Journal: Molecular Cancer

Article Title: RIPK3 activation induces TRIM28 derepression in cancer cells and enhances the anti-tumor microenvironment

doi: 10.1186/s12943-021-01399-3

Figure Lengend Snippet: RIPK3 activation induces TRIM28 phosphorylation at serine 473. (A) HT-29 cells were treated with TSZ in a time-dependent manner. Cell lysates were analyzed by western blotting. (B) TSZ-treated HT-29 cells were stained with S824 and S473 phospho-TRIM28 antibodies and analyzed by confocal fluorescence microscopy (green: S824 or S473 phospho-TRIM28; blue: DAPI). (C) HT-29 cells were treated for 4 h with doxorubicin (2 μM), etoposide (100 μM), TSZ, TNF-α + CHX (5 μg/ml) + zVAD, or TRAIL (100 ng/ml) + SZ. (D) HT-29 cells were treated with TRAIL, TRAIL+ SZ, or TSZ for the indicated times, and cell lysates were analyzed by western blotting. (E) HT-29, SNU620, and ML-1 cells were treated with TSZ for the indicated times, and cell lysates were analyzed by western blotting. (F) HT-29 cells were pretreated with Nec-1 and GSK’872 for 1 h and with TSZ for 4 h. Cell lysates were analyzed by western blotting (left). Cells were also stained and visualized by confocal fluorescence microscopy (green: phospho-MLKL; red: S473 phospho-TRIM28; blue: DAPI) (right). (G) HT-29 cells stably expressing RIPK3 shRNA or the non-silencing control were treated with TSZ for the indicated times. (H) Cells from (G) were treated with TSZ for 4 h, stained, and visualized by confocal fluorescence (green: S473 phospho-TRIM28; blue: DAPI)

Article Snippet: Cells were washed twice with DPBS, fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.25% Triton X-100 for 15 min. After incubation in a blocking buffer for 30 min, the cells were incubated overnight at 4 °C with the following primary antibodies: anti-p-TRIM28 (S473) (BioLegend, 654,102), anti-p-TRIM28 (S824) (Cell Signaling Technology, 4127), anti-TRIM28 (Cell Signaling Technology, 4123), and anti-p-MLKL (Abcam, ab187091).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Staining, Fluorescence, Microscopy, Stable Transfection, Expressing, shRNA, Control

Journal: bioRxiv

Article Title: TRIM28 is a target for paramyxovirus V proteins

doi: 10.1101/2025.05.19.654852

Figure Lengend Snippet: (A) V proteins from 11 different paramyxoviruses can co-precipitate TRIM28. HEK293T cells were transfected with plasmids expressing the indicated FLAG-tagged V constructs or FLAG-tagged mCherry. FLAG-tagged proteins were subsequently immunoprecipitated from cell lysates, and the indicated proteins were detected by western blotting. IP: immunoprecipitated fraction; INPUT: soluble input fraction; TR28: TRIM28. Data are representative of n=3 independent experiments. Black arrows indicate predicted sizes of selected proteins of interest. (B) Quantification shows TRIM28 co-precipitation levels differ between V proteins. Band intensities of co-precipitated TRIM28 from experiments shown in (A) were normalized to their input levels and expressed relative (rel.) to the levels of the respective immunoprecipitated FLAG-tagged protein. Data points correspond to results from individual experiments, and results from n=3 independent experiments are shown. Bars represent mean values, with error bars representing standard deviation (SD). Statistical significance was assessed by ordinary one-way ANOVA (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, non-significant). (C) and ( D ) V and TRIM28 proteins can be co-precipitated from infected cells. A549 cells were infected, or mock, with PIV5 or PIV5-VΔC (MOI = 2 PFU/cell) for 24 hours, and soluble cell lysates were prepared. (C) Immunoprecipitations were performed using an anti-V5 antibody, and the indicated co-precipitated proteins were analyzed by western blot. (D) Immunoprecipitations were performed using an anti-TRIM28 antibody (TR28) or an anti-Ffluc antibody (control), and the indicated co-precipitated proteins were analyzed by western blot. Input fractions were also analyzed. Data are representative of n=3 independent experiments.

Article Snippet: Membranes were blocked in 5% milk-TBS-T (5% milk in TBS supplemented with 0.1% Tween 20), or 5% BSA-TBS-T for phosphorylated protein detection, and incubated with the following primary antibodies: rabbit anti-TRIM28 polyclonal antibody (A-300-274A, Bethyl); rabbit anti-phospho-TRIM28 (S824) polyclonal antibody (A-300-767A, Bethyl); mouse anti-FLAG M2 monoclonal antibody (F1804, Sigma Aldrich); rabbit anti-FLAG polyclonal antibody (F7425, Sigma Aldrich); mouse anti-V5 monoclonal antibody (MCA1360, BioRad); mouse anti-DDB1 monoclonal antibody (2B12D1, Invitrogen); rabbit anti-HA monoclonal antibody (C29F4, Cell Signaling Technologies); rabbit anti-GFP polyclonal antibody (GTX113617); rabbit anti-PA polyclonal antibody (GTX118991, Genetex); rabbit anti-NS1 polyclonal antibody (GTX125990, Genetex); rabbit anti-SUMO2/3 polyclonal antibody (ab3742, abcam); or mouse anti-actin monoclonal antibody (sc-47778, Santa-Cruz).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, Western Blot, Standard Deviation, Infection, Control

Journal: bioRxiv

Article Title: TRIM28 is a target for paramyxovirus V proteins

doi: 10.1101/2025.05.19.654852

Figure Lengend Snippet: (A) The C-terminal domain of V proteins is necessary and sufficient for TRIM28 engagement. HEK293T cells were transfected with FLAG-tagged constructs expressing mCherry or the MeV-V, MuV-V, or PIV5-V proteins in their full-length (FL), C-terminal domain (CTD), or N-terminal domain (NTD) forms. FLAG-tagged proteins were subsequently immunoprecipitated and the indicated co-precipitated proteins analyzed by western blot. Input fractions were also analyzed. Data are representative of n=3 independent experiments. Black arrows indicate predicted sizes of selected proteins of interest. (B) Comparison of TRIM28 co-precipitation abilities between PIV2-V and PIV5-V. HEK293T cells were transfected with plasmids expressing the indicated FLAG-tagged constructs prior to anti-FLAG immunoprecipitations and analyses as in (A). Data are representative of n=3 independent experiments. (C) Schematic representation of CTD differences between PIV2-V and PIV5-V. Conserved motifs, serving as cut-off points for chimeras, are highlighted in red, and differential amino acids (aa) are noted. Chimeric constructs (Chim 1–6) were designed by swapping segments between PIV2-V (orange) and PIV5-V (blue) as indicated. (D) Comparison of TRIM28 and DDB1 co-precipitation abilities between chimeric V constructs. HEK293T cells were transfected with plasmids expressing the indicated FLAG-tagged constructs and chimeras depicted in (C) prior to anti-FLAG immunoprecipitations and analyses as in (A). Data are representative of n=3 independent experiments. (E) Quantification of TRIM28 co-precipitation by chimeric V constructs. Band intensities of co-precipitated TRIM28 from experiments shown in (D) were normalized to their input levels and expressed relative (rel.) to the levels precipitated by PIV2-V. Data points correspond to results from individual experiments, and results from n=3 independent experiments are shown. Bars represent mean values, with error bars representing standard deviation (SD). Statistical significance was assessed using a one-sample t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, non-significant). (F) Comparison of TRIM28 and DDB1 co-precipitation abilities between PIV2-V mutants. HEK293T cells were transfected with plasmids expressing the indicated FLAG-tagged constructs and mutants prior to anti-FLAG immunoprecipitations and analyses as in (A). Data are representative of n=3 independent experiments. (G) Quantification of TRIM28 co-precipitation by PIV2-V mutants. Band intensities of co-precipitated TRIM28 from experiments shown in (F) were normalized to their input levels and expressed relative (rel.) to the levels precipitated by PIV2-V. Data points correspond to results from individual experiments, and results from n=3 independent experiments are shown. Bars represent mean values, with error bars representing standard deviation (SD). Statistical significance was assessed using a one-sample t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant). (H) Comparison of TRIM28 and DDB1 co-precipitation abilities between PIV5-V and PIV5-V Y175W. HEK293T cells were transfected with plasmids expressing the indicated FLAG-tagged constructs prior to anti-FLAG immunoprecipitations and analyses as in (A). Data are representative of n=3 independent experiments. (I) Quantification of TRIM28 co-precipitation by PIV5-V and PIV5-V Y175W. Band intensities of co-precipitated TRIM28 from experiments shown in (H) were normalized to their input levels and expressed relative (rel.) to the levels precipitated by PIV5-V. Data points correspond to results from individual experiments, and results from n=3 independent experiments are shown. Bars represent mean values, with error bars representing standard deviation (SD). Statistical significance was assessed using a one-sample t test (*, P ≤ 0.05; ns, non-significant). (J) Sequence alignment of the V protein CTDs from different paramyxoviruses. The sequences are ordered by their TRIM28 co-precipitation efficiency (top: most efficient; bottom: least efficient). The residue identified as critical for differential PIV2-V/PIV5-V TRIM28 engagement (W178/Y175) is indicated with an orange arrow. Three additional residues in close structural proximity to this residue, based on the PIV5-V structure are indicated with blue arrows.

Article Snippet: Membranes were blocked in 5% milk-TBS-T (5% milk in TBS supplemented with 0.1% Tween 20), or 5% BSA-TBS-T for phosphorylated protein detection, and incubated with the following primary antibodies: rabbit anti-TRIM28 polyclonal antibody (A-300-274A, Bethyl); rabbit anti-phospho-TRIM28 (S824) polyclonal antibody (A-300-767A, Bethyl); mouse anti-FLAG M2 monoclonal antibody (F1804, Sigma Aldrich); rabbit anti-FLAG polyclonal antibody (F7425, Sigma Aldrich); mouse anti-V5 monoclonal antibody (MCA1360, BioRad); mouse anti-DDB1 monoclonal antibody (2B12D1, Invitrogen); rabbit anti-HA monoclonal antibody (C29F4, Cell Signaling Technologies); rabbit anti-GFP polyclonal antibody (GTX113617); rabbit anti-PA polyclonal antibody (GTX118991, Genetex); rabbit anti-NS1 polyclonal antibody (GTX125990, Genetex); rabbit anti-SUMO2/3 polyclonal antibody (ab3742, abcam); or mouse anti-actin monoclonal antibody (sc-47778, Santa-Cruz).

Techniques: Transfection, Construct, Expressing, Immunoprecipitation, Western Blot, Comparison, Standard Deviation, Sequencing, Residue

Journal: bioRxiv

Article Title: TRIM28 is a target for paramyxovirus V proteins

doi: 10.1101/2025.05.19.654852

Figure Lengend Snippet: (A) Schematic representation of TRIM28 domains, as well as the respective constructs subsequently used. Numbers indicate full-length (FL) TRIM28 amino acid positions. Constructs retain various combinations of functional domains: Really Interesting New Gene (RING); two BBoxes (B1 and B2); Coiled-Coil (CC); Linker (LNK); PHD; and Bromodomain (BRD). PBdS refers to a construct lacking residues surrounding the S824 phosphorylation site. The smallest domains identified as important for V co-precipitation in subsequent experiments are colored orange. (B) and ( C ) TRIM28 domains targeted by V. HEK293T cells were co-transfected with plasmids expressing FLAG-tagged mCherry or FLAG-tagged MeV-V together with the indicated HA-tagged TRIM28 construct. FLAG-tagged proteins were subsequently immunoprecipitated and the co-precipitated HA-tagged proteins were analyzed by western blot. Input fractions were also analyzed. Data are representative of n=3 independent experiments. Black arrows indicate predicted sizes of selected proteins of interest. (D) Sequence conservation of key domains from TRIM28 orthologs among selected known paramyxovirus host species. Percentage amino acid identity and similarity scores between the PHD-Bromodomain (PB) or Coiled-Coil (CC) domain of the indicated TRIM28 proteins were determined using Clustal Omega. (E) TIF-1 family member PB domains are also potential targets for V proteins. HEK293T cells were co-transfected with plasmids expressing FLAG-tagged mCherry or FLAG-tagged MeV-V together with the indicated HA-tagged PB domains from TRIM28, TRIM24, TRIM33, or TRIM66. FLAG-tagged proteins were subsequently immunoprecipitated and the co-precipitated HA-tagged proteins were analyzed by western blot. Input fractions were also analyzed. Data are representative of n=2 independent experiments.

Article Snippet: Membranes were blocked in 5% milk-TBS-T (5% milk in TBS supplemented with 0.1% Tween 20), or 5% BSA-TBS-T for phosphorylated protein detection, and incubated with the following primary antibodies: rabbit anti-TRIM28 polyclonal antibody (A-300-274A, Bethyl); rabbit anti-phospho-TRIM28 (S824) polyclonal antibody (A-300-767A, Bethyl); mouse anti-FLAG M2 monoclonal antibody (F1804, Sigma Aldrich); rabbit anti-FLAG polyclonal antibody (F7425, Sigma Aldrich); mouse anti-V5 monoclonal antibody (MCA1360, BioRad); mouse anti-DDB1 monoclonal antibody (2B12D1, Invitrogen); rabbit anti-HA monoclonal antibody (C29F4, Cell Signaling Technologies); rabbit anti-GFP polyclonal antibody (GTX113617); rabbit anti-PA polyclonal antibody (GTX118991, Genetex); rabbit anti-NS1 polyclonal antibody (GTX125990, Genetex); rabbit anti-SUMO2/3 polyclonal antibody (ab3742, abcam); or mouse anti-actin monoclonal antibody (sc-47778, Santa-Cruz).

Techniques: Construct, Functional Assay, Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Western Blot, Sequencing

Journal: bioRxiv

Article Title: TRIM28 is a target for paramyxovirus V proteins

doi: 10.1101/2025.05.19.654852

Figure Lengend Snippet: (A) and ( B ) Analysis of TRIM28 phosphorylation at S824 during virus infections. A549 cells were infected (or mock) with PIV2-GFP or PIV5-GFP (MOI = 2 PFU/cell), or IAV (MOI = 5 PFU/cell). Cell lysates were collected at the indicated times post-infection and western blot analysis was used to assess levels of the indicated proteins (phosphorylation of TRIM28 at S824: pS824 TR28). As a parallel positive control for TRIM28 phosphorylation at S824, cells were treated for 2 hours with 10 µM etoposide (etp). Data are representative of n=3 independent experiments. ( C ) and ( D ) Analysis of SUMO-modified TRIM28 levels during virus infections. A549 cells were infected similarly to experiments in (A), but cell lysates were subjected to immunoprecipitation (IP) using an anti-SUMO2/3 antibody or an anti-Ffluc antibody (control). The indicated co-precipitated proteins were analyzed by western blot. Input fractions were also analyzed. Data are representative of n=2 independent experiments. ( E ) and ( F ) Quantification of SUMO-modified TRIM28 levels during virus infections. Band intensities of immunoprecipitated SUMO2/3-modified TRIM28 from experiments shown in (C) and (D) were normalized to input levels of TRIM28 and expressed relative (rel.) to the SUMO2/3-modified TRIM28 levels in the mock-infected condition. Data points correspond to results from individual experiments, and results from n=2 independent experiments are shown. Bars represent mean values, with error bars representing standard deviation (SD).

Article Snippet: Membranes were blocked in 5% milk-TBS-T (5% milk in TBS supplemented with 0.1% Tween 20), or 5% BSA-TBS-T for phosphorylated protein detection, and incubated with the following primary antibodies: rabbit anti-TRIM28 polyclonal antibody (A-300-274A, Bethyl); rabbit anti-phospho-TRIM28 (S824) polyclonal antibody (A-300-767A, Bethyl); mouse anti-FLAG M2 monoclonal antibody (F1804, Sigma Aldrich); rabbit anti-FLAG polyclonal antibody (F7425, Sigma Aldrich); mouse anti-V5 monoclonal antibody (MCA1360, BioRad); mouse anti-DDB1 monoclonal antibody (2B12D1, Invitrogen); rabbit anti-HA monoclonal antibody (C29F4, Cell Signaling Technologies); rabbit anti-GFP polyclonal antibody (GTX113617); rabbit anti-PA polyclonal antibody (GTX118991, Genetex); rabbit anti-NS1 polyclonal antibody (GTX125990, Genetex); rabbit anti-SUMO2/3 polyclonal antibody (ab3742, abcam); or mouse anti-actin monoclonal antibody (sc-47778, Santa-Cruz).

Techniques: Phospho-proteomics, Virus, Infection, Western Blot, Positive Control, Modification, Immunoprecipitation, Control, Standard Deviation

Journal: bioRxiv

Article Title: TRIM28 is a target for paramyxovirus V proteins

doi: 10.1101/2025.05.19.654852

Figure Lengend Snippet: (A) Immunofluorescence microscopy of paramyxovirus and IAV single- or co-infections. A549 cells were infected (or mock) with PIV2-GFP or PIV5-GFP for 24 hours at MOI = 5 PFU/cell, before superinfection (or mock) with IAV for 10 hours at MOI = 5 PFU/cell. Cells were subsequently fixed, permeabilized, and stained with DAPI (nuclei, blue), anti-GFP antibody (paramyxovirus marker, green), and anti-NP antibody (IAV nucleoprotein, magenta). Images were acquired by confocal microscopy. Scale bars: 50 µm. Data are representative of n=2 independent experiments. (B) Analysis of SUMO-modified TRIM28 levels during paramyxovirus and IAV co-infections. A549 cells were infected similarly to experiments in (A), but cell lysates were subjected to immunoprecipitation (IP) using an anti-SUMO2/3 antibody or an anti-Ffluc antibody (control). The indicated co-precipitated proteins were analyzed by western blot. Input fractions were also analyzed. Data are representative of n=2 independent experiments. ( C – H ) RT-qPCR analysis of viral and host ERE transcripts during paramyxovirus and IAV single- or co-infections. A549 cells were infected similarly to experiments in (A), but the IAV superinfection lasted 16 hours. RNA was extracted from cell lysates and subsequently analyzed by RT-qPCR. (C–E) Viral transcript levels (PIV2 P, PIV5 M, and IAV M) were measured relative to mock-infected cells. (F–H) Host ERE levels (HERVK14C, ERV9.1, and LTR13) were measured relative to the IAV only infected condition. Relative quantities were calculated according to the ΔΔCt method in relation to 18S-rRNA. Data points correspond to results from individual experiments, and results from n=3 independent experiments are shown. Bars represent mean values, with error bars representing standard deviation (SD). Appropriate comparisons were tested for statistical significance using an unpaired t test (*, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, non-significant).

Article Snippet: Membranes were blocked in 5% milk-TBS-T (5% milk in TBS supplemented with 0.1% Tween 20), or 5% BSA-TBS-T for phosphorylated protein detection, and incubated with the following primary antibodies: rabbit anti-TRIM28 polyclonal antibody (A-300-274A, Bethyl); rabbit anti-phospho-TRIM28 (S824) polyclonal antibody (A-300-767A, Bethyl); mouse anti-FLAG M2 monoclonal antibody (F1804, Sigma Aldrich); rabbit anti-FLAG polyclonal antibody (F7425, Sigma Aldrich); mouse anti-V5 monoclonal antibody (MCA1360, BioRad); mouse anti-DDB1 monoclonal antibody (2B12D1, Invitrogen); rabbit anti-HA monoclonal antibody (C29F4, Cell Signaling Technologies); rabbit anti-GFP polyclonal antibody (GTX113617); rabbit anti-PA polyclonal antibody (GTX118991, Genetex); rabbit anti-NS1 polyclonal antibody (GTX125990, Genetex); rabbit anti-SUMO2/3 polyclonal antibody (ab3742, abcam); or mouse anti-actin monoclonal antibody (sc-47778, Santa-Cruz).

Techniques: Immunofluorescence, Microscopy, Infection, Staining, Marker, Confocal Microscopy, Modification, Immunoprecipitation, Control, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: MAGEC2 interacts with TRIM28 in melanoma cell lines. a Endogenous MAGEC2 binds with TRIM28 in A375 cells. A375 cells (1 × 10 8 ) were lysed and immunoprecipitated with mIgG, anti-MAGEC2 or anti-TRIM28 antibodies and immunoblotted with anti-MAGEC2 or anti-TRIM28 antibodies. Mouse IgG (mIgG) was used as a negative control for the immunoprecipitation. b Endogenous MAGEC2 binds with TRIM28 in Hs 695 T cells. Hs 695 T cells (1 × 10 8 ) were lysed and immunoprecipitated with anti-mIgG or anti-TRIM28 antibodies and immunoblotted with anti-MAGEC2 or anti-TRIM28 antibodies. Mouse IgG (mIgG) was used as a negative control for the immunoprecipitation. The experiments were repeated at least three times

Article Snippet: Antibodies used in this study were as follows: anti-GAPDH (AP0066, Bioworld, USA), anti-β-actin (AP0060, Bioworld), anti-TRIM28 (#4124, CST, USA; MB0014, Bioworld), anti-MAGEA1 (BS1217, Bioworld), anti-MAGEA3/6 (TA800918, Origene, USA), anti-MAGEA10 (15295–1-AP, Proteintech, USA), anti-MAGEC3(21491–1-AP, Proteintech), anti-FLAG (M185–3, MBL, Japan), and anti-mouse or rabbit IgG (H + L) HRP Conjugate (W4021/W4011, Promega).

Techniques: Immunoprecipitation, Negative Control

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: Knockdown of TRIM28 reduces MAGEC2 protein level and does not affect MAGEC2 mRNA level in melanoma cells. a , b Knockdown of TRIM28 decreases MAGEC2 protein level in A375 and Hs 695 T cells. TRIM28-specific siRNAs or control siRNA (si-NC) were transfected into A375 ( a ) or Hs 695 T cells ( b ) for 48 h, and cell lysates were immunoblotted with anti-MAGEC2 or anti-TRIM28 antibodies. Expression levels of GAPDH are indicated as an internal control. c , d Knockdown of TRIM28 does not affect the expression levels of other members of MAGE family in A375 cells ( c ) or Hs 695 T cells ( d ). A375 or Hs 695 T cells were transfected with TRIM28-specific siRNA for 48 h, and cell lysates were immunoblotted with indicated antibodies. e , f , g , h Knockdown of TRIM28 does not affect MAGEC2 mRNA level in A375 ( e , f ) or Hs 695 T cells ( g , h ). e , f Expression of MAGEC2 mRNA was examined in TRIM28-specific siRNA transfected-A375 cells by conventional PCR (E) or quantitative real-time PCR ( f ). g , h Knockdown of TRIM28 does not affect MAGEC2 mRNA level in Hs 695 T cells. Expression of MAGEC2 mRNA was examined in TRIM28-specific siRNA transfected-Hs 695 T cells by conventional PCR ( g ) or quantitative real-time PCR ( h ). Data are represented as mean ± SD. Each experiment was repeated three times

Article Snippet: Antibodies used in this study were as follows: anti-GAPDH (AP0066, Bioworld, USA), anti-β-actin (AP0060, Bioworld), anti-TRIM28 (#4124, CST, USA; MB0014, Bioworld), anti-MAGEA1 (BS1217, Bioworld), anti-MAGEA3/6 (TA800918, Origene, USA), anti-MAGEA10 (15295–1-AP, Proteintech, USA), anti-MAGEC3(21491–1-AP, Proteintech), anti-FLAG (M185–3, MBL, Japan), and anti-mouse or rabbit IgG (H + L) HRP Conjugate (W4021/W4011, Promega).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: Induced or exogenous expression of MAGEC2 is regulated by TRIM28. a Knockdown of TRIM28 decreases the expression level of MAGEC2 induced by demethylating agent 5-aza in A549 cells. TRIM28 specific siRNAs or control (si-NC) were transfected into A549 cells for 12 h, followed by adding 5-aza (10 μM) for 4 days, and cell lysates were immunoblotted with anti-MAGEC2 or anti-TRIM28 antibodies. Expression levels of GAPDH are indicated as an internal control. b Knockdown of TRIM28 decreases the exogenous expression level of MAGEC2 in Hs 695 T cells. FLAG-tagged MAGEC2 expression vector and TRIM28 specific siRNAs or si-NC were co-transfected into Hs 695 T cells for 48 h, and cell lysates were immunoblotted with indicated antibodies. In MAGEC2 blots, the upper band represents FLAG-tagged MAGEC2 and the lower band represents endogenously expressed MAGEC2. *, unspecific bands. c Overexpression of TRIM28 increases exogenous MAGEC2 protein level in AsPC1 cells. pCL-TRIM28 and FLAG-MAGEC2 or FLAG-MAGEA3 (as a control) expression vectors were co-transfected into AsPC1 cells for 48 h, and cell lysates were immunoblotted with indicated antibodies. Each experiment was repeated three times

Article Snippet: Antibodies used in this study were as follows: anti-GAPDH (AP0066, Bioworld, USA), anti-β-actin (AP0060, Bioworld), anti-TRIM28 (#4124, CST, USA; MB0014, Bioworld), anti-MAGEA1 (BS1217, Bioworld), anti-MAGEA3/6 (TA800918, Origene, USA), anti-MAGEA10 (15295–1-AP, Proteintech, USA), anti-MAGEC3(21491–1-AP, Proteintech), anti-FLAG (M185–3, MBL, Japan), and anti-mouse or rabbit IgG (H + L) HRP Conjugate (W4021/W4011, Promega).

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: MAGEC2 expression correlates with TRIM28 in human hepatocellular carcinoma tissues. a The expression of MAGEC2 and TRIM28 in representative positive and negative samples. Scale (left, 200×), 50 μm; Scale (right, 400×), 20 μm. b Spearman correlation analysis of the expression levels of MAGEC2 and TRIM28 in MAGEC2-positive samples. p = 0.0011

Article Snippet: Antibodies used in this study were as follows: anti-GAPDH (AP0066, Bioworld, USA), anti-β-actin (AP0060, Bioworld), anti-TRIM28 (#4124, CST, USA; MB0014, Bioworld), anti-MAGEA1 (BS1217, Bioworld), anti-MAGEA3/6 (TA800918, Origene, USA), anti-MAGEA10 (15295–1-AP, Proteintech, USA), anti-MAGEC3(21491–1-AP, Proteintech), anti-FLAG (M185–3, MBL, Japan), and anti-mouse or rabbit IgG (H + L) HRP Conjugate (W4021/W4011, Promega).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: TRIM28 protects MAGEC2 from proteasomal degradation. a , b Downregulation of TRIM28 promotes the proteasome-mediated degradation of MAGEC2 in A375 cells ( a ) or Hs 695 T cells ( b ). TRIM28 specific siRNAs or si-NC were transfected into Hs695 T or A375 cells. Twenty-four hours after transfection, cells were treated with PS-341 (10 μM), MG132 (20 μM) or vehicle (DMSO) for 8 h before immunoblotting with indicated antibodies. c Lysosome inhibitor does not affect the level of MAGEC2 protein in TRIM28 knockdown Hs 695 T cells or A375 cells. TRIM28 specific siRNAs or si-NC was transfected into A375 (left panel) or Hs695 (right panel) T cells. Twenty-four hours after transfection, cells were treated with CQ (20 μM) or vehicle (DMSO) for 24 h before immunoblotting with indicated antibodies. Detection of p62 is served as a positive control for treatment with CQ. Each experiment was repeated at least three times

Article Snippet: Antibodies used in this study were as follows: anti-GAPDH (AP0066, Bioworld, USA), anti-β-actin (AP0060, Bioworld), anti-TRIM28 (#4124, CST, USA; MB0014, Bioworld), anti-MAGEA1 (BS1217, Bioworld), anti-MAGEA3/6 (TA800918, Origene, USA), anti-MAGEA10 (15295–1-AP, Proteintech, USA), anti-MAGEC3(21491–1-AP, Proteintech), anti-FLAG (M185–3, MBL, Japan), and anti-mouse or rabbit IgG (H + L) HRP Conjugate (W4021/W4011, Promega).

Techniques: Transfection, Western Blot, Positive Control

Journal: Cell Communication and Signaling : CCS

Article Title: ZMAT2 condensates regulate the alternative splicing of TRIM28 to reduce cellular ROS accumulation, thereby promoting the proliferation of HCC cells

doi: 10.1186/s12964-024-01790-9

Figure Lengend Snippet: ZMAT2 regulates the progression of HCC through mRNA splicing. ( A ) Knockdown of ZMAT2 could influence mRNA splicing and processing. Red represents upregulation, and blue represents downregulation. ( B ) The pie chart displays the number of splicing and processing alteration events. ( C ) A pie chart demonstrates the proportion of different splicing types. Skipped Exon, SE; Alternative 5’ Splice Site, A5SS; Alternative 3’ Splice Site, A3SS; Mutually Exclusive Exons, MXE; retained intron, RI. ( D ) The dot plot displays the results of ZMAT2 RIPseq, revealing that 871 mRNAs are bound to ZMAT2. ( E ) The venn diagram illustrates the combined analysis results of RNAseq and RIPseq. A total of 25 genes possess binding sites for ZMAT2, and skipping exon occurs in these genes upon ZMAT2 knockdown. ( F ) RIP-qPCR is used to detect the binding efficiency between ZMAT2 and TRIM28 mRNA. ( G ) The visual peak plot displays the binding sites of ZMAT2 on the TRIM28 mRNA. ( H ) The binding motif of ZMAT2 on the TRIM28 mRNA. Bar charts show mean ± s.d. P < 0.05, *; P < 0.01,**; P < 0.001,***; P < 0.0001, ****

Article Snippet: Primary antibodies used were listed as follows unless otherwise specified: Anti-ZMAT2 antibody (A14354; Abclonal), anti-TRIM28 antibody (A2245; Abclonal), anti-p473-TRIM28 antibody (654102; Biolegend), anti-β-actin antibody (AC004; Abclonal), anti-DDDDK-Tag antibody (AE092; Abclonal), anti-Caspase-3 antibody (A2156; Abclonal), anti-NOX1 antibody (A8527; Abclonal), anti-GPX4 antibody (A21440; Abclonal), anti-Cleaved-GSDMD antibody (36425; CST), anti-GSDMD antibody (39754; CST).

Techniques: Knockdown, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: ZMAT2 condensates regulate the alternative splicing of TRIM28 to reduce cellular ROS accumulation, thereby promoting the proliferation of HCC cells

doi: 10.1186/s12964-024-01790-9

Figure Lengend Snippet: ZMAT2 regulates the alternative splicing of TRIM28. ( A ) Left: The Wiggle plot displays the skipping deletion of exon 11 of TRIM28 after knocking down ZMAT2. Right: RNAseq data indicated that knockdown of ZMAT2 results in skipping exon of exon 11 in TRIM28 mRNA, leading to a deletion of 25 bases. The graph was created with BioRender.com. The deletion of bases leads to translational frameshift mutation, causing the premature appearance of a stop codon in exon 12 and the production of a truncated mutant. ( B ) RT-PCR was used to detect the truncated fragment of TRIM28 resulting from skipping exon after ZMAT2 knockdown. The numbered boxes represent exons, and the red box indicates that skipping exon occurs in that exon. 259 bp (wild type) and 234 bp (truncated mutant) represent the lengths of the respective products. Specific primers were used in the experiment to detect wild-type and truncated TRIM28 mRNA with specificity. (C and D) Actinomycin D is used to inhibit mRNA transcription, and then qPCR is employed to measure the changes in the half-life of TRIM28 mRNA. HUH7 ( C ), HepG2 ( D ). Calculate the p-value using the growth data on the 6 h. TRIM28-NMD signifies the nonsense-mediated mRNA decay process involving the truncated mutant of TRIM28. ( E and F ) Detecting the expression level of TRIM28 after knocking down ZMAT2. HUH7 ( E ), HepG2 ( F ). ( G ) Intracellular ROS in HUH7 cells was detected with a red fluorescent probe dihydroethidium (DHE). ( H ) The box plot displays the trend of fluorescence intensity changes in Fig.G. ( I ) Intracellular ROS in HepG2 cells was detected with DHE. ( J ) The box plot displays the trend of fluorescence intensity changes in Fig.I. ( K ) Intracellular ROS in HUH7 cells was detected with DHE. ( L ) The box plot displays the trend of fluorescence intensity changes in Fig.K. ( M ) Intracellular ROS in HepG2 cells was detected with DHE. ( N ) The box plot displays the trend of fluorescence intensity changes in Fig.M. Bar charts show mean ± s.d. P < 0.05, *; P < 0.01,**; P < 0.001,***; P < 0.0001, ****

Article Snippet: Primary antibodies used were listed as follows unless otherwise specified: Anti-ZMAT2 antibody (A14354; Abclonal), anti-TRIM28 antibody (A2245; Abclonal), anti-p473-TRIM28 antibody (654102; Biolegend), anti-β-actin antibody (AC004; Abclonal), anti-DDDDK-Tag antibody (AE092; Abclonal), anti-Caspase-3 antibody (A2156; Abclonal), anti-NOX1 antibody (A8527; Abclonal), anti-GPX4 antibody (A21440; Abclonal), anti-Cleaved-GSDMD antibody (36425; CST), anti-GSDMD antibody (39754; CST).

Techniques: Alternative Splicing, Knockdown, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: ZMAT2 condensates regulate the alternative splicing of TRIM28 to reduce cellular ROS accumulation, thereby promoting the proliferation of HCC cells

doi: 10.1186/s12964-024-01790-9

Figure Lengend Snippet: ZMAT2 promotes the proliferation of HCC cells through TRIM28. ( A ) QPCR was used to detect the overexpression efficiency of TRIM28. ( B ) Western blot was used to detect the overexpression efficiency of TRIM28. ( C and D ) CCK-8 assay was used to detect the compensatory effect of TRIM28 on the inhibition of HCC cell proliferation caused by shZMAT2. HUH7( C ), HepG2 ( D ). ( E and F ) CCK-8 assay was used to detect the compensatory effect of uric acid on the inhibition of HCC cell proliferation caused by shZMAT2. HUH7(E), HepG2 ( F ). ( G ) Clone formation assay was used to detect the compensatory effect of TRIM28 on the inhibition of liver cancer cell proliferation caused by shZMAT2. The right histogram statistics show the difference in the number of clone formations. ( H ) The subcutaneous tumor in nude mice showed that knocking down ZMAT2 could inhibit tumor growth, while overexpression of TRIM28 could effectively alleviate the inhibitory effect. ( I ) Line chart demonstrates the growth rate of subcutaneous tumors in nude mice. Calculate the p-value using the growth data on the 33th day. ( J ) Knockdown of ZMAT2 resulted in a reduction in the weight of subcutaneous tumors in nude mice, whereas overexpression of TRIM28 effectively mitigated the suppressive effect triggered by shZMAT2. ( K ) DHE is used to detect ROS content in subcutaneous tumors. The histogram statistics on the right show the difference in ROS levels. Bar charts show mean ± s.d. P < 0.05, *; P < 0.01,**; P < 0.001,***; P < 0.0001, ****

Article Snippet: Primary antibodies used were listed as follows unless otherwise specified: Anti-ZMAT2 antibody (A14354; Abclonal), anti-TRIM28 antibody (A2245; Abclonal), anti-p473-TRIM28 antibody (654102; Biolegend), anti-β-actin antibody (AC004; Abclonal), anti-DDDDK-Tag antibody (AE092; Abclonal), anti-Caspase-3 antibody (A2156; Abclonal), anti-NOX1 antibody (A8527; Abclonal), anti-GPX4 antibody (A21440; Abclonal), anti-Cleaved-GSDMD antibody (36425; CST), anti-GSDMD antibody (39754; CST).

Techniques: Over Expression, Western Blot, CCK-8 Assay, Inhibition, Tube Formation Assay, Knockdown

Journal: Cell Communication and Signaling : CCS

Article Title: ZMAT2 condensates regulate the alternative splicing of TRIM28 to reduce cellular ROS accumulation, thereby promoting the proliferation of HCC cells

doi: 10.1186/s12964-024-01790-9

Figure Lengend Snippet: ZMAT2 forms liquid droplets with TRIM28 mRNA. ( A ) Observation of ZMAT2 condensate formation in HCC cells. ( B ) Prediction and distribution of ZMAT2 disorder sequence. Prediction website: http://d2p2.pro . ( C ) Expression and purification of recombinant ZMAT2 protein in E.coli . ( D ) Observation of phase separation of ZMAT2 recombinant protein in vitro with concentration gradients of 15 µM, 25 µM, 35 µM, and 45 µM. ( E ) Detection of ZMAT2 phase-separated condensate formation under different temperature conditions. ZMAT2 concentration 30 µM. ( F ) Detection of ZMAT2 (30 µM) phase-separated condensate formation under different NaCl concentration conditions. NaCl concentration: 150 mM, 300 mM, 500 mM, 800 mM. ( G ) FRAP is used to detect the fluidity of ZMAT2 condensates. ( H ) Detection of the formation of mixed condensates between ZMAT2 recombinant protein (20 µM) and truncated TRIM28 mRNA (with ZMAT2 binding motif). ( I ) Detection of the formation of mixed condensates between ZMAT2 recombinant protein (20 µM) and truncated TRIM28 mRNA (without ZMAT2 binding motif). ( J ) Observation of phase separation of ZMAT2 DR recombinant protein in vitro with concentration gradients of 15 µM, 25 µM, 35 µM, and 45 µM. ( K ) RT-PCR was used to detect the truncated fragment of TRIM28 resulting from skipping exon after ZMAT2 knockdown and ZMAT2/ZMAT2 DR overexpression. The numbered boxes represent exons, and the red box indicates that skipping exon occurs in that exon. 259 bp (wild type) and 234 bp (truncated mutant) represent the lengths of the respective products. Specific primers were used in the experiment to detect wild-type and truncated TRIM28 mRNA with specificity

Article Snippet: Primary antibodies used were listed as follows unless otherwise specified: Anti-ZMAT2 antibody (A14354; Abclonal), anti-TRIM28 antibody (A2245; Abclonal), anti-p473-TRIM28 antibody (654102; Biolegend), anti-β-actin antibody (AC004; Abclonal), anti-DDDDK-Tag antibody (AE092; Abclonal), anti-Caspase-3 antibody (A2156; Abclonal), anti-NOX1 antibody (A8527; Abclonal), anti-GPX4 antibody (A21440; Abclonal), anti-Cleaved-GSDMD antibody (36425; CST), anti-GSDMD antibody (39754; CST).

Techniques: Sequencing, Expressing, Purification, Recombinant, In Vitro, Concentration Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Knockdown, Over Expression, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: ZMAT2 condensates regulate the alternative splicing of TRIM28 to reduce cellular ROS accumulation, thereby promoting the proliferation of HCC cells

doi: 10.1186/s12964-024-01790-9

Figure Lengend Snippet: Schematic diagram: ZMAT2 and TRIM28 mRNA coalesce into protein-nucleic acid condensates, modulating the splicing of TRIM28 mRNA. Enhanced expression of ZMAT2 in HCC cells prompts an increase in TRIM28 expression, inhibits ROS accumulation, ultimately stimulating the proliferation of these HCC cells. Conversely, downregulation of ZMAT2 leads to the deletion of 25 bases from exon 11 of TRIM28, ultimately triggering nonsense-mediated decay (NMD). This in turn induces the accumulation of ROS within the cell, inhibiting cell proliferation. The graph was created with BioRender.com

Article Snippet: Primary antibodies used were listed as follows unless otherwise specified: Anti-ZMAT2 antibody (A14354; Abclonal), anti-TRIM28 antibody (A2245; Abclonal), anti-p473-TRIM28 antibody (654102; Biolegend), anti-β-actin antibody (AC004; Abclonal), anti-DDDDK-Tag antibody (AE092; Abclonal), anti-Caspase-3 antibody (A2156; Abclonal), anti-NOX1 antibody (A8527; Abclonal), anti-GPX4 antibody (A21440; Abclonal), anti-Cleaved-GSDMD antibody (36425; CST), anti-GSDMD antibody (39754; CST).

Techniques: Expressing