Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Sumoylation of La increases STAT3 mRNA binding by La. (A) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in GFP-LaWT (Wt)- and GFP-LaSD (K41/200R)-expressing cells. The values are normalized against GAPDH mRNA levels (n = 3). (B) RT-PCR on RNA samples prepared from RIP experiments using HEK293 cells stably overexpressing GFP-LaWT (Wt) or GFP-LaSD (K41/200R). Significantly less STAT3 mRNA was associated with GFP-LaSD than with GFP-LaWT. The asterisks indicate a significant difference (P < 0.01), as determined by Student's t test (n = 3). The data represent means and SD of the results of independent experiments.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Stable Transfection

Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Sumoylation of La promotes cell proliferation via a STAT3-mediated mechanism. (A) Representative immunoblot of STAT3 in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. GAPDH was used as a loading control. (B) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. The values are normalized against GAPDH mRNA levels (n = 3). (C) Representative immunoblot showing STAT3 protein levels in GFP-LaWT- and GFP-LaSD-expressing cells. GAPDH was used as a loading control. (D) Densitometry analysis showing significantly lower STAT3 protein expression in GFP-LaSD-expressing cells than in GFP-LaWT-expressing cells. The values are normalized against GAPDH protein levels (n = 3). (E) Representative fluorescence images showing transient transfection of control (GFP) and RFP-STAT3 in cells transduced with lentiviral constructs expressing shC or sh5. The transfection efficiency was ∼30%. (F) Overexpression of STAT3 (RFP-STAT3) restored the numbers of La-depleted (sh5) cells (n = 4; *, P = 0.0358). (G) Representative fluorescence images showing transient transfection of control and RFP-STAT3 in GFP-, GFP-LaWT-, and GFP-LaSD-expressing cells. The transfection efficiency was ∼30%. (H) Overexpression of STAT3 (RFP-STAT3) restored GFP-LaSD cell numbers (n = 3; *, P = 0.015). The asterisks indicate significant differences (P < 0.05), as determined by Student's t test. NS, not significant. The data represent means and SD of the results of independent experiments.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Western Blot, Transduction, Construct, Quantitative RT-PCR, Expressing, Fluorescence, Transfection, Over Expression

Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Global translation is not impaired in GFP-LaWT or GFP-LaSD cells, and sumoylation of La has a minor impact on STAT3 mRNA translation in HEK293 cells. (A and B) Autoradiography (A) and corresponding Coomassie-stained gel (B) of [35S]methionine-labeled total proteins of GFP-LaWT- and GFP-LaSD-expressing cells (30 min and 60 min). (C) Overlay of polyribosome fractionation profiles of two gradients loaded with extracts from GFP-LaWT (Wt-I/II) cells and two gradients loaded with extracts from GFP-LaSD (SD-I/II) cells. (D) STAT3 mRNA distribution in polyribosomal gradients from GFP-LaWT or GFP-LaSD cells as analyzed by RT-qPCR. (E) GADPH mRNA distribution in polyribosomal gradients from GFP-LaWT or GFP-LaSD cells as analyzed by RT-qPCR. The results are representative of three independent experiments.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Autoradiography, Staining, Labeling, Expressing, Fractionation, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Sumoylation of La promotes STAT3 protein stability. (A) GFP-LaWT- and GFP-LaSD-expressing cells were treated with CHX (20 μM) for the indicated times and analyzed for STAT3 expression by immunoblot analysis. GAPDH protein levels were analyzed as a loading control. (B) Quantification of immunoblots revealed that STAT3 stability was reduced in GFP-LaSD cells compared to GFP-LaWT (n = 3; *, P = 0.022 at 6 h and P = 0.023 at 12 h). The asterisks indicate significant differences (P < 0.05), as determined by Student's t test. (C) GFP-LaWT- and GFP-LaSD-expressing cells were treated with CHX (20 μM) and the proteasome inhibitor MG132 (10 μg/ml) for the indicated times and analyzed for STAT3 expression by immunoblot analysis. GAPDH protein levels were analyzed as a loading control. (D) Quantification of immunoblots revealed no significant difference in STAT3 stability in GFP-LaSD and GFP-LaWT cells (n = 3; P = 0.24 at 6 h; P = 0.51 at 12 h). (E) Representative immunoblot showing global ubiquitination in GFP-LaWT and GFP-LaSD cells treated or not treated with the proteasome inhibitor MG132 (10 μg/ml). GAPDH protein levels were analyzed as a loading control. (F) Representative immunoblot showing ubiquitination of STAT3 in GFP-LaSD cells in the absence or presence of MG132. GFP-LaWT- and GFP-LaSD-expressing cells were cotransfected with Flag-tagged STAT3 (STAT3-Flag) and HA-tagged ubiquitin (UB-HA). After 24 h, the cells were treated or not treated with MG132 (10 μg/ml) and subjected to immunoprecipitation applying a Flag-specific antibody. The immunoblots were analyzed with HA-specific (detection of ubiquitin) or Flag-specific (detection of STAT3) antibody. Protein levels in the extracts (Input) used for IP (bottom) were also assessed.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: International journal of oncology

Article Title: TIP60 governs the auto‑ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex.

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: Figure 1. TIP60 and ubiquitin co‑transfection induces the downregulation of UHRF1. Cells were co‑transfected with either TIP60‑eGFP (green) and RFP‑Ubiquitin (red) or eGFP and RFP‑Ubiquitin. Immunostaining of UHRF1 in HeLa cells without (A) or with treatment by MG‑132 (B). Cells were fixed following transfection and labeled with anti‑UHRF1 antibody. Endogenous UHRF1 protein was labeled with Alexa 647‑labeled secondary antibody before visualization with confocal microscopy. Scale bar, 10 µm. (C and D) Represent mean fluorescence intensities levels of UHRF1 in the different samples. Values are the mean ± SEM for three independent experiments; *P<0.05; ****P<0.0001 (vs. control group), determined by one‑way ANOVA with Tukey's post hoc test. UHRF1, ubiquitin‑like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa.

Article Snippet: Other antibodies used included rabbit polyclonal anti‐HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti‐DNMT1 (1:5,000; cat. no. PTG‐MAB0079, ProteoGenix), mouse monoclonal anti‐ubiquitin (1:500; cat. no. 05‐944, Sigma‐Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002‐1‐Ig, Proteintech Group, Inc.; and cat. no. A‐11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti‐GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti‐GFP (1:1,000; cat. no. 66002‐1‐Ig, Proteintech Group, Inc.), mouse mono‐ clonal anti‐p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti‐caspase‐3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti‐BCL2 (1:1,000; cat. no. 05‐826, Merck KGaA), mouse monoclonal anti‐poly(ADP‐ribose) polymerase (PARP; 1:1,000; cat. no. 51‐6639GR, BD Biosciences) and rabbit polyclonal anti‐BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Ubiquitin Proteomics, Immunostaining, Transfection, Labeling, Confocal Microscopy, Fluorescence, Control

Journal: International journal of oncology

Article Title: TIP60 governs the auto‑ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex.

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: Figure 3. TIP60 induces auto‑ubiquitination of UHRF1 in HeLa cells. Cells stably expressing either UHRF1 WT or UHRF1 C724A‑H741A mutant were transfected with either TIP60-eGFP WT or TIP60ΔMYST‑eGFP mutant. All samples were treated with 10 µM of MG‑132, 8 h before harvesting the cells. Whole cell lysates and immunoprecipitated samples were analyzed by SDS‑PAGE and then immunoblotted with anti‑GFP and anti‑Ubiquitin antibodies. Inputs and IP gels were processed in parallel under similar conditions. UHRF1, ubiquitin‑like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa.

Article Snippet: Other antibodies used included rabbit polyclonal anti‐HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti‐DNMT1 (1:5,000; cat. no. PTG‐MAB0079, ProteoGenix), mouse monoclonal anti‐ubiquitin (1:500; cat. no. 05‐944, Sigma‐Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002‐1‐Ig, Proteintech Group, Inc.; and cat. no. A‐11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti‐GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti‐GFP (1:1,000; cat. no. 66002‐1‐Ig, Proteintech Group, Inc.), mouse mono‐ clonal anti‐p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti‐caspase‐3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti‐BCL2 (1:1,000; cat. no. 05‐826, Merck KGaA), mouse monoclonal anti‐poly(ADP‐ribose) polymerase (PARP; 1:1,000; cat. no. 51‐6639GR, BD Biosciences) and rabbit polyclonal anti‐BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Stable Transfection, Expressing, Mutagenesis, Transfection, Immunoprecipitation