Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through direct binding. (a) After HEK293FT cells were co-transfected with the indicated plasmids, cells were immunoprecipitated (IP) with anti-HA antibody and subsequently immunoblotted (IB) with the anti-Flag or anti-HA antibody. (b) After HCT116 cells were treated with 100 ng/ml BMP6, endogenous PSMD14 was immunoprecipitated with anti-PSMD14 antibody and subsequently immunoblotted with the indicated antibodies against endogenous ALK2 and PSMD14 proteins. (c) Expression of endogenous ALK2 and ID3 proteins in PSMD14 -depleted or control (siCON) HCT116 cells were examined by immunoblot analysis (left). Expression of ALK2 mRNA in PSMD14 -depleted or control HCT116 cells was analyzed by qRT-PCR (right). (d) After a plasmid encoding HA-ALK2 was co-transfected into 293FT cells with dose-dependent expression of Flag-PSMD14, cells were immunoblotted with the indicated antibodies. (e) PSMD14-depleted or control (siCON) HCT116 cells were treated with 20 μM cycloheximide (CHX; protein synthesis inhibitor) for the indicated times. Cells were immunoblotted with anti-ALK2 and anti-PSMD14 antibodies. (f) After plasmids encoding wild-type (WT) Flag-PSMD14 and catalytic inactive DUB mutants of PSMD14 were respectively transfected into HCT116 cells, cells were treated with 20 μM CHX for the indicated times and immunoblotted. In (e) and (f) , ALK2 levels were quantified by using ImageJ software and normalized to β-actin expression. The data were statistically analyzed and the bars represent the mean ± s .d. from three independent experiments. * P <0.05, *** P < 0.001 (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (g) PSMD14 -depleted and control (siCON) HCT116 cells were treated with 100 ng/ml BMP6 for 10 min and immunostained with the indicated antibodies to detect endogenous PSMD14 (green) and ALK2 (red). PKH26 molecule (yellow) and DAPI (blue) were used to stain the cell membrane and nuclei. Scale bar; 10 μm. (h) After HCT116 cells were treated with BMP6 for 10 min, cells were fractionated into membrane (Mem), cytoplasmic (Cyt) and nuclear (Nuc) extracts, which were subsequently immunoblotted with the indicated antibodies. Expressions of α-Tubulin, Lamin B1 and E-cadherin were used as markers and loading controls of cytosolic, nuclear and membrane fractions, respectively. Except for ( h ), expression of β-actin was used as a loading control of immunoblot analysis. The images in this figure are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Expressing, Control, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Software, Staining, Membrane

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through deubiquitinating Smurf1-mediated polyubiquitination of ALK2 (a, b) Plasmids encoding Flag-ALKs, Flag-PSMD14 or HA-Ubi were co-transfected into HEK293FT cells according to the indicated combinations. Ubiquitination of Flag-ALK2, Flag-ALK3, and Flag-ALK6 were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. (c) Flag-ALK2 and Flag-PSMD14 were co-transfected into HEK293FT cells with a plasmid encoding wild-type or lysine mutant (K48 or K63) HA-Ubi in the indicated combinations. When the K48 mutant of His-Ubi was transfected, MG132 was treated to prevent ALK2 degradation. (d) The catalytically inactive DUB mutant of Flag-PSMD14 (H113Q, C120A or C120S) and wild-type Flag-PSMD14 were co-transfected into HEK293FT cells with Flag-ALK2 and HA-Ubi in the indicated combinations. (e) After PSMD14-depleted and control HCT116 cells were treated with 100 ng/ml BMP6 for the indicated times, ubiquitination of endogenous ALK2 protein was examined by IP and IB with the indicated antibodies. IgG was used as a negative control for IP. (f) After a plasmid encoding HA-ALK2 was co-transfected into HEK293FT cells with plasmids encoding Flag-Smurf1, Flag-Smurf2, Flag-CHIP, or Flag-TRIM33, respectively, co-immunoprecipitation assays were performed with the indicated antibodies. (g) Flag-Smurf1 or Flag-Smurf2 was co-transfected into HEK293FT cells with HA-Ubi and Flag-ALK2 in the indicated combinations. (h) Flag-ALK2 was co-transfected into HEK293FT cells with wild-type His-Ubi or a lysine mutant (K48 or K63) of HA-Ubi in the absence or presence of Flag-Smurf1. (i) Flag-Smurf1 and the K48 lysine mutant of HA-Ubi were co-transfected into HEK293FT cells with wild-type Flag-PSMD14 or the catalytic inactive DUB mutant of PSMD14 (H113Q). In (a) - (d) , ubiquitination of Flag-ALK proteins were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. In (g) - (i) , ubiquitination of the ALK2 protein was analyzed by IP and IB with the indicated antibodies. In (h) and (i) , cells were pre-treated with MG132 to prevent protein degradation. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Plasmid Preparation, Mutagenesis, Control, Negative Control, Expressing

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 and ALK2 are required for BMP-mediated colon cancer tumorigenesis. (a) For the analysis of cell proliferation, 1 × 10 3 cells for each cell line were cultured in 12-well plates in the absence or presence of 100 ng/ ml BMP6 for the indicated times. Cell numbers were counted at the indicated time points. (b) BrdU incorporation assays were used to detect cell proliferation. (c) MTT assays were performed to analyze cell viability. (d) For colony forming assays, cells were seeded in 6-well plates with soft-agar media and incubated for 14 days. Colonies were counted and described as graphs demonstrating the percentage of colonies in the field. In (a) - (d) , the data were statistically analyzed and the error bras represent the mean ± s .d. * P <0.05, ** P <0.01, *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 3 per group, compared to the indicated controls). (e) 1 × 10 6 PSMD14 -depleted, ALK2 -depleted or control HCT116 cells were injected in the NOD-SCID mouse group ( n = 5 per group). The tumorigenesis experiment was processed for 30 days. (f) Cell lysates isolated from each tumor xenograft were immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (g) Tumor sizes ( n = 5 per group) were calculated every 5 days. (h) After isolating each tumor from mice ( n = 5 per group), tumor weights were measured and calculated as the average per group. In (g) and (h) , the data were statistically analyzed and the error bras represent the mean ± s .d. *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 5 per group, compared to the indicated controls). In (a) - (h) , PSMD14 - and ALK2 -depleted HCT116 cells were generated by the infection of recombinant lentiviruses expressing shRNAs targeting ALK2 or PSMD14 .

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Cell Culture, BrdU Incorporation Assay, Incubation, Control, Injection, Isolation, Expressing, Western Blot, Generated, Infection, Recombinant

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 depletion reduces BMP6-mediated colorectal cancer stemness. (a, b) FACS analysis of CD133 + /CD44 + cells in PSMD14 - or ALK2 -depleted HCT116 cells, which were treated with 100 ng/ml BMP6 for 48 h. The proportion of the CD133 + /CD44 + fraction was described with the density plots (a) and a bar graph (b) . shGFP-expressing HCT116 cells were used as a control. (c) Sphere forming assay of PSMD14 - or ALK2 - depleted HCT116 cells. Spheres with a diameter above 50 μm were counted and described in a bar graph. Scale bars, 50 μm. (d) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6. Expression of pluripotent transcription factors were analyzed by immunoblotting with the indicated antibodies. siCON-expressing HCT116 cells were used as a control. (e, h) 2 × 10 4 cells of PSMD14 -, ALK2 -, ABCA7 - or ABCC4 -depleted HCT116 were respectively treated with 20 μM oxaliplatin and 30 μM DAPT and their viabilities were measured at 6 h. shGFP-expressing HCT116 cells were used as a control. (f, g) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6 for 6 h. Expressions of ABCA7 and ABCC4 were measured by quantitative RT-PCR. The data were statistically analyzed by two-way ANOVA followed by Bonferroni's multiple comparison test ( n = 3, *** P < 0.001 compared to the indicated controls. ns; not significant). The bars represent the mean ± s .d. The images in this figure are representative of three independent experiments. In (b), (c), (e) and (h) were statistically analyzed by one-way ANOVA followed by Dunnett's test ( n = 3, ** P <0.01, *** P < 0.001 compared to the indicated controls. ns; not significant).

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Comparison

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Higher expression of PSMD14 and ALK2 confer poor prognosis in human colorectal cancer. (a) Cell lysates isolated from the indicated colorectal cancer cell lines were immunoblotted with the ALK2 and PSMD14 antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (b, c) Using the Kaplan-Meier (KM) plotter tool, expression of PSMD14 and ALK2 mRNA between normal and tumor tissues of colorectal cancer patients in a public GEO dataset (GSE21510; n = 148) were analyzed. (d) To verify the correlation of ALK2 mRNA expression in normal colon tissues and tumor tissues with the expression levels of PSMD14, a public GSE dataset (GSE21510) was analyzed by the KM plotter tool. In (b) - (d) , the boxes represent the interquartile range, centre is the median, and the minimum and maximum values are represented in the whiskers. *** P <0.001, ns; not significant (Student t -test, compared to normal tissues or PSMD14-low samples). (e) Correlation of ALK2 and PSMD14 mRNAs in normal colon tissues, polyps in colon, and colorectal tumor tissues of human colorectal cancer patient samples (GSE68468, n = 288). Samples were classified into three groups (double low expression of ALK2 and PSMD14 , single high expression of ALK2 or PSMD14 , and double high expression of ALK2 and PSMD14 ) in normal tissues, polyps and tumors. (f, g) Scatter dot plot analysis represents the scores of the expression of PSMD14 and ALK2 protein in the matched normal and tumor tissues of human colon cancer patients (normal tissue n = 70, cancer tissue n = 70) analyzed by immunohistochemistry. *** P <0.001 (Student t -test, compared to normal tissues). (h) Scatter dot plot shows Spearman correlations between ALK2 and PSMD14 protein expression according to scoring within all samples. *** P < 0.001 (Student t -test). The Spearman r indicates the Spearman correlation coefficients. (i, j) Correlation of PSMD14 or ALK2 mRNA expression with overall survival rates of human colorectal cancer patients were analyzed by a KM plot analysis in a public GSE dataset (GSE17538; n = 224 patients). P = 0.0372, P = 0.0021 (Log-rank test). HR = hazard ratio.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Isolation, Control, Western Blot, Immunohistochemistry

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Schematic representation of the proposed mechanism of BMP6 signaling pathway regulated by PSMD14-ALK2 axis in colorectal cancers. ALK2 type I receptor is polyubiquitinated by E3 ligase Smurf1 and subsequently degraded in the absence of BMP6. Upon treatment of BMP6, PSMD14 binds to ALK2 protein and deubiquitinates the K48-linked polyubiquitin chains of ALK2 protein, resulting in the increase of ALK2 stability and thus leads to the initiation of BMP6 signaling pathway. This initiation of BMP6 signaling pathway facilitates tumor growth, cancer stemness and chemoresistance in colorectal cancers.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques:

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Characterization of DUB enzymes involved in regulation of BMP signaling in human colorectal cancer cells. (a) After HCT116 and RKO cells were reverse-transfected with specific siRNA against PSMD14 and control siRNA (siCON) and treated with 100 ng/ml BMP6 for the indicated times, immunoblotting assays were performed with the indicated antibodies. (b) Expressions of ID1, ID3 , and SMAD6 mRNAs were analyzed by quantitative real-time RT-PCR (qRT-PCR) in PSMD14 -knockdown and control HCT116 or RKO cells. Expression of the mRNAs detected by qRT-RCR were normalized to Gapdh mRNA. (c) BRE-Luc luciferase reporter assays were performed in PSMD14 -knockdown and control HCT116 and RKO cells. In (b) and (c) , bar graphs show the mean ± s .d. from three independent experiments. ** P <0.01 (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (d) After Smad4-null HT29 cells were reverse-transfected with PSDM14 -specific siRNA or siCON and treated with BMP6, immunoblots were performed with the indicated antibodies. (e) HCT116 or RKO cells overexpressing Flag-PSMD14 were immunoblotted with the indicated antibodies upon treatment of BMP6. (f) Expressions of ID1, ID3 , and SMAD6 mRNAs were analyzed by qRT-PCR in PSMD14-overexpressing HCT116 and RKO cells. In all immunoblot analyses, β-actin expression was used as a loading control and the images are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Knockdown, Expressing, Luciferase

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: The role of PSMD14 in the BMP6 signaling pathway is independent of the 26S proteasome system. (a) After PSMD14, PSMB5 or PSMB4 expression was depleted by specific siRNA against each mRNA in HCT116 cells and cells were subsequently treated with 100 ng/ml BMP6, cells were immunoblotted with the indicated antibodies. (b) Expression of ID3 mRNA was analyzed by qRT-PCR in PSMD14, PSMB4 or PSMB5-knockdown HCT116 cells, which were treated with BMP6. Expressions of ID3 mRNA were normalized to Gapdh mRNA. Bar graphs show the mean ± s .d. from three independent experiments. * P <0.05, ** P <0.01, *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (c) After PSMD14 -knockdown HCT116 cells were pre-treated with protease inhibitor MG132 or bortezomib, cells were treated with BMP6 for the indicated times and subsequently immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The images in all immunoblot analyses are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Protease Inhibitor, Control, Western Blot

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through direct binding. (a) After HEK293FT cells were co-transfected with the indicated plasmids, cells were immunoprecipitated (IP) with anti-HA antibody and subsequently immunoblotted (IB) with the anti-Flag or anti-HA antibody. (b) After HCT116 cells were treated with 100 ng/ml BMP6, endogenous PSMD14 was immunoprecipitated with anti-PSMD14 antibody and subsequently immunoblotted with the indicated antibodies against endogenous ALK2 and PSMD14 proteins. (c) Expression of endogenous ALK2 and ID3 proteins in PSMD14 -depleted or control (siCON) HCT116 cells were examined by immunoblot analysis (left). Expression of ALK2 mRNA in PSMD14 -depleted or control HCT116 cells was analyzed by qRT-PCR (right). (d) After a plasmid encoding HA-ALK2 was co-transfected into 293FT cells with dose-dependent expression of Flag-PSMD14, cells were immunoblotted with the indicated antibodies. (e) PSMD14-depleted or control (siCON) HCT116 cells were treated with 20 μM cycloheximide (CHX; protein synthesis inhibitor) for the indicated times. Cells were immunoblotted with anti-ALK2 and anti-PSMD14 antibodies. (f) After plasmids encoding wild-type (WT) Flag-PSMD14 and catalytic inactive DUB mutants of PSMD14 were respectively transfected into HCT116 cells, cells were treated with 20 μM CHX for the indicated times and immunoblotted. In (e) and (f) , ALK2 levels were quantified by using ImageJ software and normalized to β-actin expression. The data were statistically analyzed and the bars represent the mean ± s .d. from three independent experiments. * P <0.05, *** P < 0.001 (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (g) PSMD14 -depleted and control (siCON) HCT116 cells were treated with 100 ng/ml BMP6 for 10 min and immunostained with the indicated antibodies to detect endogenous PSMD14 (green) and ALK2 (red). PKH26 molecule (yellow) and DAPI (blue) were used to stain the cell membrane and nuclei. Scale bar; 10 μm. (h) After HCT116 cells were treated with BMP6 for 10 min, cells were fractionated into membrane (Mem), cytoplasmic (Cyt) and nuclear (Nuc) extracts, which were subsequently immunoblotted with the indicated antibodies. Expressions of α-Tubulin, Lamin B1 and E-cadherin were used as markers and loading controls of cytosolic, nuclear and membrane fractions, respectively. Except for ( h ), expression of β-actin was used as a loading control of immunoblot analysis. The images in this figure are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Expressing, Control, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Software, Staining, Membrane

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through deubiquitinating Smurf1-mediated polyubiquitination of ALK2 (a, b) Plasmids encoding Flag-ALKs, Flag-PSMD14 or HA-Ubi were co-transfected into HEK293FT cells according to the indicated combinations. Ubiquitination of Flag-ALK2, Flag-ALK3, and Flag-ALK6 were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. (c) Flag-ALK2 and Flag-PSMD14 were co-transfected into HEK293FT cells with a plasmid encoding wild-type or lysine mutant (K48 or K63) HA-Ubi in the indicated combinations. When the K48 mutant of His-Ubi was transfected, MG132 was treated to prevent ALK2 degradation. (d) The catalytically inactive DUB mutant of Flag-PSMD14 (H113Q, C120A or C120S) and wild-type Flag-PSMD14 were co-transfected into HEK293FT cells with Flag-ALK2 and HA-Ubi in the indicated combinations. (e) After PSMD14-depleted and control HCT116 cells were treated with 100 ng/ml BMP6 for the indicated times, ubiquitination of endogenous ALK2 protein was examined by IP and IB with the indicated antibodies. IgG was used as a negative control for IP. (f) After a plasmid encoding HA-ALK2 was co-transfected into HEK293FT cells with plasmids encoding Flag-Smurf1, Flag-Smurf2, Flag-CHIP, or Flag-TRIM33, respectively, co-immunoprecipitation assays were performed with the indicated antibodies. (g) Flag-Smurf1 or Flag-Smurf2 was co-transfected into HEK293FT cells with HA-Ubi and Flag-ALK2 in the indicated combinations. (h) Flag-ALK2 was co-transfected into HEK293FT cells with wild-type His-Ubi or a lysine mutant (K48 or K63) of HA-Ubi in the absence or presence of Flag-Smurf1. (i) Flag-Smurf1 and the K48 lysine mutant of HA-Ubi were co-transfected into HEK293FT cells with wild-type Flag-PSMD14 or the catalytic inactive DUB mutant of PSMD14 (H113Q). In (a) - (d) , ubiquitination of Flag-ALK proteins were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. In (g) - (i) , ubiquitination of the ALK2 protein was analyzed by IP and IB with the indicated antibodies. In (h) and (i) , cells were pre-treated with MG132 to prevent protein degradation. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Plasmid Preparation, Mutagenesis, Control, Negative Control, Expressing

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 and ALK2 are required for BMP-mediated colon cancer tumorigenesis. (a) For the analysis of cell proliferation, 1 × 10 3 cells for each cell line were cultured in 12-well plates in the absence or presence of 100 ng/ ml BMP6 for the indicated times. Cell numbers were counted at the indicated time points. (b) BrdU incorporation assays were used to detect cell proliferation. (c) MTT assays were performed to analyze cell viability. (d) For colony forming assays, cells were seeded in 6-well plates with soft-agar media and incubated for 14 days. Colonies were counted and described as graphs demonstrating the percentage of colonies in the field. In (a) - (d) , the data were statistically analyzed and the error bras represent the mean ± s .d. * P <0.05, ** P <0.01, *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 3 per group, compared to the indicated controls). (e) 1 × 10 6 PSMD14 -depleted, ALK2 -depleted or control HCT116 cells were injected in the NOD-SCID mouse group ( n = 5 per group). The tumorigenesis experiment was processed for 30 days. (f) Cell lysates isolated from each tumor xenograft were immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (g) Tumor sizes ( n = 5 per group) were calculated every 5 days. (h) After isolating each tumor from mice ( n = 5 per group), tumor weights were measured and calculated as the average per group. In (g) and (h) , the data were statistically analyzed and the error bras represent the mean ± s .d. *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 5 per group, compared to the indicated controls). In (a) - (h) , PSMD14 - and ALK2 -depleted HCT116 cells were generated by the infection of recombinant lentiviruses expressing shRNAs targeting ALK2 or PSMD14 .

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Cell Culture, BrdU Incorporation Assay, Incubation, Control, Injection, Isolation, Expressing, Western Blot, Generated, Infection, Recombinant

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 depletion reduces BMP6-mediated colorectal cancer stemness. (a, b) FACS analysis of CD133 + /CD44 + cells in PSMD14 - or ALK2 -depleted HCT116 cells, which were treated with 100 ng/ml BMP6 for 48 h. The proportion of the CD133 + /CD44 + fraction was described with the density plots (a) and a bar graph (b) . shGFP-expressing HCT116 cells were used as a control. (c) Sphere forming assay of PSMD14 - or ALK2 - depleted HCT116 cells. Spheres with a diameter above 50 μm were counted and described in a bar graph. Scale bars, 50 μm. (d) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6. Expression of pluripotent transcription factors were analyzed by immunoblotting with the indicated antibodies. siCON-expressing HCT116 cells were used as a control. (e, h) 2 × 10 4 cells of PSMD14 -, ALK2 -, ABCA7 - or ABCC4 -depleted HCT116 were respectively treated with 20 μM oxaliplatin and 30 μM DAPT and their viabilities were measured at 6 h. shGFP-expressing HCT116 cells were used as a control. (f, g) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6 for 6 h. Expressions of ABCA7 and ABCC4 were measured by quantitative RT-PCR. The data were statistically analyzed by two-way ANOVA followed by Bonferroni's multiple comparison test ( n = 3, *** P < 0.001 compared to the indicated controls. ns; not significant). The bars represent the mean ± s .d. The images in this figure are representative of three independent experiments. In (b), (c), (e) and (h) were statistically analyzed by one-way ANOVA followed by Dunnett's test ( n = 3, ** P <0.01, *** P < 0.001 compared to the indicated controls. ns; not significant).

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Comparison

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Higher expression of PSMD14 and ALK2 confer poor prognosis in human colorectal cancer. (a) Cell lysates isolated from the indicated colorectal cancer cell lines were immunoblotted with the ALK2 and PSMD14 antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (b, c) Using the Kaplan-Meier (KM) plotter tool, expression of PSMD14 and ALK2 mRNA between normal and tumor tissues of colorectal cancer patients in a public GEO dataset (GSE21510; n = 148) were analyzed. (d) To verify the correlation of ALK2 mRNA expression in normal colon tissues and tumor tissues with the expression levels of PSMD14, a public GSE dataset (GSE21510) was analyzed by the KM plotter tool. In (b) - (d) , the boxes represent the interquartile range, centre is the median, and the minimum and maximum values are represented in the whiskers. *** P <0.001, ns; not significant (Student t -test, compared to normal tissues or PSMD14-low samples). (e) Correlation of ALK2 and PSMD14 mRNAs in normal colon tissues, polyps in colon, and colorectal tumor tissues of human colorectal cancer patient samples (GSE68468, n = 288). Samples were classified into three groups (double low expression of ALK2 and PSMD14 , single high expression of ALK2 or PSMD14 , and double high expression of ALK2 and PSMD14 ) in normal tissues, polyps and tumors. (f, g) Scatter dot plot analysis represents the scores of the expression of PSMD14 and ALK2 protein in the matched normal and tumor tissues of human colon cancer patients (normal tissue n = 70, cancer tissue n = 70) analyzed by immunohistochemistry. *** P <0.001 (Student t -test, compared to normal tissues). (h) Scatter dot plot shows Spearman correlations between ALK2 and PSMD14 protein expression according to scoring within all samples. *** P < 0.001 (Student t -test). The Spearman r indicates the Spearman correlation coefficients. (i, j) Correlation of PSMD14 or ALK2 mRNA expression with overall survival rates of human colorectal cancer patients were analyzed by a KM plot analysis in a public GSE dataset (GSE17538; n = 224 patients). P = 0.0372, P = 0.0021 (Log-rank test). HR = hazard ratio.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Isolation, Control, Western Blot, Immunohistochemistry

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Schematic representation of the proposed mechanism of BMP6 signaling pathway regulated by PSMD14-ALK2 axis in colorectal cancers. ALK2 type I receptor is polyubiquitinated by E3 ligase Smurf1 and subsequently degraded in the absence of BMP6. Upon treatment of BMP6, PSMD14 binds to ALK2 protein and deubiquitinates the K48-linked polyubiquitin chains of ALK2 protein, resulting in the increase of ALK2 stability and thus leads to the initiation of BMP6 signaling pathway. This initiation of BMP6 signaling pathway facilitates tumor growth, cancer stemness and chemoresistance in colorectal cancers.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: