Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 2. AMPK Phosphorylates PTPS and Promotes LTBP1-PTPS Interaction (A) HT29 cells with stable expression of Flag-PTPS were cultured for 12 h under normoxia or hypoxia. Coomassie brilliant blue staining analysis of the immunoprecipitates was performed. (B) HT29 cells transfected with or without PTPS shRNA were cultured for 12 h under normoxia or hypoxia. (C) HT29 cells were cultured for 12 h under hypoxia. The immunoprecipitates were treated with CIP (10 units) and analyzed by immunoblotting. (D) HT29 cells were pretreated with Compound C (10 mM), SP600125 (20 mM), and SB203580 (10 mM) for 1 h before being cultured for 12 h under hypoxia. (E) Immunoprecipitated Myc-LTBP1 from HT29 cells were treated with CIP (10 units) and incubated with cell lysates of HT29 cell cultured for 12 h under normoxia or hypoxia. (F) In vitro phosphorylation analyses were performed by mixing the purified AMPK complex with purified His-PTPS proteins in the presence of [g-32P] ATP. (G) Thr 58 of PTPS is evolutionarily conserved in the indicated species (upper panel). In vitro phosphorylation analyses were performed by mixing the purified AMPK complex with the indicated purified His-PTPS proteins in the presence of [g-32P]ATP (bottom panel). (H) HT29 cells with transient expression of WT PTPS or PTPS T58A were cultured for 12 h under hypoxia. (I) The indicated purified His-PTPS proteins were mixed with CIP (10 units) treated immunoprecipitated Myc-LTBP1 from HT29 cells in presence or absence of purified AMPK. His pull down analyses was performed. Immunoblotting and immunoprecipitation were performed with the indicated antibodies. See also Figure S2.

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Expressing, Cell Culture, Staining, Transfection, shRNA, Western Blot, Immunoprecipitation, Incubation, In Vitro, Phospho-proteomics

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 3. AMPK-Phosphorylated PTPS Regulates LTBP1 S-Nitrosylation and Protein Stability (A) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS T58A were cultured for 24 h under normoxia or hypoxia condition. Cells were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia treatment. Immunoblotting analysis was performed (left panel). Relative intensity of LTBP1 to beta-actin was analyzed (right panel). (B) HT29 cells expressing Flag-PTPS were cultured for 12 h under hypoxia. Immunoprecipitation was performed using the Flag antibody, and the extracts were analyzed by mass spectrometry. The results of a mass spectrometric analysis of a tryptic fragment at m/z 715.54 (upper panel) and 1,432.58 (bottom panel), revealing the cysteine residues with S-nitrosylation. (C and D) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS T58A (C) or HT29 cells expressed with WT Myc-LTBP1 and indicated Myc-LTBP1 mutants (D) were cultured for 12 h under hypoxia. The S-nitrosylation detection assay was performed. (E) HT29 cells with depleted LTBP1 and reconstituted expression of WT rLTBP1 or rLTBP1 2C/S were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia. Immunoblotting analysis was performed (left panel). Relative intensity of LTBP1 to beta-actin was analyzed (right panel).

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Expressing, Cell Culture, Western Blot, Immunoprecipitation, Mass Spectrometry, Detection Assay

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 4. BH4 Production Is Indispensable for the Regulation of LTBP1 S-Nitrosylation and Protein Stability (A–C) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q cultured for 24 h (A) or 12h (B) under hypoxia. The S-nitrosylation detection assay was performed. Cells were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia treatment (C). (D and E) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q were cultured for 12 h under hypoxia. Immunoprecipitation and immunoblotting (D) and NOS activity assay against Myc-LTBP1 immunoprecipitates (E) were performed. (F) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q were cultured for 24 h under hypoxia and were added with indicated concentration of exogenous BH4. Immunoblotting analysis was performed (upper panel). Relative intensity of LTBP1 to beta-actin was analyzed (bottom panel). (G and H) HT29 cells with depleted PTPS and reconstituted expression of rPTPS R25Q were cultured for 12 h under hypoxia and were added with indicated concentration of exogenous BH4 in presence or absence of 1400W (100 mM). The S-nitrosylation detection assay was performed (G). Cells were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia treatment. Immunoblotting analysis was performed (H, upper panel). Relative intensity of LTBP1 to beta-actin was analyzed (H, bottom panel).

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Expressing, Cell Culture, Detection Assay, Immunoprecipitation, Western Blot, Nos Activity Assay, Concentration Assay

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 1. PTPS-Mediated BH4 Production Is Required for Tumor Cell Growth under Hypoxia (A) Immunohistochemical staining with PTPS was performed on 161 human colorectal cancer specimens. Representative photos of early stage and late stage tumor versus the adjacent normal tissues were shown (magnification: 3100 and 3400). Scale bars: 100 mm. (B) The t test indicating significant difference of PTPS level between tumor and the adjacent normal tissues (the upper panel). The chi-square test indicated a significant association of high PTPS level with the number of early and non-metastasis tumors (two middle panels). The t test indicated a significant difference of tumor size between low PTPS level-group and high PTPS level-group (bottom panel). (C) Protein levels of PTPS, GTPCH, and SR were examined in indicated cell lines. (D) HT29 cells were expressed with a vector for control shRNA or PTPS shRNA and reconstituted with expression of WT rPTPS or rPTPS R25Q. (E) HT29 cells with or without depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q were cultured for 24 h under normoxia or hypoxia condition. Cellular viability was examined by CCK8 assay at 48 h post hypoxia treatment. (F) A cartoon showing that PTPS depletion can lead to impairment of cellular BH4 production from biosynthesis and salvage pathways. (G) HT29 cells with or without depleted PTPS were cultured for 24 h under hypoxia and were added with indicated concentration of exogenous BH4. Cellular viability was examined by CCK8 assay at 48 h post hypoxia treatment. (H) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q were cultured for 24 h under hypoxia and were added with indicated concentration of exogenous BH4. Cellular viability was examined by CCK8 assay at 48 h post hypoxia treatment. Immunoblotting analyses were performed using the indicated antibodies (C and D). The values are presented as mean ± SEM (n = 3 independent experiments). **p < 0.01 and #p > 0.05 between indicated groups (E, G, and H). See also Figure S1.

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Immunohistochemical staining, Staining, Plasmid Preparation, Control, shRNA, Expressing, Cell Culture, CCK-8 Assay, Concentration Assay, Western Blot

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 2. AMPK Phosphorylates PTPS and Promotes LTBP1-PTPS Interaction (A) HT29 cells with stable expression of Flag-PTPS were cultured for 12 h under normoxia or hypoxia. Coomassie brilliant blue staining analysis of the immunoprecipitates was performed. (B) HT29 cells transfected with or without PTPS shRNA were cultured for 12 h under normoxia or hypoxia. (C) HT29 cells were cultured for 12 h under hypoxia. The immunoprecipitates were treated with CIP (10 units) and analyzed by immunoblotting. (D) HT29 cells were pretreated with Compound C (10 mM), SP600125 (20 mM), and SB203580 (10 mM) for 1 h before being cultured for 12 h under hypoxia. (E) Immunoprecipitated Myc-LTBP1 from HT29 cells were treated with CIP (10 units) and incubated with cell lysates of HT29 cell cultured for 12 h under normoxia or hypoxia. (F) In vitro phosphorylation analyses were performed by mixing the purified AMPK complex with purified His-PTPS proteins in the presence of [g-32P] ATP. (G) Thr 58 of PTPS is evolutionarily conserved in the indicated species (upper panel). In vitro phosphorylation analyses were performed by mixing the purified AMPK complex with the indicated purified His-PTPS proteins in the presence of [g-32P]ATP (bottom panel). (H) HT29 cells with transient expression of WT PTPS or PTPS T58A were cultured for 12 h under hypoxia. (I) The indicated purified His-PTPS proteins were mixed with CIP (10 units) treated immunoprecipitated Myc-LTBP1 from HT29 cells in presence or absence of purified AMPK. His pull down analyses was performed. Immunoblotting and immunoprecipitation were performed with the indicated antibodies. See also Figure S2.

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Expressing, Cell Culture, Staining, Transfection, shRNA, Western Blot, Immunoprecipitation, Incubation, In Vitro, Phospho-proteomics

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 3. AMPK-Phosphorylated PTPS Regulates LTBP1 S-Nitrosylation and Protein Stability (A) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS T58A were cultured for 24 h under normoxia or hypoxia condition. Cells were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia treatment. Immunoblotting analysis was performed (left panel). Relative intensity of LTBP1 to beta-actin was analyzed (right panel). (B) HT29 cells expressing Flag-PTPS were cultured for 12 h under hypoxia. Immunoprecipitation was performed using the Flag antibody, and the extracts were analyzed by mass spectrometry. The results of a mass spectrometric analysis of a tryptic fragment at m/z 715.54 (upper panel) and 1,432.58 (bottom panel), revealing the cysteine residues with S-nitrosylation. (C and D) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS T58A (C) or HT29 cells expressed with WT Myc-LTBP1 and indicated Myc-LTBP1 mutants (D) were cultured for 12 h under hypoxia. The S-nitrosylation detection assay was performed. (E) HT29 cells with depleted LTBP1 and reconstituted expression of WT rLTBP1 or rLTBP1 2C/S were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia. Immunoblotting analysis was performed (left panel). Relative intensity of LTBP1 to beta-actin was analyzed (right panel).

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Expressing, Cell Culture, Western Blot, Immunoprecipitation, Mass Spectrometry, Detection Assay

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 4. BH4 Production Is Indispensable for the Regulation of LTBP1 S-Nitrosylation and Protein Stability (A–C) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q cultured for 24 h (A) or 12h (B) under hypoxia. The S-nitrosylation detection assay was performed. Cells were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia treatment (C). (D and E) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q were cultured for 12 h under hypoxia. Immunoprecipitation and immunoblotting (D) and NOS activity assay against Myc-LTBP1 immunoprecipitates (E) were performed. (F) HT29 cells with depleted PTPS and reconstituted expression of WT rPTPS or rPTPS R25Q were cultured for 24 h under hypoxia and were added with indicated concentration of exogenous BH4. Immunoblotting analysis was performed (upper panel). Relative intensity of LTBP1 to beta-actin was analyzed (bottom panel). (G and H) HT29 cells with depleted PTPS and reconstituted expression of rPTPS R25Q were cultured for 12 h under hypoxia and were added with indicated concentration of exogenous BH4 in presence or absence of 1400W (100 mM). The S-nitrosylation detection assay was performed (G). Cells were treated with cycloheximide (CHX; 30 mg/mL) for the indicated length of time at 6 h under hypoxia treatment. Immunoblotting analysis was performed (H, upper panel). Relative intensity of LTBP1 to beta-actin was analyzed (H, bottom panel).

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Expressing, Cell Culture, Detection Assay, Immunoprecipitation, Western Blot, Nos Activity Assay, Concentration Assay

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 5. PTPS Maintains Tumor Cell Growth through Inhibition of TGF-b Secretion (A and B) HT29 cells with PTPS depletion and reconstituted expression of WT rPTPS, rPTPS T58A, or rPTPS R25Q (A) or HT29 cells with PTPS depletion and reconstituted expression of WT rLTBP1 or rLTBP1 2C/S (B) were transfected with LTBP1 shRNA or added with exogenous BH4 (40 mM). TGF-b secretion was analyzed at 12 h after cells were cultured for 24 h under hypoxia. (C and D) HT29 cells with PTPS depletion and reconstituted expression of WT rPTPS, rPTPS T58A, or rPTPS R25Q (C) or HT29 cells with depleted LTBP1 with reconstituted expression of WT rLTBP1 or rLTBP1 2C/S (D) were transfected with LTBP1 shRNA or added with exogenous BH4 (40 mM). Cells were cultured for 24 h under hypoxia, and cellular viability was examined by CCK8 assay at 48 h post hypoxia treatment. (E and F) HT29 cells with PTPS depletion and reconstituted expression of WT rPTPS, rPTPS T58A, or rPTPS R25Q (E) or HT29 cells with PTPS depletion and reconstituted expression of WT rLTBP1 or rLTBP1 2C/S (F) were added with TGF-b antibody. Cells were cultured for 24 under hypoxia, and then cellular viability was examined by CCK8 assay at 48 h post hypoxia treatment. The values are presented as mean ± SEM (n = 3 independent experiments). *p < 0.05, **p < 0.01, and #p > 0.05 between indicated groups. See also Figure S5.

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Inhibition, Expressing, Transfection, shRNA, Cell Culture, CCK-8 Assay

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 6. PTPS-T58 Phosphorylation Promotes Tumorigenesis and Displays High Levels in the Early Stage of Colorectal Cancer (A and B) HT29 cells with PTPS depletion and reconstituted expression of the WT rPTPS or rPTPS T58A were transfected with control shRNA or LTBP1 shRNA (A). HT29 cells with LTBP1 depletion were reconstituted with expression of the WT rLTBP1 or rLTBP1 2C/S (B). A total of 3 3 106 HT29 cells were subcutaneously injected into the athymic nude mice. Representative tumor xenografts were shown (left panel). Tumor volumes were measured by using length ‘‘a’’ and width ‘‘b’’ and calculated using the following equation: V = ab2/2. Data represent the means ± SEM (n = 6, right panel). (C) Immunohistochemical staining with anti-PTPS pT58 was performed on 161 human colorectal cancer specimens. Representative photos of early stage and late stage tumor versus the adjacent normal tissues were shown (magnification: 3100 and 3400). Scale bars: 100 mm. (D) The t test indicating significant difference of PTPS pT58 level between tumor and the adjacent normal tissues of (the upper panel). The chi-square test indicating significant association of high PTPS pT58 level with the number of early and non-metastasis tumors (two middle panels). The t test indicating significant difference of tumor size between low PTPS pT58 level-group and high PTPS pT58 level-group (bottom panel). (E) Immunohistochemical staining with anti-LTBP1 was performed on colorectal cancer specimens. Representative photos of early stage and late stage tumor versus the adjacent normal tissues were shown (magnification: 3100 and 3400). Scale bars: 100 mm. (F) The t test indicating significant difference of LTBP1 level between tumor and the adjacent normal tissues of (the upper panel). The chi-square test indicating significant association of low LTBP1 level with the number of early and non-metastasis tumors (two middle panels). The t test indicating significant difference of tumor size between low LTBP1 level-group and high LTBP1 level-group (bottom panel).

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Phospho-proteomics, Expressing, Transfection, Control, shRNA, Injection, Immunohistochemical staining, Staining

Journal: Molecular cell

Article Title: PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

doi: 10.1016/j.molcel.2019.09.018

Figure Lengend Snippet: Figure 7. The Diagram Displaying the Regu- lation of LTBP1-TGF-b Signaling by PTPS PTPS protein level is upregulated in early colo- rectal tumors. Hypoxia-induced AMPK activation leads to PTPS T58-phosphorylation, which pro- motes PTPS-LTBP1-iNOS complex formation. Within the complex, BH4 production mediated by PTPS facilitates iNOS-dependent LTBP1 S-nitro- sylation and the subsequent LTBP1 degradation mediated by FBXO6. In consequence, the negative regulation of LTBP1 by PTPS under hypoxia prevents LTBP1-TGF-b secretion and TGF- b-induced growth arrest of tumor cells and thus drives tumorigenesis during early stage of tumor development.

Article Snippet: For immunoprecipitation of endogenous PTPS, PTPS antibody from Santa Cruz Biotechnology (sc-514628, 1:300) were used for IP and antibody from proteintech (#12150-1-AP, 1:1000) were used for immunoblotting.

Techniques: Activation Assay, Phospho-proteomics