasing concentrations of cisplatin. The IC 50 were calculated following 5 (MB002 and MIC) or 3 days (Daoy) of treatment. Data are mean + standard deviation from three independent repeats, each containing five technical replicates. Cisplatin dose–response curves of SOX9‐expressing Daoy cells (left) and MICs (right) in the absence or presence of FBW7α. Experiments and data analysis were performed as described in (A). Overall survival analysis of mice bearing Daoy cells or Daoy cells expressing dox‐inducible SOX9 treated with cisplatin. The dox‐preconditioned cells (10 5 cells) were orthotopically xenografted to nude Foxn1 nu mice and left for 1 week prior to being treated with vehicle control or cisplatin (2 mg/kg) intraperitoneally every other day for a total of six doses. Heat map of the row‐wise z‐scores of 11 genes associated with cisplatin resistance in MB002 cells expressing Sox9‐WT or Sox9‐T236/T240A. Heat map was generated using the GenePattern software. Quantitative analysis of ATP7A, DUSP2, and TTK mRNAs in MB002 cells following expression of SOX9‐WT or SOX9‐T236/240A. Total RNA was collected 24 h following doxycycline treatment, from which cDNA was generated for qPCR. Data are mean mRNA level (normalized to B2M transcript) + standard deviation from three independent experiments with statistical significance determined by multiple‐comparison two‐way ANOVA with Bonferroni's post‐test. Time‐course Western blotting of HA‐SOX9, ATP7A, DUSP2, ERK1/2 pThr202/Tyr204, and total ERK1/2 in MB002 cells following doxycycline induction of either EV, SOX9‐WT, or SOX9‐T236/240A. GAPDH was used as a loading control.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Alamar Blue Assay, Expressing, Standard Deviation, Control, Generated, Software, Comparison, Western Blot

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Overall survival of nude Foxn1 nu mice orthotopically engrafted with Daoy cells expressing SOX 9‐T236/240A treated with cisplatin (red curve) or a vehicle control (blue curve). The experiment was carried out as described in Fig C.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Expressing, Control

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Western blotting of pT236‐SOX9 and total SOX9 following treatment of Daoy cells with a panel of inhibitors targeting PI3K/AKT/mTOR pathway. The whole‐cell lysates were collected for gel electrophoresis following 6‐h treatment with 1 μM of AZD5363, AZD8186, or AZD2014. The blots shown are representative of three independent repeats. Western blotting of endogenous SOX9 protein turnover in the presence of PI3K/mTOR dual inhibitor, AZD2014. Daoy cells were transfected with either siScr or siFBW7 for 48 h prior to further treatment with 1 μM AZD2014. SOX9 protein turnover was examined 2 h following the inhibitor treatment by cycloheximide chase assay. Immunoblotting of cyclin E protein was used to indicate the efficacy of FBW7 knockdown. Changes in SOX9 protein level were quantified relative to GAPDH protein using ImageJ. The blots shown are representative of three independent experiments. Quantification of resazurin‐based Daoy and MB002 cell viability following treatment with cisplatin (blue bars), AZD2014 (red bars), or their combination (green bars). Daoy cells were treated for 72 h, while MB002 cells were subjected to 5‐day course of treatment. The cell viability was calculated relative to the vehicle‐treated cells. The synthetic lethality combination index (CI) for each treatment was calculated from the mean of three independent experiments using the Compusyn software. Error bars indicate standard deviation. Time‐course Western blotting of endogenous SOX9 and apoptotic markers including cleaved PARP and caspase‐3 following MB002 treatment with cisplatin, AZD2014, or their combination. β‐Actin was used as a loading control. The blots shown are representative of three independent experiments. Western blotting profiling of MB002 cells expressing EV, SOX9‐WT, or SOX9‐T236/240A following 48‐h treatment with cisplatin, AZD2014, and their combination. The set of blots shown is representative of three independent experiments. Quantification of cell viability of MB002 cells expressing EV, SOX9‐WT, or SOX9‐T236/240A following 5‐day treatment with either 10 μM cisplatin (black bars), 0.5 μM AZD2014 (blue bars), or their combination (red bars). Data are mean + standard deviation from three independent experiments, from which statistical significance was analyzed by two‐way ANOVA multiple comparisons with Bonferroni's post‐test. Immunoblots of ATP7A and DUSP2 protein following doxycycline treatment of Daoy‐SOX9/T‐FBW7α cells. The cells were either untreated or incubated with 5 μg/ml doxycycline for 72 h to induce FBW7a prior to being harvested for gel electrophoresis. The blots shown are representative of two independent experiments.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Western Blot, Nucleic Acid Electrophoresis, Transfection, Knockdown, Software, Standard Deviation, Control, Expressing, Incubation

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: Immunoblots of PI3K/AKT/mTOR pathway activity to assess inhibition upon treatment of Daoy cells with 1 μM AZD5363, AZD8186, or AZD2014. Western blotting of cycloheximide chase from Daoy cells treated with 1 μM PI3K inhibitor AZD8186. Cells were transfected with either non‐targeting or FBW7‐specific siRNA for 48 h prior to cycloheximide chase. siRNA depletion of FBW7 leads to stabilization of SOX9 protein and was calculated using Compusyn software. Cytotoxicity assay for Daoy medulloblastoma cells treated with a combination of cisplatin with (i) BEZ235 or (ii) KU0063794. The synthetic lethality combination index (CI) for each treatment was calculated using Compusyn software. Data are expressed as mean + standard deviation from two independent experiments. Quantitative PCR analysis of endogenous SOX9 mRNAs in MB002 following 24 and 72 h treatment with cisplatin, AZD2014, or their combination. The mRNAs levels were normalized relative to GAPDH and expressed relative to the DMSO‐treated samples. Data are expressed as mean + standard deviation from two independent experiments, with statistical significance determined by multiple‐comparison two‐way ANOVA with Bonferroni's post‐test.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Western Blot, Activity Assay, Inhibition, Transfection, Software, Cytotoxicity Assay, Standard Deviation, Real-time Polymerase Chain Reaction, Comparison

Journal: The EMBO Journal

Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

doi: 10.15252/embj.201693889

Figure Lengend Snippet: SCF FBW 7α together with GSK 3 regulate SOX 9 protein levels through the ubiquitin–proteasome system. FBW 7α mutations, or transcriptional downregulation, lead to stabilization of SOX 9 protein and SOX 9‐driven EMT ‐like reprogramming, migration and drug resistance in medulloblastoma. Small‐molecule inhibitors of the PI 3K/ mTOR pathway can be used to stimulate GSK 3/ FBW 7α‐mediated SOX 9 turnover and may provide a novel strategy to target SOX 9‐driven medulloblastoma.

Article Snippet: Rabbit polyclonal antibody specific against SOX9 pT236&240 was produced by Innovagen AB (Lund, Sweden; Project # 92528).

Techniques: Ubiquitin Proteomics, Migration