Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and integrin b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or a5 subunits (E). See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Expressing, Immunoprecipitation, Control, Silver Staining, Mass Spectrometry, Stable Transfection

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 5. Tinagl1 Inhibits integrin/FAK and EGFR Signaling Pathways (A) Gene set enrichment analysis of lung metastatic nodules formed by LM2 cells stably expressing the vector control or Tinagl1 were dissected and digested. n = 3 per group. (B) Heatmap representation of microarray data displaying the expression of EGFR or integrin/FAK regulated genes in the control versus Tinagl1-expressing LM2 cells. (C) Heatmap representation of microarray data displaying the expression of genes compensated by integrin/FAK (left) or EGFR (right) in control versus Tinagl1- expressing LM2 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Stable Transfection, Expressing, Plasmid Preparation, Control, Microarray

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 6. Tinagl1 Inhibits EGFR Dimerization and Blocks the Interaction between the Integrin b1 Subunit and FN (A) LM2 cells were transfected with plasmids to overexpress GFP-EGFR and EGFR-Myc. 48 hr after transfection, the cells were treated with or without 1 mg/mL of r-Tinagl1 for 1 hr, followed by 10 min of 1 ng/mL EGF treatment. The cells were then collected and immunoprecipitated with either IgG or anti-Myc antibody. IP samples were subjected to WB analysis (top), and the amount of EGFR-GFP that interacts with EGFR-Myc was quantified and normalized to the PBS treatment group (bottom). (B) LM2 cells stably expressing EGFR-Myc were pre-treated with PBS or 1 mg/mL of r-Tinagl1 for 1 hr and then treated with 1 ng/mL of EGF for another 10 min. Next, the cells were collected and the dimers were crosslinked with disuccinimidyl suberate (DSS) treatment, followed by WB analysis (top) and quantification of the ratio of dimerized EGFR in each treatment group (bottom). (C) HEK293T cells overexpressing the integrin b1 subunit were lysed. 20 mg of FN was added into the lysate, and the lysate was divided into eight groups. PBS or the indicated amount of proteins were added into each group followed by IP with IgG or anti-b1 antibody. The IP samples were then subjected to WB analysis. (D) HEK293T cells overexpressing both integrin b1 subunit and Tinagl1-HA were lysed and divided into eight groups. PBS or the indicated amount of proteins were added into the lysate, followed by IP and WB analysis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Transfection, Immunoprecipitation, Stable Transfection, Expressing

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 7. Tinagl1 Inhibits TNBC Progression by Simultaneously Targeting the Integrin/FAK and EGFR Signaling Pathways (A) 104 LM2 cells was injected into the MFP of NSG mice. Mice were intravenously treated with the indicated reagents twice per week when tumors reached 2 mm in diameter. n = 6 mice per group. (B) WB analysis for the activation of EGFR and FAK in primary tumor of each group after 5 weeks of the treatments as in (A). (C) Quantification of tumor volumes of each treatment group of (A). n = 12 tumors per group. (D and E) Lungs were collected and spontaneous metastasis was examined by ex vivo BLI at the endpoint. n = 6 lungs per group. Representative lungs (D) and quantitative data (E) is shown. Data represent means ± SEM. n.s., not significant; p > 0.05, **p < 0.001, ***p < 0.0001. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Injection, Activation Assay, Ex Vivo

Journal: Molecular and cellular endocrinology

Article Title: SDHB and SDHD silenced pheochromocytoma spheroids respond differently to tumour microenvironment and their aggressiveness is inhibited by impairing stroma metabolism.

doi: 10.1016/j.mce.2022.111594

Figure Lengend Snippet: Fig. 1. Characterization of stable SDHB and SDHD silenced cell lines. (A) Representative blots of the expression of mitochondrial SDHA, B and D subunits. (B) Densitometric analysis of western blot bands, performed by Bio-Rad imaging and analysis software (Quantity One), showed significant differences in the SDHB and SDHD subunit expression levels in SDHB (light grey) and in SDHD silenced cells (dark grey) respectively, compared to Wt (black). Bars are the means of three independent preparations ± SD, ***p < 0.001. (C) Representative traces of SDH enzymatic activity measured in cell homogenates. The silenced SDHB and SDHD cells (dotted and continuous lines, respectively) showed a similar decrease of the SDH activity, significantly different compared with Wt (dashed line). (D) Histogram represents the SDH activity expressed as the percentages. SDHB and SDHD silenced cells (light and dark grey, respectively) showed a significantly decreased of SDH activity compared to Wt (black). Bars are the means of three independent experiments (each of them conducted in duplicate samples) ± SD, ***p < 0.01. (E) The bar graph represents the means of intracellular succinate/fumarate ratio ± SD, measured by GC/MS, in three independent experiments with two replicates. SDHB silenced cells (light grey) showed a significant increase of the metabolites ratio compared with both Wt (black) and to SDHD silenced cells (dark grey), *p < 0.05, **p < 0.01. (F) Representative immunoblot of HIF1α expression in Wt, SDHB and SDHD silenced cells. (G) Optical density analysis of western blot bands. Actin im munoblots was used as loading control. For all the analyses One-way ANOVA post-test Bonferroni was used.

Article Snippet: Bound antibodies were detected using ECL reagents (Immobilon) and analysed with a Bio-Rad ChemiDoc Imaging System (Quantity One) for dedicated chemiluminescent image acquisition.

Techniques: Expressing, Western Blot, Imaging, Software, Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 1 Decreased PICT1 protein expression in ATII cells in emphysema patients. Lung tissue and ATII cells were obtained from control non-smoker (N) and smoker (S) organ donors and emphysema patients (E). Panel I: A—PICT1 mRNA levels in lung tissue by RT-PCR. B—Representative Western blot images of PICT1 expression in lung tissue. C—Quantification of protein expression normalized to β-actin is shown. Panel II: A—PICT1 mRNA levels in ATII cells by RT-PCR. B—Representative Western blot images of PICT1 expression in ATII cells. C—Quantification of protein expression. Panel III: A – PICT1 was immunoprecipitated in lung tissue, followed by mass spectrometry analysis. Representative PICT1 (A) and TRIM22 spectrum (B) are shown. C TRIM22 mRNA expression in ATII cells by RT-PCR. D ATII cells were stained in lung tissue sections using SP-C (magenta), PICT1 (red), and TRIM22 (green) antibodies and DAPI (blue) followed by analysis by immunofluorescence (scale bar—5 μm). PICT1 fluorescence intensity in the nucleus (E) and cytoplasm (F) was quantified. G The ratio of nuclear to cytoplasmic PICT1 fluorescence intensity. H Pearson’s correlation coefficient for PICT1 and MRE11 fluorescence co-localization in ATII cells. Data are shown as means ± SEM (N = 3—14 lungs per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Mass Spectrometry, Staining, Immunofluorescence, Fluorescence

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 3 Decreased MRE11 protein levels in ATII cells in a murine model of emphysema. Wild-type mice were exposed to cigarette smoke for 8 months, as described in the Methods section, to induce emphysema. A Hematoxylin and eosin staining in murine lung tissue (scale bar 50 μm). Minimum (B), maximum (C), and mean (D) alveolar diameters were measured in lung tissue sections. E Representative micro-CT of the murine lung (scale bar-100 μm). F The intersection surface was quantified using micro-CT images. G Pict1 and H Mre11 mRNA levels were evaluated in lung tissue by RT-PCR. I Representative Western blotting images of PICT1 and MRE11 expression in lung tissue. PICT1 (J) and MRE11 expression (K) are quantified. L ATII cells in lung tissue sections were identified using SP-C (green). PICT1 (magenta), and MRE11 (red) antibodies, and DAPI (blue) by immunofluorescence (scale bar—5 μm). Quantification of PICT1 (M) and MRE11 (N) fluorescence intensity in ATII cells is shown. O Pearson’s correlation coefficient for PICT1 and MRE11 fluorescence co-localization. Data are shown as means ± SEM (N = 3 – 8 mice per group). p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques: Staining, Micro-CT, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 6 Mitochondrial dysfunction in human primary ATII cells, A549 cells, and MLE15 cells. A PICT1 (red), TOM20 (green), and DAPI (blue) staining in lung tissue sections obtained from non-smokers (N), smokers (S), and emphysema patients (E) by immunofluorescence (scale bar—5 μm). ATII cells were identified using SP-C (magenta). B Pearson’s correlation coefficient for PICT1 and TOM20 co-localization in ATII cells is shown (N = 3 lungs per group). C Quantification of mitochondrial networks in ATII cells. D Mitochondrial respiration analysis in wild-type A549 cells and cells with PICT1 deletion treated with 20% cigarette smoke extract (CSE) for 24 h. Quantification of basal respiration (E) and maximum respiration (F) in A549 cells. G ATP-linked respiration after exposure to CSE relative to controls in A549 cells. QPCR was used to determine mtDNA amount (H), mtDNA damage (I), and common deletions (CD, J) in A549 cells. Representative histograms using MitoSOX staining and flow cytometry analysis (K) and the quantification of fluorescence intensity (L) in A549 cells. M Representative Western blotting images of MLE15 cells treated with NT (non-target) or PICT1 siRNA. N Histograms of MitoSOX staining by flow cytometry analysis (N) and quantification (O) in MLE15 cells. Data are shown as means ± SEM (KD – knockdown, N = 3 – 10 experimental replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques: Staining, Immunofluorescence, Flow Cytometry, Fluorescence, Western Blot, Knockdown

Journal: Cell communication and signaling : CCS

Article Title: Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema.

doi: 10.1186/s12964-024-01896-0

Figure Lengend Snippet: Fig. 7 The role of PICT1 in nuclear DNA damage and mitochondrial (mt) function. Increased PICT1/TRIM22 interaction induced by smoking leads to decreased PICT1 levels and high ROS production. This caused nuclear and mtDNA damage, common deletions, mitochondrial superoxide generation, and reduced mtDNA amount and respiration, contributing to ATII cell death and emphysema development

Article Snippet: PICT1 knockout A549 cell line was generated using PICT-1 CRISPR plasmid (Santa Cruz Biotechnology) and CRISPR-Cas9 technology, as we previously described [18].

Techniques:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 1 Identification of potential genes implicated in colorectal cancer (CRC) and cancer metabolism-associated biological processes. (A) A screening procedure to find putative gene candidates. (B) Colorectal cancer (CRC) samples were found to differ from adjacent controls in terms of physiopathology and biological processes related to metabolism in a number of databases, including TCGA, ICGC, and the NCBI Gene Expression Omnibus (GEO) datasets (GEO: GSE254054, GSE231943, GSE252858, GSE234804, GSE236678, GSE231436, GSE197088, and GSE239549). (C) Following gene differential expression analysis, the total number of differentially expressed genes that crossed over into various databases was counted. (D) Six upregulated and four down regulated DEGs were identified based on a survival analysis of differentially expressed genes across six databases.In the databases of TCGA and ICGC, P < 0.05 was deemed statistically significant. (E) Six upregulated and four downregulated DEGs represent the molecular mechanisms impacting the onset of colorectal cancer and metabolic reprogramming. (F) Palmitoyltransferase ZDHHC6 expression in the ICGC and TCGA databases. (G) Pancarcinoma analysis using TCGA datasets to measure ZDHHC6 expression levels in various malignancies. (H) The overall survival (OS) of colorectal cancer patients in the TCGA and ICGC databases according to different ZDHHC6 expression levels. (I) After dividing the TCGA and ICGC samples’ ZDHHC6 expression levels into groups of high and low expression levels, the grouped samples underwent GSEA analysis. The data were expressed as the mean ± SEM. A P value less than 0.05 was considered statistically significant. ***P < 0.001

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Gene Expression, Quantitative Proteomics, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 2 Increased ZDHHC6 is positively associated with the development of human colorectal cancer (CRC). (A) ZDHHC6 mRNA expression levels in 73 pairs of CRC sample pairs (T) and their corresponding adjacent sample pairs (N). n = 73 pairs. (B) ZDHHC6 protein expression levels in sixteen pairs of similar adjacent tissues and colorectal cancer tissues selected at random. For each group, n = 3. (C) ZDHHC6 mRNA expression levels in relation to a range of CRC-associated cell lines, such as SNU-C2A, SW48, HT-29, LS1034, HCT116, and Caco-2, as well as the matching human normal colonic epithelial cell line (FHC), are displayed in qPCR analysis. For each group, n = 5. (D, E) ZDHHC6 protein expression in SNU-C2A, SW48, HT-29, LS1034, HCT116, Caco-2, and FHC cell line as demonstrated by western blotting (D) and immunofluorescence analysis (E). 200 μm; each group has n = 5. (F, G) qPCR analysis (F) and western blotting experiment (G) demonstrate the effect of the gradually increased dosage of 2-bromopalmitate (2-BP) on the relative ZDHHC6 mRNA and protein expression levels in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. For each group, n = 3. (H) An immunofluorescence assay demonstrating the co-expression of ZDHHC6 and Ki67 in response to 40 µM 2-bromopalmitate (2-BP) in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. 200 μm; each group has n = 3. Data are expressed as mean ± SEM. The relevant experiments presented in this section were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 4 ZDHHC6 facilitates lipid deposition and carcinogenesis in CRC cells. (A) A venn diagram shows the variations in metabolites produced by HCT116 cells with ZDHHC6 knockout (KO) and wild-type (WT) phenotypes. ZDHHC6 and fatty acid synthesis pathways have a significant association, according to pathway enrichment analysis of the 36 metabolites. Total peak area was used to correct the LC-MS-based untargeted metabolomic study and its findings. (B) Using these 36 differential metabolites, pathway analysis showed enhanced signaling pathways. (www.metaboanalyst.ca). (C) A heatmap showing how these 36 significantly altered metabolites changed. Student’s t-test, unpaired, two-tailed, P < 0.05. The fold change is indicated by -2.0 ~ 2.0 (Fc). (D, E) The ratios of various isotopic forms of FFA C16:0 (palmitate) in ZDHHC6 (KO) (D) and AdZDHHC6 (E) HCT116 cells after a brief exposure to glucose [U-13C]. When the cell density was around 85%, the media was changed to RPMI 1640 containing 2 g/L glucose tagged with [U-13C]. Following a 24-hour period, the PBS-rinsed cell culture plates were quickly frozen in liquid nitrogen and subjected to an LC-MS assay analysis (n = 4 per group). (F) Representative im munofluorescence pictures of HCT116 cells with ZDHHC6 (WT) and ZDHHC6 (KO) phenotypic, demonstrating ZDHHC6 expression, lipid accumulation (Bodipy staining), and corresponding intracellular triglyceride (TG) levels (n = 4 per group). (G, H) ZDHHC6 (WT) and ZDHHC6 (KO) HCT116 cells were injected into the right flanks of nude mice. Every two days, tumor volumes were measured. On day 22 following dissection, tumor pictures (G), growth curves, and weight (H) were recorded (n = 4 per group). Scale bars, 1 cm. (I) A heatmap utilizing untargeted metabolomic analysis comparing significantly changed metabolites between tumors originating from ZDHHC6 (KO) HCT116 cells and ZDHHC6 (WT) cell lines. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Produced, Knock-Out, Liquid Chromatography with Mass Spectroscopy, Protein-Protein interactions, Two Tailed Test, Cell Culture, Expressing, Staining, Injection, Dissection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 5 ZDHHC6 specifically binds to the lipid metabolism key transcription factor of PPARγ. (A) After 24 h of SFB-ZDHHC6 transfection in HCT116 cells, ZDHHC6-interacting proteins were identified by tandem affinity purification and mass spectrometry (MS). This was accomplished by removing S-protein, Flag, and streptavidin binding peptide (SFB). (B) ZDHHC6 or IgG antibodies were used to immunoprecipitate HCT116 cell lysates, and PPARγ, PPARα, PPARδ, SREBP1, and ZDHHC6 antibodies were used for western blotting experiments. (C) ZDHHC6 or IgG antibodies were used to immunoprecipitate cellular lysates of SNU-C2A, SW48, HT-29, LS1034, and Caco-2 cells, and ZDHHC6 or PPARγ antibodies were used for western blotting experiments. (D) GST pulldown assay using GST-PPARγ and purified His-ZDHHC6 in HCT116 cells. (E) Schematic of the experimental procedure showing the genes expression in HCT116, Caco-2, SNU-C2A and HT-29 after adenovirus-mediated ZDHHC6 overactivation (AdZDHHC6). The lower schematic diagram showing the inter section of the results from the proteomics and IP-MS analyses. (F) For a duration of 24 h, plasmids expressing Flag-PPARγ or Myc-ZDHHC6 individually or in combination were transfected into HCT116, Caco-2, SNU-C2A and HT-29 cells, respectively. His or Flag antibodies were used for immunoblotting after cellular lysates had been immunoprecipitated with Flag and/or His antibodies. (G) GST pulldown assay using GST-PPARγ and purified Flag-ZDHHC6 in Caco-2 and SNU-C2A cells, respectively. (H) Assay for immunofluorescence staining demonstrating ZDHHC6 and PPARγ co-expression in HCT116, Caco-2, and SNU-C2A cells. 20 μm. (I) In HCT116 cells, vectors containing the hinge-LBD domain, full length (FL), AF-1, DBD, and PPARγ were co-expressed with SFB-ZDHHC6. S-bead pulldown was used to immunoprecipitate cellular lysates. (J) Based on GSEA signaling pathway analysis, an assay of the TCGA-CRC and ICGC-CRC datasets showed a significant connection between ZDHHC6 and the PPARγ pathway in CRC. Data are expressed as mean ± SEM. The rel evant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Transfection, Affinity Purification, Mass Spectrometry, Binding Assay, Western Blot, GST Pulldown Assay, Purification, Expressing, Protein-Protein interactions, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 6 Identification of the palmitoylation site on PPARγ at evolutionarily conserved cysteine residues. (A) For a duration of 24 h, HCT116 cells were exposed to 60 µM 2-BP, 1 µM ABD957, 6 µM palmostatin B (Palm B), and 10 µM palmostatin M (Palm M) treatments. The slices that were fixed underwent immunofluorescence labeling using PPARγ (red) and pan-palmitoylation (green). 10 μm scale bars; n = 5 per group. (B) Schematic diagram of the Click-iT assay for palmitoylation measurement of PPARγ. HCT116 cells were treated with 100 µM Click-iT PA and azides for five hours. The resulting lysates were then submitted to Click-iT detection as per the product instructions, and PPARγ antibody western blotting analysis was performed. The indicated group’s expression of PPARγ is indicated by the western blotting bands on the right. (C) Using the GPS-Palm program (MacOS_20200219) (The CUCKOO Work group, http://gpspalm.biocuckoo.cn/) and the MDD-Palm algorithm (http://csb.cse.yzu.edu.tw/MDDPalm/), the palmitoylation site on PPARγ in Homo sapiens (upper) and Mus musculus (lower) is predicted to be located. PPARγ’s lower palmitoylation site contains conserved cysteine residues shared by Rattus norvegicus, Bos taurus, Canis familiaris, Mus musculus, and Homo sapiens. (D) After incubating Click-iT PA and azides for five hours on HCT116 cells overexpressing either PPARγ WT or PPARγ C313S mutant, the corresponding cellular lysates were obtained and Click-iT detection was performed in com pliance with the product’s instructions. After the palmitoylated proteins were added to the streptavidin-sepharose bead conjugate for pull-down detec tion, PPARγ and ACTIN antibodies were used in a western blotting examination. While PPARγ C313S was not palmitoylated in top gel, lane 6, or the control groups, it was for PPARγ WT in lane 5. Three separate runs of this experiment were conducted. (E) CHX was cultured with HCT116 cells overexpressing either the PPARγ WT or PPARγ C313S mutant for a specific amount of time. PPARγ and ACTIN antibodies were used in immunoblotting detection of the obtained cellular lysates. The relative PPARγ remaining ratio (n = 4 per group) is displayed in the right curve graph at the specified time point. (F) PPARγ WT or PPARγ C313S mutant overexpression was observed in the upper HCT116 cells. Pan-palmitoylation (green) and PPARγ (red) immunofluorescent label ing were applied to the cell sections. Lower, AdZDHHC6 + PPARγ C313S mutant or PPARγ C313S alone were overexpressed in HCT116 cells, respectively. The bar graph displays the intensity of PPARγ fluorescence in each of the indicated groups (n = 5 pictures; P < 0.05 vs. PPARγ C313S + AdControl or PPARγ WT). Scale bars, 20 μm. (G) In HCT116 cells, PPARγ-Flag and ZDHHC6-HA plasmids were transfected. Alk16 labeling was used to determine the palmi toylated PPARγ expression contents in the presence or absence of hydroxylamine therapy. (H) PPARγ-Flag was used to transfect SNU-C2A cells (WT) or ZDHHC6-deleted SNU-C2A cells, and Alk16 was used to label the cells. Subcellular fraction was extracted, and the levels of PPARγ protein were adjusted to verify that the input cells from the wild type and the knockout cell had the same quantity of PPARγ. Immunoblotting analysis was used to evaluate the palmitoylated PPARγ expression contents in the cell membrane (Mem.), cell cytoplasm (Cyto.), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Immunofluorescence, Labeling, Western Blot, Expressing, Mutagenesis, Control, Cell Culture, Over Expression, Fluorescence, Transfection, Knock-Out, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 7 ZDHHC6-mediated palmitoylated PPARγ enhances its nucleus translocalization. (A) ZDHHC6 and PPARγ expression were examined in the ZDH HC6-deleted HCT116, SNU-C2A and SW48 cells, respectively (n = 3 per group). (B) ZDHHC6 and PPARγ co-expression in AdshZDHHC6-transfected HCT116 cells, along with the matching fluorescence density as determined by Pearson’s analysis (n = 4 per group; P < 0.05 vs. AdshRNA). The scale bars are 20 μm. (C) In ZDHHC6-deleted HCT116 or ZDHHC6-deleted SW48 cells, palmitoylation levels and PPARγ expression were analyzed using western blotting assay (n = 4 per group). (D) Western blotting assay using PPARγ, ACTIN, and HA antibodies, followed by PPARγ overexpressing the HA-tagged ZDHHC6 construct in various CRC cell lines (n = 3 per group). (E) Immunofluorescence pictures demonstrating the co-expression of PPARγ and ZDHHC6 in ZDHHC6-overex pressed HCT116 cells, together with the matching fluorescence density as determined by Pearson’s analysis (n = 4 per group; P < 0.05 compared to empty vector). The scale bars are 20 μm. (F) HCT116 cells underwent IP of HA after co-transfecting with PPARγ and HA-ZDHHC6. ZDHHC6 and PPARγ Mutual Co-IP shows that endogenous ZDHHC6 and PPARγ bind to each other in HCT116 cells. (G) Using various alkyl-labeled fatty acylation, such as alk-C14, alk- C16, alk-C18, and alk-C20, the palmitoylation of PPARγ in the indicated cells was detected. By using streptavidin bead pulldown to identify acylated PPARγ, an immunoblotting experiment using PPARγ and ACTIN antibodies (n = 6 per group) was performed. (H) To identify acylated PPARγ in SW48, LS1034, and HT-29 cells, the same methodology as in (G) was applied. Following that, the lysates (n = 6 per group) were subjected to western blotting analysis using PPARγ and ACTIN antibodies. (I) Using Click reaction-associated streptavidin pulldown, the palmitoylation levels of Flag-labeled PPARγ WT, PPARγ C313S, PPARγ C156S, PPARγ C176S, and PPARγ C159S mutants were examined. Three individuals per group underwent an immunoblotting experiment using Flag and ACTIN antibodies on the relevant lysates. (J) ZDHHC6-HA and PPARγ-Flag were the vectors used to transfect the HCT116 cells. Using alk-C16 labeling, higher, palmitoylated PPARγ levels were demonstrated in both the presence and absence of hydroxylamine therapy. The corresponding fluorescence density and ACLY and PPARγ co-expression in HCT116 WT or HCT116 ZDHHC6 (KO) cells are depicted in the lower representative immunofluorescence images, which were analyzed using Pearson’s method (n = 5 per group; P < 0.05 vs. WT). The scale bars are 20 μm. (K) After transfecting the HCT116 WT or HCT116 ZDHHC6 (KO) cells with PPARγ-Flag, the cells were labeled with alk-C16. To verify that the wild type and knockout cell components for input had the same quantity of PPARγ, subcellular fraction was obtained and PPARγ protein levels were adjusted. Western blotting analysis was used to assess palmitoylated PPARγ levels in the cell membrane (Mem.), cell cytoplasm (Cyto. ), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Transfection, Fluorescence, Western Blot, Construct, Immunofluorescence, Plasmid Preparation, Co-Immunoprecipitation Assay, Labeling, Knock-Out, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 9 ZDHHC6-driven lipid biosynthesis contributes to CRC carcinogen esis by upregulating PPARγ. (A, B) In HCT116-related stable cells (Control, ZDHHC6, and ZDHHC6 + shPPARγ) (A) and HCT116-related stable cells (shControl, shZDHHC6, and shZDHHC6 + PPARγ) (B), the percentages of different isotopomers of FFA C16:0 following exposure to [U-13C] glucose are shown. Each group has n = 5. (C, D) The relative TG content and PPARγ expression abundance in the aforementioned cell lines from (A) and (B) are displayed in representative immunofluorescence pictures. Each group has n = 5. The scale bars are 20 μm. (E) In null mice, right flanks were in jected with ZDHHC6 + shPPARγ, ZDHHC6, and Control, stable cells related to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n = 5. (F) The right flanks of null mice were injected with shCon trol, shZDHHC6, and shZDHHC6 + PPARγ, stable cells linked to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n = 5. (G) Kaplan-Meier curves representing the survival analysis based on TCGA CRC prognostic data for ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-positive patients. (H) Based on the prognosis information from the ICGC CRC database, Kaplan-Meier curves were used to analyze the sur vival of ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-posi tive patients. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Control, Expressing, Immunofluorescence, Dissection, Injection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 10 Palmitoylation stabilizes PPARγ by ZDHHC6 via blocking its lysosomal degradation to promotes lipid biosynthesis-associated CRC development. As a palmitoyltransferase enzyme, ZDHHC6 regulates the synthesis of fatty acids. To be more precise, ZDHHC6 directly attaches palmitoyl groups to PPARγ, a protein that controls the expression of genes. By stabilizing PPARγ and blocking its lysosomal degradation, the palmitoylation mechanism triggers the production of ACLY and subsequently leads to the development of lipid buildup-related CRC carcinogenesis

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Blocking Assay, Expressing

Journal: Oncotarget

Article Title: Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation.

doi: 10.18632/oncotarget.2639

Figure Lengend Snippet: Figure 1: Overexpression of cPLA2α increases p-AKT and cell proliferation in DLD-1 cells. (A) Immunoblot in DLD-1 cells stably transfected with cPLA2α (DLD-1/cPLA2α) or empty vector (DLD-1/CMV) with or without EGF treatment (20 ng/mL, 30 min). (B) Densitometry quantification of (A). *P<0.05 vs. DLD-1/CMV, #P<0.05 vs. DLD-1/CMV+EGF, ^P<0.05 DLD-1/cPLA2α vs. DLD-1/ cPLA2α+EGF, n=3. (C) Arachidonic acid concentration in intracellular compartments and the supernatant measured by Mass Spectrometer. *P <0.05 vs. DLD-1/CMV. (D) DNA content analysis by PI-Flow cytometry. *P <0.05 vs. DLD-1/CMV, n=3. All data was expressed as Mean ± SD.

Article Snippet: Antibodies against cPLA2α (Cat. #: SC-454) and phospho- Oncotarget12312www.impactjournals.com/oncotarget cPLA2α at Ser 505 (Cat. #: SC-34391), AKT (Cat. #: SC8312) and phospho-AKT at Ser473 (Cat. #: SC-7985) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX); Anti-Ki-67 (Cat. #: RM-9106) was from Thermo Fisher Scientific (Scoresby, VIC, Australia); Human EGF (Cat. #: E9644), BrdU (Cat. #: B5002), propidium iodide (Cat. #: P4170), antibody against BrdU (Cat. #: B8434) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Over Expression, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Concentration Assay, Mass Spectrometry, Flow Cytometry

Journal: Oncotarget

Article Title: Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation.

doi: 10.18632/oncotarget.2639

Figure Lengend Snippet: Figure 2: Silence of cPLA2α decreases EGF-stimulated p-AKT and cell proliferation in HT-29 cells. (A) immunoblot and (B) quantification of cPLA2α and AKT in cells transfected with cPLA2α siRNA or scramble control (10 nM for 72 h) with or without EGF treatment (20 ng/mL, 30 min). *P <0.05 vs. cells transfected with scramble control without EGF; #P <0.05 vs. cells transfected with scramble control with EGF. ^P <0.05 cPLA2α siRNA vs. cPLA2α siRNA+EGF. (C) Arachidonic acid concentration in the intracellular and supernatant compartments measured by Mass Spectrometry. *P <0.05 vs. cells transfected with scramble control. (D) DNA content analysis by PI-Flow cytometry. *P <0.05 vs. cells transfected with scramble control.; (E) Immunoblot of HT-29 cells treated with 25 µM Efipladib for 72 h and/or 20 ng/mL EGF for 30 min before harvesting. (F) Densitometry quantification. *P <0.05 vs. DMSO, #P <0.05 vs. DMSO+EGF, n=3. All data expressed as Mean ± SD.

Article Snippet: Antibodies against cPLA2α (Cat. #: SC-454) and phospho- Oncotarget12312www.impactjournals.com/oncotarget cPLA2α at Ser 505 (Cat. #: SC-34391), AKT (Cat. #: SC8312) and phospho-AKT at Ser473 (Cat. #: SC-7985) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX); Anti-Ki-67 (Cat. #: RM-9106) was from Thermo Fisher Scientific (Scoresby, VIC, Australia); Human EGF (Cat. #: E9644), BrdU (Cat. #: B5002), propidium iodide (Cat. #: P4170), antibody against BrdU (Cat. #: B8434) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Western Blot, Transfection, Control, Concentration Assay, Mass Spectrometry, Flow Cytometry

Journal: Oncotarget

Article Title: Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation.

doi: 10.18632/oncotarget.2639

Figure Lengend Snippet: Figure 3: Pharmacological blockade of cPLA2α by Efipladib results in decreased cell proliferation. DLD-1 (A) or HT- 29 cells (B) were plated in 96-well plates and treated with vehicle control (DMSO) or Efipladib for 72 h. The viable cell number was determined by the MTS assay. DLD-1 (C) or HT-29 (D) cells were plated in 6-well plates and treated with control (DMSO) or Efipladib for 72 h. BrdU was added for 3 h prior to harvesting. BrdU incorporation was determined by immunocytochemistry. Percentage of BrdU positive cells was determined as the average of 10 high-power fields (X40) per sample. *P <0.05 vs. vehicle-treated control, n=3. (E) DLD- 1 cells were treated with Efipladib at 25 µM for 1 or 2 days, followed by staining with PI and subsequent analysis with flow cytometry. *P<0.05 vs. vehicle-treated control, n=3. (F) HT-29 cells were treated with Efipladib at indicated doses for 3 days, followed by PI-staining and DNA content analysis. *P<0.05 vs. vehicle-treated control, n=3. All data expressed as Mean ± SD.

Article Snippet: Antibodies against cPLA2α (Cat. #: SC-454) and phospho- Oncotarget12312www.impactjournals.com/oncotarget cPLA2α at Ser 505 (Cat. #: SC-34391), AKT (Cat. #: SC8312) and phospho-AKT at Ser473 (Cat. #: SC-7985) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX); Anti-Ki-67 (Cat. #: RM-9106) was from Thermo Fisher Scientific (Scoresby, VIC, Australia); Human EGF (Cat. #: E9644), BrdU (Cat. #: B5002), propidium iodide (Cat. #: P4170), antibody against BrdU (Cat. #: B8434) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Control, MTS Assay, BrdU Incorporation Assay, Immunocytochemistry, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation.

doi: 10.18632/oncotarget.2639

Figure Lengend Snippet: Figure 4: Pharmacological blockade of cPLA2α by Efipladib impedes the growth of DLD-1 xenografts and decreases p-AKT levels in vivo. (A) DLD-1 cells were inoculated into the flanks of nude mice. When xenograft tumours had reached 50 mm3 in volume, mice were randomised to control (n=7) or Efipladib treatment (7 mice/group) at a dose of 10 mg/kg i.p. daily for 14 days. Inhibition of tumour growth in the Efipladib-treated mice compared with the controls (p<0.001 by two way ANOVA with repeat measurement). *p<0.05 vs. control at the same day. (B) The fraction of Ki-67 positive cells was determined from the average number of positive cells in 10 high-power fields (×40). *p < 0.05 vs. control. (C) Xenografts were harvested, fixed and paraffin-embedded, and stained for Ki- 67 by immunohistochemistry. Scale bar = 50 µm, magnification 200×. (D) Immunoblot of DLD-1 xenograft tumour and densitometry quantification. *p<0.05 vs. control, n=3. All data expressed as Mean ± SD.

Article Snippet: Antibodies against cPLA2α (Cat. #: SC-454) and phospho- Oncotarget12312www.impactjournals.com/oncotarget cPLA2α at Ser 505 (Cat. #: SC-34391), AKT (Cat. #: SC8312) and phospho-AKT at Ser473 (Cat. #: SC-7985) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX); Anti-Ki-67 (Cat. #: RM-9106) was from Thermo Fisher Scientific (Scoresby, VIC, Australia); Human EGF (Cat. #: E9644), BrdU (Cat. #: B5002), propidium iodide (Cat. #: P4170), antibody against BrdU (Cat. #: B8434) were from Sigma-Aldrich (St. Louis, MO).

Techniques: In Vivo, Control, Inhibition, Staining, Immunohistochemistry, Western Blot

Journal: Oncotarget

Article Title: Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation.

doi: 10.18632/oncotarget.2639

Figure Lengend Snippet: Figure 5: Immunohistochemical analysis of total and phospho-cPLA2α at Ser505 in human CRC tissue array. Inset AC: normal colon mucosa exhibited relatively low levels of total cPLA2α (A) and phospho-cPLA2α (C). Inset BD: CRC tissue had stronger total cPLA2α (B) and phospho-cPLA2α (D) in malignant epithelial cells. Low magnification 100×. Scale bar = 100 µm. High magnification 400×. Scale bar = 10 µm.

Article Snippet: Antibodies against cPLA2α (Cat. #: SC-454) and phospho- Oncotarget12312www.impactjournals.com/oncotarget cPLA2α at Ser 505 (Cat. #: SC-34391), AKT (Cat. #: SC8312) and phospho-AKT at Ser473 (Cat. #: SC-7985) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX); Anti-Ki-67 (Cat. #: RM-9106) was from Thermo Fisher Scientific (Scoresby, VIC, Australia); Human EGF (Cat. #: E9644), BrdU (Cat. #: B5002), propidium iodide (Cat. #: P4170), antibody against BrdU (Cat. #: B8434) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Immunohistochemical staining

Journal: PLoS ONE

Article Title: Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

doi: 10.1371/journal.pone.0077288

Figure Lengend Snippet: ( A ) Extracts from ZR-75 cells transfected with vectors expressing GST or LOX-PP-GST were precipitated with Glutathione-Sepharose 4B beads, resolved by SDS-PAGE and silver stained. The band(s) at ∼85 kDa was analyzed by LC-MS/MS mass spectrometry and identified as CIN85 and CD2AP. *, non-specific proteins. The positions of the co-precipitated CD2AP/CIN85 and LOX-PP proteins are indicated by the solid and large hatched arrows, respectively, and of GST by the dashed line. ( B–C ) GST or LOX-PP-GST (PP-GST) was co-expressed with GFP-CIN85 WT (B) or FLAG-CD2AP (C) in HEK293T cells, and LOX-PP associated proteins isolated by GST-pull down assays and subjected to WB for GFP (B) or FLAG (C) and GST. Input, 4% of lysates (4%). ( D ) Recombinant LOX-PP-myc-His (0.5 µM) was subjected to a GST-pull down assay using 0.5 µM of either GST or GST (G)-CIN85, and WB for the His or CIN85 (Calbiochem) antibody. Input, 5%. ( E ) Samples of whole cell extracts (10 µg) of the indicated human and mouse cells were subjected to WB for CIN85 (Upstate). ( F ) (Left) TX-100 extracts of Hs578T (Upper) or ZR-75 (Lower) cells were immunoprecipitated with mouse IgG or CIN85 (Upstate) antibody, and analyzed for CIN85 (Upstate) and LOX-PP. (Right) TX-100 extracts of Hs578T (upper) or ZR-75 (lower) cells were immunoprecipitated with rabbit IgG or LOX-PP antibodies, and subjected to WB.

Article Snippet: Antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) included GST (B-14), CD2AP (H-290), and CD2AP (B-4), normal rabbit IgG (sc-2027), and normal mouse IgG (sc-2025).

Techniques: Transfection, Expressing, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Recombinant, Pull Down Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

doi: 10.1371/journal.pone.0077288

Figure Lengend Snippet: ( A ) GST or LOX-PP-GST (PP-GST) was expressed in ZR-75 (left panel) or Hs578T (right panel) cells. GST and associated proteins were precipitated as described in and subjected to WB for CIN85 (Upstate Biotechnology and Calbiochem antibodies for ZR-75 and Hs578T cells, respectively), CD2AP (H-290), c-Cbl, AMAP1, EGFR, p130Cas and GST. Input, 4%. ( B–C ) HEK293T cells were transfected with AMAP1-FLAG, CIN85, HA-c-Cbl, HA-ubiquitin and LOX-PP-GST (PP-GST) as indicated and subjected to a ubiquitination assay. FLAG-tagged AMAP1 was immunoprecipitated and total whole cell extracts were subjected to WB with an HA antibody (upper panel), or the indicated antibodies (lower panel). (B). Data were quantified and relative mono-ubiquitination of AMAP1 with and without LOX-PP was determined by averaging the results of three independent experiments (C). P value was calculated using Student's t -test. *, P<0.03. ( D ) FLAG-CIN85 was expressed in Hs578T (left panel) or MCF-7 (right panel) cells. After lysis, the indicated amount of recombinant LOX-PP-myc-His was added and the mixture incubated at 4°C for 2 h. Proteins were then immunoprecipitated with a FLAG antibody and subjected to WB with FLAG and c-Cbl antibodies. (L Exp, longer exposure).

Article Snippet: Antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) included GST (B-14), CD2AP (H-290), and CD2AP (B-4), normal rabbit IgG (sc-2027), and normal mouse IgG (sc-2025).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Lysis, Recombinant, Incubation

Journal: PLoS ONE

Article Title: Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

doi: 10.1371/journal.pone.0077288

Figure Lengend Snippet: ( A ) Schematic representation of the full length CIN85 (WT) and the amino terminal-containing deletion mutant (NT). The SH3, Src homology 3 domains A, B, and C, PR, proline-rich region, CC, coiled-coil domain are indicated. ( B ) GST or LOX-PP-GST (PP-GST) protein was co-expressed with FLAG-tagged CIN85 WT or CIN85 NT in HEK293T cells. GST-pull down assays were performed and bound or whole cell extracts subjected to WB with FLAG and GST antibodies. ( C ) Recombinant LOX-PP-myc-His (0.5 µM) was incubated with GST, GST (G-)-tagged SH3-A, SH3-B or SH3-C peptides (0.5 µM each) and subjected to a GST-pull down assay. The precipitated proteins were analyzed by Coomassie staining (lower panel) and with antibodies against LOX-PP (upper panel). ( D ) Schematic representation of LOX-PP deletion mutants prepared in the pcDNA3-GST vector. SP, Signal peptide. The lengths of the constructs are indicated on the left. ( E–F ) HA-CIN85 was co-transfected with either LOX-PP-WT (1-162) or the indicated deletion mutants (from part D) or empty vector (EV) DNA into HEK293T cells. Extracts were prepared and samples (4%) were analyzed directly as a measure of input (E) or subjected to GST-pull down assays and WB for HA and GST (F). ( G ) Amino acid sequences of LOX-PP from various species are shown. The positions of aa 111 and aa 116 and of aa 113 and aa 117 are indicated by black and grey boxes, respectively.

Article Snippet: Antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) included GST (B-14), CD2AP (H-290), and CD2AP (B-4), normal rabbit IgG (sc-2027), and normal mouse IgG (sc-2025).

Techniques: Mutagenesis, Recombinant, Incubation, Pull Down Assay, Staining, Plasmid Preparation, Construct, Transfection

Journal: PLoS ONE

Article Title: Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

doi: 10.1371/journal.pone.0077288

Figure Lengend Snippet: ( A ) To begin to identify the critical amino acids in LOX-PP, vectors expressing individual point mutants in which residues aa 111 to aa 120 were replaced with alanine, or WT LOX-PP protein were co-transfected with FLAG-CIN85 WT in HEK293T cells. (right panel) The mutated proteins compared with the WT LOX-PP for their ability to interact with CIN85 using GST-pull down assays and WB with FLAG and GST antibodies. (left panel) Input, 4%. EV, empty vector DNA. ( B ) To confirm the interaction of CIN85 and endogenous c-Cbl, GFP or GFP-CIN85 proteins were expressed in HEK293T cells and TX-100 extracts prepared. Following immunoprecipitation with a GFP antibody, the precipitated proteins subjected to WB with antibodies against c-Cbl (upper panel) and GFP (lower panel). ( C ) To test whether LOX-PP mutants unable to interact with CIN85 compete for its binding with c-Cbl, GFP-tagged-full-length CIN85 was co-expressed with GST, or GST-tagged LOX-PP-WT (PP-WT-G), LOX-PP-P111A (PP-P111A-G), or LOX-PP-R116A (PP-R116A-G) in HEK293T cells. TX-100 extracts were immunoprecipitated with GFP antibody and the precipitated proteins detected with antibodies against c-Cbl, GFP and GST. Input, 4%. ( D ) To test the specificity of the changes in binding of the mutant LOX-PP proteins, vectors expressing GST tagged LOX-PP-WT (WT), or mutants LOX-PP-P111A (P111A) or LOX-PP-R116A (R116A) or GST (EV) were transfected into ZR-75 cells and their ability to interact with c-Raf, which maps to aa 26-100, monitored by GST-pull down assays. WB for CIN85 (Upstate: clone 84), c-Raf and GST was performed.

Article Snippet: Antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) included GST (B-14), CD2AP (H-290), and CD2AP (B-4), normal rabbit IgG (sc-2027), and normal mouse IgG (sc-2025).

Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Mutagenesis

Journal: PLoS Biology

Article Title: The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

doi: 10.1371/journal.pbio.1001635

Figure Lengend Snippet: (A) Recombinant hnRNP L by itself does not drive the VEGFA RNA switch and restore in vitro translation of the GAIT-element-bearing reporter. In vitro translation of capped and poly(A)-tailed firefly luciferase ( FLuc )- VEGFA HSR-A 30 reporter transcript was determined in a wheat germ extract in the presence of [ 35 S]Met, cytosolic extracts from IFN-γ-treated U937 cells, and recombinant hnRNP L. FLuc expression was determined by activity assay, normalized by RLuc expression, and reported as mean ± standard deviation (SD, n = 3). (B) Schematic of HSR in VEGFA 3′UTR. CARE (red), GAIT element (green), extended CARE (CARE-E, dotted line), AUSL-A (dashed line), and AUSL-D (dashed and dotted line) are indicated. (C) Mass spectrometric analysis of CARE-binding proteins. U937 cells were treated with normoxia (Nmx.) or hypoxia (Hpx.) for 24 h and the S100 extracts, precleared, and incubated with biotinylated CARE-E (extended CARE, sequences in ) and magnetic streptavidin microbeads. Specifically bound proteins were subjected to SDS-PAGE and Coomassie staining. Bands specifically enriched in affinity-purified lysates from hypoxia-treated cells were trypsinized, and peptide sequences of hnRNP L, DRBP76, and hnRNP A2/B1 detected by mass spectrometry. (D) Hypoxia-inducible binding of hnRNP L, DRBP76, and hnRNP A2/B1 to CARE. Cells were treated with Nmx. or Hpx. for 24 h, and the precleared S100 extracts incubated with biotinylated, wild-type, or antisense (A.S.) CARE-E, and then with magnetic streptavidin microbeads. Specifically bound proteins were subjected to immunoblot analysis. (E) DRBP76 and hnRNP A2/B1 form a complex with hnRNP L in vivo . Cells were treated with IFN-γ in Nmx. or Hpx. for 24 h. Cell lysates were incubated with or without RNase A, immunoprecipitated with anti-hnRNP L antibody, and subjected to immunoblot analysis (left panel). Total expression of hnRNP L, hnRNP A2/B1, and DRBP76 was determined by immunoblot as input control (right panel). (F) Interprotein interactions of HILDA constituents. Recombinant hnRNP A2/B1 and DRBP76 were incubated with GST-hnRNP L or GST immobilized to glutathione (GSH)-agarose beads. After washing, binding was detected by immunoblot (left). Recombinant hnRNP L and DRBP76 were incubated with GST-hnRNP A2/B1 or GST immobilized to GSH-agarose beads (right). (G) hnRNP L domain mapping. In vitro synthesized S 35 -Met-labeled hnRNP L segments (top) were incubated with cytosol from U937 cells. hnRNP A2/B1 (left) and DRBP76 (right) were immunoprecipitated, and the interacting hnRNP L segments detected by autoradiorgraphy. Key hnRNP L domains are shown above.

Article Snippet: Human U937 monocytic cells (ATCC, Rockville, MD) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin at 37°C and 5% CO 2 .

Techniques: Recombinant, In Vitro, Luciferase, Expressing, Activity Assay, Standard Deviation, Binding Assay, Incubation, SDS Page, Staining, Affinity Purification, Mass Spectrometry, Western Blot, In Vivo, Immunoprecipitation, Control, Synthesized, Labeling

Journal: PLoS Biology

Article Title: The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

doi: 10.1371/journal.pbio.1001635

Figure Lengend Snippet: (A) Condition-dependent binding of VEGFA mRNA to GAIT and HILDA complexes. U937 cells were treated with IFN-γ for 24 h under Nmx. or Hpx. Lysates were subjected to IP with anti-hnRNP L or -EPRS antibodies (or IgG control) coupled with qRT-PCR using gene-specific primers. VEGFA mRNA was normalized to GAPDH mRNA, and results normalized to amount of VEGFA mRNA in EPRS IP of cells treated with IFN-γ under Nmx. (B) hnRNP A2/B1 and DRBP76 are essential for hnRNP L binding to VEGFA mRNA in vivo . U937 cells were transfected with hnRNP A2/B1- and DRBP76-specific (or scrambled) siRNA, and lysates immunoprecipitated with anti-hnRNP L antibody. Extracted RNA was subjected to RT-PCR using primers specific for VEGFA or β-actin mRNA. Efficiency of hnRNP L IP was shown by immunoblot. (C) Protein binding domains of VEGFA HSR. Recombinant hnRNP A2/B1, hnRNP L, and DRBP76 were incubated with [ 32 P]UTP-labeled VEGFA HSR, CARE, AUSL, AUSL-A, and AUSL-D RNA and subjected to UV crosslinking. Crosslinked products were treated with RNase A and detected by SDS-PAGE and autoradiography. (D) HSR region required for RNA switch activity. Reporter constructs containing FLuc upstream of wild-type or mutant VEGFA HSR were transfected into U937 cells with a plasmid expressing RLuc driven by the SV40 promoter. Luciferase activity was measured after treatment with IFN-γ under Nmx. or Hpx. for 8 or 24 h. Relative luciferase activity (FLuc/RLuc) was determined from three independent experiments and reported as mean ± SD ( n = 3). FLuc mRNA expression was determined by semi-quantitative RT-PCR (inset). (E) Permissible spacer length between GAIT element and CARE. Reporter constructs with FLuc upstream of wild-type or mutant VEGFA HSR containing poly(C) spacers were transfected into U937 cells together with a RLuc -bearing plasmid. Luciferase activity was measured after treatment with IFN-γ under Nmx. or Hpx. for 0, 8, and 24 h. Cells were treated as in (D) and luciferase activity determined in three independent experiments, and reported as mean ± SD ( n = 3). FLuc mRNA expression was determined by semiquantitative RT-PCR (inset). (F) Schematic of heterotrimeric HILDA complex binding VEGFA HSR RNA in hypoxia.

Article Snippet: Human U937 monocytic cells (ATCC, Rockville, MD) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin at 37°C and 5% CO 2 .

Techniques: Binding Assay, Control, Quantitative RT-PCR, In Vivo, Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Protein Binding, Recombinant, Incubation, Labeling, SDS Page, Autoradiography, Activity Assay, Construct, Mutagenesis, Plasmid Preparation, Expressing, Luciferase

Journal: PLoS Biology

Article Title: The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

doi: 10.1371/journal.pbio.1001635

Figure Lengend Snippet: (A) hnRNP A2/B1, DRBP76, and hnRNP L are required for hypoxia-inducible RNA switch activity in vitro . Effectiveness of knockdown by siRNA targeting hnRNP A2/B1, DRBP76, and hnRNP L, and scrambled (scramb.) control was determined by immunoblot analysis; β-actin was probed as loading control (top). FLuc reporter RNA bearing the VEGFA HSR and RLuc control transcripts were subjected to in vitro translation in RRL in presence of lysates from U937 cells transfected with scrambled or gene-specific siRNA and incubated with IFN-γ under hypoxia; lysates from IFN-γ-treated normoxic cells shown as control (bottom). (B) hnRNP A2/B1 and DRBP76 are required for robust in vivo expression of endogenous VEGF-A in hypoxia. Lysates from siRNA-treated cells as in (A) were probed with anti-VEGF-A and anti-GAPDH antibodies, and normalized VEGF-A expression was quantified by densitometry. Expression of VEGF-A mRNA was determined by qRT-PCR and normalized by GAPDH mRNA. Results are reported as mean ± SEM ( n = 3). (C) hnRNP A2/B1 and DRBP76 are required for efficient VEGFA mRNA translation in presence of IFN-γ and Hpx. U937 cells were transfected with siRNA targeting hnRNP A2/B1 and DRBP76 (or scrambled siRNA), and then subjected to IFN-γ and Hpx. Cell lysates were fractionated on a sucrose gradient, and total RNA in translationally active and inactive pools subjected to qRT-PCR with VEGFA - and GAPDH -specific primers. Results are reported as mean ± SD ( n = 3).

Article Snippet: Human U937 monocytic cells (ATCC, Rockville, MD) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin at 37°C and 5% CO 2 .

Techniques: Activity Assay, In Vitro, Knockdown, Control, Western Blot, Transfection, Incubation, In Vivo, Expressing, Quantitative RT-PCR

Journal: PLoS Biology

Article Title: The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

doi: 10.1371/journal.pbio.1001635

Figure Lengend Snippet: (A) Steady-state amount of hnRNP L mRNA is not regulated by IFN-γ. U937 cells were treated with IFN-γ under Nmx. or Hpx. for 0, 8, and 24 h. HnRNP L and β-actin mRNA were determined by semiquantitative RT-PCR. (B) IFN-γ induces hnRNP L degradation in normoxic cells. U937 cells were treated with CHX for up to 16 h under Nmx. and lysates subjected to immunoblot and quantitated by densitometry. (C) IFN-γ-inducible degradation of hnRNP L is proteasome-mediated. U937 cells were treated with CHX or CHX plus MG132 in presence of IFN-γ for up to 12 h under Nmx. (left panel) or Hpx. (right panel), and lysates subjected to immunoblot and quantitated by densitometry. (D) IFN-γ induces polyubiquitination of endogenous hnRNP L in vivo . U937 cells were treated with IFN-γ for up to 24 h in the absence or presence of MG132, and lysates subjected to IP with mouse-derived hnRNP L antibody followed by immunoblot with rabbit-derived hnRNP L antibody. (E) IFN-γ induces normoxia-dependent ubiquitination of hnRNP L. U937 cells were transfected with HA-ubiquitin, treated with MG132 in Nmx. or Hpx., immunoprecipitated with anti-hnRNP L antibody, and subjected to immunoblot with anti-HA antibody. (F) IFN-γ induces interaction of hnRNP L with VHL. Lysates from U937 cells treated with IFN-γ and MG132 for up to 24 h were immunoprecipitated with anti-VHL antibody and subjected to immunoblot with anti-hnRNP L, -VHL, -hnRNP A2/B1, and -DRBP76 antibodies. (G) IFN-γ-induced polyubiquitination and degradation of hnRNP L is mediated by VHL. U937 cells were transfected with VHL-specific (or scrambled) siRNA. After recovery, cells were treated with IFN-γ in the presence or absence of MG132 and lysates immunoblotted with anti-VHL, -hnRNP L, -ubiquitin, and -GAPDH antibodies.

Article Snippet: Human U937 monocytic cells (ATCC, Rockville, MD) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin at 37°C and 5% CO 2 .

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, In Vivo, Derivative Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: PLoS Biology

Article Title: The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

doi: 10.1371/journal.pbio.1001635

Figure Lengend Snippet: (A) Hypoxia increases cytoplasmic localization of hnRNP L. U937 cells treated with IFN-γ for 24 h under Nmx. or Hpx. were immunostained using rabbit anti-hnRNP L and -β-actin antibodies. Cell nuclei were stained with DAPI. (B) Analysis of hypoxia-stimulated translocation of hnRNP L by cell fractionation. U937 cells treated with IFN-γ in Nmx. or Hpx. were fractionated and subjected to immunoblot. (C) Hypoxia induces hnRNP L phosphorylation in vivo . U937 cells were incubated under Hpx. and then with a 4-h pulse of 32 P-orthophosphate between 6 and 10 h (denoted as 8 h) or between 22 and 26 h (denoted as 24 h). Lysates were immunoprecipitated with anti-hnRNP L antibody (or pre-immune IgG), and 32 P-labeled protein detected by autoradiography. (D) Hypoxia induces tyrosine phosphorylation of hnRNP L. Lysates from cells treated with Hpx. for 0, 8, and 24 h were immunoprecipitated with anti-hnRNP L antibody and immunoblotted with antibodies targeting phosphoserine (P-Ser), phosphothreonine (P-Thr), or phosphotyrosine (P-Tyr). (E) Time course of hypoxia-inducible hnRNP L phosphorylation. U937 cells were treated with Hpx. for up to 24 h and lysates immunoprecipitated with anti-hnRNP L antibody and immunoblotted with antibodies targeting P-Tyr or hnRNP L. (F) Hypoxia induces cytoplasmic accumulation of P-Tyr-hnRNP L. Cytosolic and nuclear fractions from U937 cells treated with Hpx. for 24 h were immunprecipitated with anti-hnRNP L antibody and immunoblotted with anti-P-Tyr and -hnRNP L antibodies. Western blots were done using anti-HDAC1 and anti-tubulin antibodies. (G) Hypoxia induces Tyr 359 phosphorylation of hnRNP L. pcDNA3-hnRNP L-Myc bearing selected Tyr-to-Ala mutations were transfected into U937 cells with endogenous hnRNP L knocked down by 3′UTR-targeting siRNA. After recovery, cells were treated with Hpx. for 24 h. Lysates were immunprecipitated with anti-hnRNP L antibody and immunoblotted with anti-P-Tyr and -hnRNP L antibodies. (H) Sequence conservation of hnRNP L phospho-site in vertebrate animals. Tyr phospho-site in aligned sequences is shown (red). (I) Cellular localization of phospho-mimetic and phospho-dead hnRNP L in normoxia. c-Myc-tagged, wild-type (WT), phospho-mimetic (Tyr-to-Asp, Y-D), and phospho-dead (Tyr-to-Ala, Y-A) mutant hnRNP L were transiently transfected into U937 cells and were determined in cytoplasmic and nuclear fractions by immunoblot analysis with anti-c-Myc, -HDAC1, and -tubulin antibodies. (J) Phospho-mimetic hnRNP L binds hnRNP A2/B1. c-Myc-tagged, wild-type (WT), phospho-dead (Y-A), and phospho-mimetic (Y-D) hnRNP L were expressed in U937 cells by transient transfection. Lysates were immunoprecipitated with anti-c-Myc antibody and immunoblotted with anti-hnRNP L and -hnRNP A2/B1 antibodies. Expression of c-Myc-tagged hnRNP L was determined with anti-c-Myc antibody of total lysate. (K) Phospho-mimetic hnRNP L inhibits VHL-mediated, proteasomal degradation of hnRNP L. c-Myc-tagged wild-type, phospho-dead, and phospho-mimetic hnRNP L was transiently transfected into U937 cells, and then treated with IFN-γ for 24 h. Lysates were immunoblotted with anti-c-Myc and -actin antibodies. C-Myc-tagged hnRNP L was determined by immunoblot with anti-c-Myc antibody of total lysate from cells not treated with IFN-γ as controls for transfection and expression of hnRNP L-bearing vectors.

Article Snippet: Human U937 monocytic cells (ATCC, Rockville, MD) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin at 37°C and 5% CO 2 .

Techniques: Staining, Translocation Assay, Cell Fractionation, Western Blot, Phospho-proteomics, In Vivo, Incubation, Immunoprecipitation, Labeling, Autoradiography, Transfection, Sequencing, Mutagenesis, Expressing

Journal: PLoS Biology

Article Title: The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

doi: 10.1371/journal.pbio.1001635

Figure Lengend Snippet: (A) Rapid degradation of hnRNP L in absence of hnRNP A2/B1. U937 cells were transfected with hnRNP A2/B1-specific (or scrambled) siRNA. After recovery, cells were treated with IFN-γ and Hpx. for 0, 8, and 24 h. Lysates were immunoblotted with anti-hnRNP A2/B1, -hnRNP L, and -GAPDH antibodies. (B) Time course of hnRNP L degradation in absence of hnRNP A2/B1. U937 cells were treated as in (A) for up to 24 h. Lysates were immunoblotted with anti-hnRNP L and -DRBP76 antibodies. (C) IFN-γ induces prolyl hydroxylation of hnRNP L. U937 cells were treated with IFN-γ and MG132 for up to 24 h. Lysates were immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hydroxyproline and -hnRNP L antibodies. (D) hnRNP L is not subject to prolyl hydroxylation in Hpx. U937 cells were treated with IFN-γ and Hpx. for up to 24 h. Lysates were immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hydroxyproline and -hnRNP L antibodies. (E) hnRNP A2/B1 inhibits prolyl hydroxylation of hnRNP L in hypoxia. U937 cells were transfected with hnRNP A2/B1-specific (or scrambled) siRNA. After recovery, cells were treated with IFN-γ, Hpx., and MG132 for up to 24 h. Lysates were immunoblotted with anti-hnRNP A2/B1 antibody. Lysates were also immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hydroxyproline and -hnRNP L antibodies. (F) Hypoxia induces binding of hnRNP A2/B1 to hnRNP L and prevents VHL binding. U937 cells were subjected to Nmx. or Hpx. for 24 h in the presence of IFN-γ stimulus. Lysates were immunoprecipitated with anti-hnRNP L antibody and immunoblotted with anti-hnRNP A2/B1 and -VHL antibodies. Total hnRNP L in cell lysates was determined by immunoblot. IP with pre-immune IgG of hypoxic lysate served as a control. (G) Reconstitution of RNA switch function of HILDA complex. Phospho-mimetic hnRNP L (Y359D) was pre-incubated with DRBP76 and hnRNP A2/B1 as indicated (5 pmol each) for 0.5 h on ice. In vitro translation of the FLuc reporter bearing the VEGFA HSR element (and RLuc control RNA) was determined in a wheat germ extract in the presence of 35 S-Met, cytosolic extracts from IFN-γ-treated U937 cells, and HILDA components as indicated. In a control experiment, wild-type hnRNP L replaced phospho-mimetic hnRNP L. FLuc expression was normalized by RLuc and reported as mean ± SD, n = 3.

Article Snippet: Human U937 monocytic cells (ATCC, Rockville, MD) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin at 37°C and 5% CO 2 .

Techniques: Transfection, Immunoprecipitation, Binding Assay, Western Blot, Control, Incubation, In Vitro, Expressing

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 deficiency enhances p38α, especially p-p38αprotein levels. ( A , C ). mRNA and protein level of Nedd4 are decreased in iBMDM cells with lentiviral-based shRNA. ( A ) iBMDM cells transduced with control or shRNA targeting to Nedd4 were collected and the total RNA was extracted with TRIzol, reverse transcribed, and analyzed for Nedd4 mRNA with Q-PCR. ( B , F ). The p38, especially p-p38 protein levels were increased in Nedd4 deficient iBMDM cells stimulated for 0-60 min (above lanes) with LPS. ( B ) iBMDM cells transduced with control or shRNA targeting to Nedd4 were subjected to immunoblot analysis. ( F ) Immunoblot analysis of Nedd4 knockout (Nedd4 −/− ) iBMDM cell lines. ( C – E ). ImageJ analysis for the immunoblot of Nedd4, total p38 and p-p38 levels in iBMDM cells transduced with control or shRNA targeting to Nedd4. ( G – I ) ImageJ analysis for the immunoblot of total p38 and p-p38 levels in two Nedd4 −/− iBMDM cell lines. The results are represented from at least three independent experiments.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: shRNA, Transduction, Control, Reverse Transcription, Western Blot, Knock-Out

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: p38, especially p-p38α might be involved in Nedd4 ubiquitination system. ( A ) The smear bands above 40 kDa almost disappeared in Nedd4 −/− iBMDM cells. Overexposure for immunoblot analysis of p-p38 in wild type and Nedd4 −/− iBMDM cells stimulated for 0–60 min (above lanes) with LPS treatment. ( B ) p-p38 was degraded through lysosomal and proteasomal pathways. Immunoblot analysis of p-p38 in wild-type iBMDM cells stimulated for 0–60 min (above lanes) with LPS stimulation and Chloroquine or MG132 respectively. The results are represented from at least three independent experiments. ( C , D ) ImageJ analysis for the immunoblot of total p38 ( C ) and p-p38 ( D ) levels in iBMDM cells treated with Chloroquine or MG132 respectively.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Ubiquitin Proteomics, Western Blot

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 inhibits TNF-α production and AP-1 activation mediated by p38α activation. ( A ) mRNA level of TNF-α was enhanced compared with control cells. Control and Nedd4-silenced iBMDM cells were stimulated with LPS for 90 min, and total RNA was extracted. After reverse transcription, TNF-α mRNA was analyzed with Q-PCR. ( B ) ELISA of TNF-α in supernatants from control and Nedd4-silenced iBMDM cells stimulated for 0–90 min (above lanes) with LPS. Data are representative of three independent experiments with similar results (mean ± s.d.) (*, significant difference). ( C ) Nedd4 inhibits MyD88-dependent transcription of gene encoding AP-1. Luciferase assay of the induction of gene encoding AP-1 in lysates of 293T cells transfected with 150 ng pGL3-AP-1 (firefly-luciferase) reporter plasmid and 3 ng pGL3-TK (renilla-luciferase) reporter, and 50 ng MyD88-expressing plasmid plus 0, 50, 100, 200 or 300 ng Nedd4-expressing plasmid and cultured for 24 h. Empty control vector pcDNA3.1(+) was added to each sample to ensure transfection of the same amount of DNA in each. Luciferase activity is normalized to renilla luciferase activity and is presented relative to basal luciferase activity. *, p < 0.05 compared with no Nedd4 group. Data are the mean ± s.d. of 4 samples in one experiment representative of similar results obtained in three independent experiments.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Activation Assay, Control, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Cell Culture, Activity Assay

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 can interact with endogenous and exogenous p38α. ( A ) Nedd4 interacts with endogenous p-p38α. iBMDMs cells were incubated with LPS at the indicated times (30 min and 60 min), lysed, immunoprecipitated with anti-Nedd4, gel separated and detected with anti-p-p38 antibody. ( B , C ) The interaction between Nedd4 and p38αV1 requires phosphorylation of p38αV1 by MKK6. HEK293 cells co-transfected with various molecules (above lanes), lysed, immunoprecipitated with anti-Nedd4, gel separated and detected with anti-Flag antibody ( B ); immunoprecipitated with anti-Flag, gel separated and detected with anti-Nedd4 antibody ( C ). ( D ) The interaction between Nedd4 and p38αV2 does not require phosphorylation of p38αV2 by MKK6. 293T cells co-transfected with various molecules (above lanes), lysed, immunoprecipitated with anti-Flag, gel separated and detected with anti-Nedd4 antibody. ( E ) Nedd4 interacts with p-p38αV1 and p38αV2 by its WW domains. These results are represented from three independent experiments.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Incubation, Immunoprecipitation, Phospho-proteomics, Transfection

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 mediates polyubiquitination of p38α isoforms (Left. Ubiquitination of p38αV1; Right. Ubiquitination of p38αV2). Immunoblot analysis of anti-Flag immunoprecipitates of lysates of 293T cells co-transfected with various molecules (above lanes), probed with anti-HA ( A ), anti-K48 ubiquitin ( B ), anti-K63 ubiquitin ( C ) and anti-p-p38. For whole cell lysates, p38αV1 and p38αV2 were probed with anti-Flag, MKK6 was probed with anti-HA and Nedd4 was probed with anti-Nedd4. Lane 3 just showed the signals of ubiquitinated proteins, not MKK6.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Analysis for polyubiquitination sites of p38α and conformation of p38αV1 and p38αV2. ( A ) Coomassie blue stained gel indicates the bands of p38αV1 and p38αV2 with polyubiquitination. 293T cells co-transfected with p38αV1-flag or p38αV2-flag, HA-Nedd4, MKK6-HA and ubiquitin-His were lysed, immunopricipitated with Ni-column and anti-Flag purification beads, gel-separated, and stained with Coomassie blue. The square area indicates the presence of ubiquinated-p38αV1 and p38αV2. ( B ) K53(K54) residue of p38α was identified as an common ubiquitination site in p38αV1 and p38αV2 by mass spectrometry. The protein sample with ubiquitinated p38αV1 and p38αV2 were recovered from the gel, enzyme digested, and subjected to mass analysis. Representative MS/MS spectra of peptides demonstrated ubiquitination at K53(K54) of p38αV1 and p38αV2. Peak matching expected y ions is labeled. ( C ) The conformational difference between p38αV1 and p38αV2. PDB documents (model No. 1p38 for p38αV1; model No. 3py3 for p38αV2) from SWISS-model, red arrows indicate the conformational difference between p38αV1 and p38αV2.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Staining, Transfection, Ubiquitin Proteomics, Purification, Residue, Mass Spectrometry, Tandem Mass Spectroscopy, Labeling

Journal: The Journal of Neuroscience

Article Title: Loss of Protein Arginine Methyltransferase 8 Alters Synapse Composition and Function, Resulting in Behavioral Defects

doi: 10.1523/JNEUROSCI.0591-17.2017

Figure Lengend Snippet: PRMT8 localizes to synaptic sites. A, Selected proteins from mass spectrometry analysis of the synaptosomal proteome of wild-type mouse cortex. The names, molecular weight (MW), number of unique peptides identified, and total intensity are indicated. B, Western blot of nuclear, presynaptic and postsynaptic density (PSD) fractions from DIV17 mouse primary neurons expressing tagged PRMT8 probed with antibodies recognizing the PSD protein PSD95, the nuclear protein HDAC2, and the synaptic vesicle protein Svp38, as well as HA to detect PRMT8. Equivalent cellular proportions of each fraction were loaded for direct comparison between the fractions. Molecular weights are indicated. C–F, Immunostaining of DIV17 mouse primary neurons expressing tagged PRMT8 and stained with antibodies against FLAG and the postsynaptic marker PSD95. Neurons expressing an empty vector act as a control for the specificity of FLAG staining. Scale bar, 10 μm.

Article Snippet: Antibodies used included the following: mouse-PSD95 (NeuroMAB), mouse-HDAC2 (Abcam), rabbit-HA (Santa Cruz Biotechnology), mouse-Svp38 (Sigma), rabbit-Cacna1C (Novus), mouse-Syn1 (Synaptic Systems), rabbit-Nsf-1 (Thermo Fisher), rabbit-Syn2 (Abcam), rabbit-Syn3 (Synaptic Systems), rabbit-Syt7 (Abcam), mouse-Syt12 (NeuroMAB), rabbit-Cplx1 (Proteintech), mouse-β-actin (Sigma), rabbit-NR2A (Cell Signaling Technology), rabbit-NR2B (Cell Signaling Technology), mouse-NR1 (Millipore), mouse-GluA1 (Millipore), rabbit-GluA2 (Cell Signaling Technology), mouse-CaMKIIA (Millipore Bioscience Research Reagents), mouse-Shank1 (NeuroMAB), mouse-α-tubulin (Sigma), rabbit-Homer (GeneTex), rabbit-eIF4G1 (Cell Signaling Technology), rabbit-eIF4H (Cell Signaling Technology), rabbit-eIF4E (Cell Signaling Technology), and rabbit-FMRP (Cell Signaling Technology).

Techniques: Mass Spectrometry, Molecular Weight, Western Blot, Expressing, Comparison, Immunostaining, Staining, Marker, Plasmid Preparation, Control