Journal: Breast Cancer Research : BCR

Article Title: BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion

doi: 10.1186/s13058-018-1046-3

Figure Lengend Snippet: BHLHE40 activates gene expression by sequestering histone deacetylase (HDAC)1 and HDAC2 from genome DNA binding in MDA-MB-231 cells exposed to hypoxia and low glucose (1%O 2 /LG, 4 h). a BHLHE40-knockout (KO) diminished dissociation of HDAC1 and HDAC2 from the promoter region of HBEGF in MDA-MB-231 cells exposed to 1%O 2 /LG (4 h), as determined by chromatin immunoprecipitation (ChIP) followed by qPCR of the HBEGF promoter region (−529 to −372 from the transcription start site). HBEGF promoter binding activity of HDAC1 or HDAC2 was calculated as: (DNA amount in anti-HADC IP complex – DNA amount in control IgG IP complex)/DNA amount in 1% input. * p < 0.05 ( n = 6, 1%O 2 /LG vs. control), ** p < 0.05 ( n = 6, KO vs. EV). b 1%O 2 /LG treatment increased interactions between BHLHE40 and HDAC1/2 in the soluble cellular fraction of MDA-MB-231 empty vector (EV) cells. Protein-protein interaction was detected by reciprocal co-immunoprecipitation (IP)/immunoblotting (IB) analysis. c HDAC inhibition induced expression of BHLHE40 target genes. Cells were exposed to hypoxia (1% O 2 ) or HDAC inhibitors (BRD6688 10 μM or TSA 2 μM) for 24 h. mRNA expression levels were determined by qPCR, normalized to RPL13A, and presented as mean ± SD ( n = 6). * p < 0.05 ( n = 6, treated vs. untreated control cells), one-way ANOVA followed by Tukey’s post-hoc tests

Article Snippet: To detect protein-protein interaction, soluble proteins were extracted using the Pierce IP Lysis Buffer (Thermo Scientific) supplemented with protease inhibitor cocktails and co-immunoprecipitation (CoIP) was performed using the TrueBlot Immunoprecipitation and Western Blot Kit (Rockland Immunochemicals Inc., Limerick, PA, USA).

Techniques: Gene Expression, Histone Deacetylase Assay, Binding Assay, Knock-Out, Chromatin Immunoprecipitation, Activity Assay, Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Inhibition, Expressing

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