cell mitochondria isolation buffer containing pmsf  (Beyotime)

Journal: International Journal of Biological Sciences

Article Title: SUMO E3 ligase MUL1 inhibits lymph node metastasis of bladder cancer by mediating mitochondrial HSPA9 translocation

doi: 10.7150/ijbs.98772

Figure Lengend Snippet: MUL1 regulated the translocation of HSPA9 from mitochondria to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.

Article Snippet: Cells were suspended in 2 ml of cell mitochondria isolation buffer containing 100 mM PMSF (ST506, Beyotime) for 30 minutes.

Techniques: Translocation Assay, Immunoprecipitation, Western Blot, Positive Control, Negative Control, Immunofluorescence, Knockdown, Over Expression

Journal: International Journal of Biological Sciences

Article Title: SUMO E3 ligase MUL1 inhibits lymph node metastasis of bladder cancer by mediating mitochondrial HSPA9 translocation

doi: 10.7150/ijbs.98772

Figure Lengend Snippet: MUL1 SUMOylated HSPA9 at the lysine 612 (K612) residue. (A-B). Anti-HSPA9 IP was performed in MUL1-silencing (E) and MUL1-overexpressing (F) cell lysates. SUMO2/3 and HSPA9 levels were detected via western blotting. (C). T24 cells were treated with ML792 at the concentrations indicated in the images. Mitochondrial and cytosolic cell lysates were separated and prepared. HSPA9 levels in mitochondria and mitochondria-free cell fractions were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (D). Representative IF images of HSPA9 localization in T24 cells with the treatment of ML792 are shown. Green: HSPA9; red: TOM20; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E). GPS-SUMO, JASSA and SUMOplot tools were used to predict the HSPA9 SUMOylation site. Sequences around SUMO-conjugation motifs are shown. Red: SUMO-conjugation motifs. (F). Schematic diagram of the predicted HSPA9 K612 site using SWISS-MODEL tools. (G). Wild-type HSPA9 and HSPA9-K612R were overexpressed in T24 and UM-UC-3 cells to generate OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. GFP levels were detected via western blotting. (H). Anti-GFP IP was performed in OE-HSPA9 and OE-HSPA9-K612R cell lysates. SUMO2/3 levels were detected via western blotting. (I). Mitochondrial and mitochondria-free cell fractions of OE-HSPA9 and OE-HSPA9-K612R cells were prepared. GFP expression was detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (J). Stable overexpression of exogenous MUL1 was performed in OE-HSPA9 or OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Representative IF images of GFP and TOM20 colocalization are shown. Green: GFP; red: TOM20; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. -, no statistical significance; *, P < 0.05; **, P < 0.01.

Article Snippet: Cells were suspended in 2 ml of cell mitochondria isolation buffer containing 100 mM PMSF (ST506, Beyotime) for 30 minutes.

Techniques: Residue, Western Blot, Conjugation Assay, Expressing, Over Expression

Journal: International Journal of Biological Sciences

Article Title: SUMO E3 ligase MUL1 inhibits lymph node metastasis of bladder cancer by mediating mitochondrial HSPA9 translocation

doi: 10.7150/ijbs.98772

Figure Lengend Snippet: SUMOylated HSPA9 catalyzed the degradation of SUZ12 and EZH2. (A). The identification of HSPA9-interacting proteins is shown in a schematic diagram. By comparing the qualitative MS analysis and relative quantitative proteomics results, SUZ12 and EZH2 were selected for further research. (B). Nuclear and mitochondria-free cytoplasmic faction of T24 and UM-UC-3 cells were separated. Anti-HSPA9 IP was performed in nuclear and mitochondria-free cytoplasmic protein lysates. HSPA9, SUZ12 and EZH2 levels were detected via western blotting. IgG served as a negative control. (C). Anti-GFP IP was performed in OE-HSPA9 and OE-HSPA9-K612R cell lysates. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. SUZ12 and EZH2 levels were detected via western blotting. Input served as a positive control, and IgG served as a negative control. (D). Representative IF images of GFP-SUZ12 and GFP-EZH2 colocalization in T24 OE-HSPA9 and OE-HSPA9-K612R cells are shown. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Green: GFP; red: SUZ12/EZH2; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. (E). Stable overexpression of vector or exogenous MUL1 was performed in T24 OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Representative IF images of GFP and SUZ12 colocalization are shown. Green: GFP; red: SUZ12; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. (F). SUZ12, EZH2, STAT3 and p-STAT3 levels were detected via western blotting in OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. (G). SUZ12, EZH2, STAT3 and p-STAT3 levels were detected via western blotting in MUL1-silencing or MUL1-overexpressing cells. (H). Anti-EZH2 and anti-SUZ12 IP were performed in T24 OE-HSPA9 and OE-HSPA9-K612R cell lysates. Ubiquitin-K48 levels were detected via western blotting.

Article Snippet: Cells were suspended in 2 ml of cell mitochondria isolation buffer containing 100 mM PMSF (ST506, Beyotime) for 30 minutes.

Techniques: Quantitative Proteomics, Western Blot, Negative Control, Positive Control, Over Expression, Plasmid Preparation, Ubiquitin Proteomics

Journal: Carcinogenesis

Article Title: DUT enhances drug resistance to proteasome inhibitors via promoting mitochondrial function in multiple myeloma.

doi: 10.1093/carcin/bgac071

Figure Lengend Snippet: Figure 1. DUT expression is augmented in BTZ-resistant cells. (A) Schematic illustration of the SILAC assay in the BR and WT MM.1S cells. (B) Shown were 584 differential expressed proteins including 231 downregulated and 353 upregulated proteins (FC cutoff, 1.5-folds; FDR cutoff, 0.05) in the BR MM.1S cells compared with the WT cells detected by SILAC assay. (C) GO enrichment analysis and KEGG pathway annotation indicated that nucleotide metabolism and mitochondrial homeostasis were significantly associated with the response of BTZ resistance. (D) Immunoblotting assay was performed to detect the expressions of DUT in control and BTZ-resistant MM cells. (E) Expression of DUT in MM patients with all responses (R) and non-response (non-R) from the cohort GSE9782, and (F) shows GUT expression among all responses, including complete response (CR), partial response (PR) and disease progression (PD). (G) Correlation of DUT expression with overall survival (OS) in MM patient cohorts from the multiple myeloma research foundation (MMRF) (CoMMpass trial, NCT145429).

Article Snippet: The cells were suspended in the mitochondrial isolation buffer (Mitochondria Isolation Kit for Cultured Cells, Beyotime, C3601) and placed on ice for 15 min, homogenized for 15 times, then centrifuged at 600g for 10 min at 4°C.

Techniques: Expressing, Multiplex sample analysis, Western Blot, Control, Biomarker Discovery

Journal: Carcinogenesis

Article Title: DUT enhances drug resistance to proteasome inhibitors via promoting mitochondrial function in multiple myeloma.

doi: 10.1093/carcin/bgac071

Figure Lengend Snippet: Figure 4. Mitochondrial dysfunction is involved in BTZ resistance. (A) Immunofluorescence assay showed subcellular localization of DUT. (B) The enrichment of DUT in mitochondria (Mito) and cytosol (Cyto) fractions was determined by Western blotting. Voltage-dependent anion channel (VDAC) was used as a marker for the mitochondrial fraction, and tubulin as an indicator for the total and the cytosolic fraction. (C) Real-time PCR assay (left) and Western blotting assay (right) showed the mRNA and protein levels of genes involved in mitochondrial biogenesis (PGC1α), mitochondrial dynamics (FIS1) and antioxidant system (SOD2) in BR MM cells. (D) OCR was assessed using a Seahorse XFe96 analyzer, and (E) quantifications of basal respiration, ATP synthesis and maximal respiration in WT and BR MM cells. (F) Mitochondrial ATP synthesis rate was determined based on luciferase- catalyzed ATP-dependent oxidation of luciferin.

Article Snippet: The cells were suspended in the mitochondrial isolation buffer (Mitochondria Isolation Kit for Cultured Cells, Beyotime, C3601) and placed on ice for 15 min, homogenized for 15 times, then centrifuged at 600g for 10 min at 4°C.

Techniques: Immunofluorescence, Western Blot, Marker, Real-time Polymerase Chain Reaction, Luciferase

Journal: Carcinogenesis

Article Title: DUT enhances drug resistance to proteasome inhibitors via promoting mitochondrial function in multiple myeloma.

doi: 10.1093/carcin/bgac071

Figure Lengend Snippet: Figure 5. DUT inhibition abrogates mitochondrial modulation induced by BTZ resistance. (A) The mRNA level and (B) protein level of PGC1α, FIS1 and SOD2 in DUT knockdown and control cells were determined by Real-time PCR and Western blotting assays. (C) OCRs of DUT knockdown and control cells were assessed using a Seahorse XFe96 analyzer. OCR including (D) basal respiration level, (E) ATP synthesis, (F) maximal respiration in DUT knockdown (KD) MM.1S cells with or without BTZ treatment were assessed by a Seahorse XFe96 analyzer. (G) Mitochondrial ATP synthesis rates of DUT knockdown and control cells were determined based on luciferase-catalyzed ATP-dependent oxidation of luciferin. (H) DUT knockdown and control cells were exposed to 5 nM BTZ for 24h, then ΔΨm change in the membrane potential of mitochondria was analyzed with JC-1 staining and flow cytometry.

Article Snippet: The cells were suspended in the mitochondrial isolation buffer (Mitochondria Isolation Kit for Cultured Cells, Beyotime, C3601) and placed on ice for 15 min, homogenized for 15 times, then centrifuged at 600g for 10 min at 4°C.

Techniques: Inhibition, Knockdown, Control, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Membrane, Staining, Flow Cytometry