Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: CTMP and p21 were novel PRAME-interacting proteins in MM cells. (A) Pathway enrichment results based on Metascape analysis of top 100 proteins with reduced expression followed by PRAME knockdown. Top 20 pathways were listed. Regulation of mitotic cell cycle was most significantly enriched. (B) Proteins interacting with cyclins analyzed by STRING database. In addition to proteins of cytokinin-dependent kinase (CDK) family, cytokinin-dependent kinase inhibitors (such as CDKN1A and CDKN1B ) are attractive. (C) CCND3 and p21 expression levels tested by Western blot in MM cell lines compared PRAME knockdown or overexpression groups to control groups (related to - ). LP-1 was not applied for subsequent protein detection experiments for the low protein expression level of p21 (related to - ). (D) Flow diagram of IP-MS of RPMI8226 cells. (E) Western blot verified the CTMP/Akt/p21/CCND3 pathway in MM cell lines RPMI8226, KM-3 and SKO-007 (related to - ). Protein expression levels of CTMP and p21 were increased, and p -Akt and CCND3 were decreased followed by PRAME knockdown. (F) PRAME interacted with CTMP, p21 and Cul2 at the endogenous level. Cells were harvested and subjected to immunoprecipitation with anti-PRAME Ab and immunoblotting with anti-CTMP, anti-p21 and anti-Cul2 Ab (related to - ). (G) Higher PRAME transcript levels correlated with lower THEM4 and CDKN1A transcript levels in MM patients' plasma cells.

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Expressing, Knockdown, Western Blot, Over Expression, Control, Protein-Protein interactions, Immunoprecipitation, Clinical Proteomics

Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: PRAME degrades CTMP and p21 through UPS. (A, B) CTMP and p21 were degraded by proteasomes. RPMI8226 cells were treated with 10 μmol/L MG-132 versus DMSO in combination with 50 μg/mL CHX for indicated time (0, 2h, 4h and 8h, respectively), and then were subjected to immunoblotting using anti-CTMP and anti-p21 with GAPDH as a loading control (related to - ). (C, D) The expression of CTMP and p21 in A and B was quantified by densitometric analysis using ImageJ software, respectively. Data are the means ± SD of 3 assays. ns , no significance; * P < 0.05; ** P < 0.01; *** P < 0.001. (E, F) PRAME mediated polyubiquitination of CTMP and p21 in RPMI8226 cells. PRAME knockdown and control cell models were used. Cells were extracted and subjected to immunoprecipitation with anti-CTMP and anti-p21 Ab and immunoblotting with anti-ubiquitin Ab with MG-132 for 2h and 6h, respectively (related to - ).

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Western Blot, Control, Expressing, Software, Knockdown, Immunoprecipitation, Ubiquitin Proteomics

Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: PRAME increases sensitivity of MM cells to bortezomib by targeting CTMP and p21. (A) Apoptosis analysis by flow cytometry in PRAME knockdown and overexpression cells with corresponding vector controls after bortezomib treating for 48h (LP-1, 10 nM; RPMI8226 20 nM). Data are the means ± SD of 3 assays. ns , no significance; * P < 0.05; ** P < 0.01; *** P < 0.001. (B) CTMP and p21 were degraded more intensely by proteasomes in PRAME high expression group than that in PRAME low expression group. PRAME knockdown and the control RPMI8226 cells were treated with 10 μmol/L MG-132 versus DMSO in combination with 50 μg/mL CHX for indicated time (0, 2h, 4h and 8h, respectively), and then were subjected to immunoblotting using anti-PRAME, anti-CTMP and anti-p21 with GAPDH as a loading control (related to - ). (C, D) The expression of CTMP and p21 in B was quantified by densitometric analysis using ImageJ software, respectively. Significance was shown between the fold changes of shCtrl PRAME MG132+CHX/DMSO + CHX group compared to shPRAME MG132+CHX/DMSO + CHX group at each time point. Data are the means ± SD of 3 assays. ns , no significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Flow Cytometry, Knockdown, Over Expression, Plasmid Preparation, Expressing, Control, Western Blot, Software

Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: Mechanism diagram exhibits the role of PRAME in promoting cell proliferation and increasing sensitivity to bortezomib in MM cells. On the CTMP/Akt/p21/CCND3 signal pathway, PRAME plays the role of Cul2-dependent substrate recognizing receptor protein, and interacts with its target CTMP, labeling it with ubiquitin, and making it chemotactic to proteasome and degraded through UPS. The degradation of CTMP contributes to phosphorylation and activation of Akt, which leads to blocking of p21 expression. At the same time, PRAME directly takes p21 as its ubiquitination substrate, directing it degraded through the UPS. Both of the above-mentioned two pathways decrease p21 levels, thus leading to accumulation of CCND3 and promoting the cell cycle progression and cell proliferation.

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Labeling, Ubiquitin Proteomics, Phospho-proteomics, Activation Assay, Blocking Assay, Expressing

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 1. Localization of CTMP in mitochondria. (A) Cytoplasm and mitochondria fractions were isolated from A549 cells. Total lystates, the cytosolic fraction, and mitochondria were analyzed by Western blotting (COXIV-mitochondria marker and α-tubulin-cytosolic marker). (B) A549 cells were transiently co-transfected with pEGFP-N1-CTMP and pDsRed-Mito for 24 h, and cells were observed using confocal microscopy. (C) A549 cells stably expressing pDsRed-Mito were infected with lentivirus-CTMP for 24 h and immunostained with anti-CTMP antibody. (D) The mitochondria fraction from A549 cells was isolated and treated with the indicated reagents. Samples were separated into supernatant (S) and precipitate (P) fractions, and then analyzed by immunoblotting with the indicated antibodies (cytochrome c, mitochondria membrane space protein; and COXIV, mitochondria membrane protein). (E) Immunofluorescence assay of apoptosis-inducing factor (AIF). A549 cells were grown on glass coverslips and transiently transfected with pEGFP-N1-CTMP. The cells were then stained with anti-AIF antibody (scale bar, 10 µm).

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Isolation, Western Blot, Marker, Transfection, Confocal Microscopy, Stable Transfection, Expressing, Infection, Membrane, Immunofluorescence, Staining

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 2. CTMP induces apoptosis in A549 cells. (A) A549 cells were infected with lentivirus-CTMP for 24 h and treated with 1 µM staurosporine for the indi cated times (6 and 8 h). (B) A549 cells were infected with lentivirus-CTMP for 24 h and treated with 0.2 µM actinomycin D for the indicated times (12 and 24 h). (C) Western blot analysis was performed to measure CTMP expression in A549 cells after siRNA transfection for the indicated times. Right panel, densitometric analysis of CTMP expression in A549 cells after siRNA transfection for 48 h. Each bar represents the mean ± SE (n=3), *p<0.05 and **p<0.01 compared to the corresponding control values and Western blot analysis measuring the levels of cleaved PARP and active caspase-3 was performed. (D) ELISA assay of active caspase-3. (E) ELISA assay of cleaved PARP. CON, untreated A549 cells; VEC, A549 cells infected with the lentivirus expression vector (VEC); CTMP, A549 cells infected with lentivirus-CTMP (CTMP). (F) Quantitative analysis of TUNEL-positive cells. Each bar represents the mean ± SE (n=3), *p<0.05 and **p<0.01 compared to the corresponding control values.

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Infection, Western Blot, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, TUNEL Assay

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 3. CTMP inhibits OPA1 expression and changes mitochondria morphology. (A) Western blot analysis of MFN1, MFN2, and DRP-1. A549 cells were infected with lentivirus-VEC/CTMP and cultured for 48 h before analysis. (B) A549 cells were infected with lentivirus-CTMP, treated with 0.2 µM CCCP (carbonyl cyanide 3-chlorophenylhydrazone), and transfected with siRNA specific for CTMP. (C) The mitochondrial ultrastructure was analyzed by TEM. (D) A549 cells stably expressing mitochondrion-targeted pDsRed-Mito were infected with lentivirus expression vector and lentivirus-CTMP for 48 h and observed with confocal microscopy (scale bar, 10 nm).

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Infection, Cell Culture, Transfection, Stable Transfection, Plasmid Preparation, Confocal Microscopy

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 4. CTMP reduces mitochondria membrane potential that leads to cytochrome c release. (A) CTMP-infected cells were stained using 5 µg/ml JC-1 and analyzed by flow cytometry. (B) Western blot analysis of CTMP and cytochrome c in mitochondria. (C and D) The bands of interest were further analyzed by densitometry. Each bar represents the mean ± SE (n=3), *p<0.05 and **p<0.01 compared to the corresponding control values.

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Membrane, Infection, Staining, Flow Cytometry, Western Blot, Control

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 5. CTMP inhibits heat-shock proteins. A549 cells were infected with lentivirus-VEC/CTMP and cultured for 48 h. (A) Western blot analysis of Hsp27 and Apaf-1. (B) Immunofluorescence assay of Hsp27. (C) FACS analysis of Apaf-1 in CTMP-infected cells.

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Infection, Cell Culture, Western Blot, Immunofluorescence