smad3 truncation mutants  (Addgene inc)

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 1. SMAD3 promotes the expression of AR and AR targets. (A) Volcano plot showing the differentially expressed genes between control and SMAD3-KD Rv1 cells in the RNA-seq analysis. (B) GO analysis of the downregulated genes after SMAD3 KD showing enrichment of the AR signaling pathway. (C) BART analysis of the downregulated genes after SMAD3 KD. AR is predicted to be a top transcription factor altered after SMAD3 KD. (D, E) Real-time RT-PCR results showing the reduced mRNA levels of AR, AR-V7 and example AR targets in the SMAD3-KD Rv1 (D) or C4-2 (E) cells. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (***P < 0.001). (F) Western blots showing the reduced level of AR and AR-V7 in the SMAD3-KD Rv1 or C4-2 cells. (G) Real-time RT-PCR results showing the reduced mRNA level of AR and AR-V7 in the SMAD3-KD LN95 cells. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (**P < 0.01; ***P < 0.001). (H) Western blots showing the reduced level of AR and AR-V7 in the SMAD3-KD LN95 cells.

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Expressing, Control, RNA Sequencing, Quantitative RT-PCR, Western Blot

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 2. SMAD4 or SMAD2 has little or no effect on the expression of AR and AR targets. (A, B) Volcano plot of RNA-seq results showing the differentially expressed genes upon KD of SMAD2 (A) or SMAD4 (B) in Rv1 cells. (C) Heatmap showing the altered level of AR and classic AR target genes in the RNA-seq analysis of SMAD3-KD, SMAD4-KD or SMAD2-KD Rv1 cells. The FPKM value of RNA-seq results was log2 transformed and used for the heatmap preparation using the pheatmap package in R. (D) GO analysis of the downregulated genes after SMAD2 KD. (E) BART analysis of the downregulated genes after SMAD2 KD to predict the altered transcription factors. (F) GO analysis of the downregulated genes after SMAD4 KD. (G) BART analysis of the downregulated genes after SMAD4 KD to predict the altered transcription factors.

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Expressing, RNA Sequencing, Transformation Assay

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 3. TGF- treatment has no effect on the expression of AR and AR targets. (A) TGF- did not induce phospho-SMAD3 or phospho-SMAD2 in Rv1 or C4-2 cells. Indicated cells were treated with 10 ng/ml of TGF- for 1 h. Western blot analysis was performed using the indicated antibodies. (B and C) Real-time RT-PCR results showing that TGF- treatment (10 ng/ml for 6 h) of Rv1 (B) or C4-2 (C) cells had no effect on transcript levels of AR, AR-V7 and AR targets. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (ns, not significant). (D) Real-time RT-PCR results showing the decreased transcript levels of AR, AR-V7 and AR targets in the SMAD3-KD VCaP cells. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (***P < 0.001). (E) Western blots showing the decreased level of AR and AR-V7 in the SMAD3-KD VCaP cells. (F) Western blots showing the increased levels of phospho-SMAD3 and phospho-SMAD2 in VCaP cells after TGF- treatment (10 ng/ml for 1 h). (G) Real-time RT-PCR analysis of VCaP cells showing no effect of TGF- treatment (10 ng/ml for 6 h) on transcript levels of AR, AR-V7 and AR targets relative to untreated cells. Quantification was presented as mean ± SD (n = 3), and t test was used for statistical analysis (ns, not significant; *P < 0.05). (H) Decreased levels of phospho-SMAD3 after treating PC3 cells with SIS3 for 24 h. (I) No changes of AR or AR-V7 levels after treating Rv1 cells with SIS3 for 24 h. (J) Effect of SIS3 treatment on transcript levels of AR, AR-V7 and AR target genes. Rv1 cells were treated with indicated concentration of SIS3 for 24 h before real-time RT-PCR analysis. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; ***P < 0.001).

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Concentration Assay

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 4. SMAD3 binds to intron 3 of AR gene to promote AR mRNA expression. (A) Heatmap showing two replicates of SMAD3 ChIP-seq peaks relative to IgG negative control antibody. (B) SMAD3 ChIP-seq peak annotation. (C) The Homer motif analysis showing the significant enrichment of SBE motifs on SMAD3 peaks. (D) Image showing the SMAD3 ChIP-seq peaks at the AR gene. The red lines and numbers indicate the nine regions analyzed by ChIP-PCR as described in (E). (E) ChIP-PCR of SMAD3 showing the enrichment of SMAD3 at regions 6 and 7 that are located at the center of the major SMAD3 ChIP-seq peak. ChIP assays were performed on Rv1 cells with control and SMAD3 antibodies. Precipitated chromatin was analyzed by qPCR for the nine regions of AR gene as indicated in D. % of input was calculated and presented as mean ± SD (n = 3), and t test was used for statistical analysis (***P < 0.001). The comparison at other regions (1–5,8,9) was not significant. (F) ChIP-PCR of SMAD4 showing no enrichment of SMAD4 at regions 6 and 7. ChIP assays were performed with control and SMAD4 antibodies as described in E. % of input was calculated and presented as mean ± SD (n = 3), and t test was used for statistical analysis. The comparison at each of the 9 regions was not significant. (G) ChIP-PCR of SMAD3 showing the similar SMAD3 enrichment at regions 6 and 7 of AR gene between control and SMAD4-KD Rv1 cells. % of input was calculated and presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant). Region 9 serves as a negative control region for SMAD3 binding. (H) Real-time RT-PCR results showing that CRISPRi constructs targeting region 6 or 7 reduced the mRNA levels of AR, AR-V7 and AR targets in Rv1 cells. 3 sgRNAs (sg1,sg2,sg3) were simultaneously used to target the three SBEs at or near region 6. Three sgRNAs (sg4,sg5,sg6) were simultaneously used to target the 4 SBEs at region 7. Rv1 cells expressing the indicated CRISPRi constructs were analyzed by the real-time RT-PCR analysis of indicated genes. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001). (I) ChIP-PCR of Cas9 showing the enrichment of Cas9 at AR exon 2, region 6 or 7 in the CRISPRi-expressing Rv1 cells as described in H. % of input was calculated and presented as mean ± SD (n = 3), and t test was used for statistical analysis (ns, not significant; ***P < 0.001).

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Expressing, ChIP-sequencing, Negative Control, Control, Comparison, Binding Assay, Quantitative RT-PCR, Construct

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 5. Overlaps of SMAD3 peaks and AR peaks in the ChIP-seq analysis. (A) Co-immunoprecipitation (Co-IP) of AR with SMAD3 in Rv1 or C4-2 cells. SMAD3 was immunoprecipitated from cells and analyzed by western blotting for co-precipitation of AR. Trueblot secondary antibodies were used in the western blots. (B) The C-terminal MH2 domain of SMAD3 interacted with AR. Myc-AR was co-expressed with Flag-tagged SMAD3 fragments (WT, C mutant lacking the C-terminal MH2 domain, or N mutant lacking the N-terminal MH1 domain) in 293T cells. Flag IP was performed and analyzed by western blotting with Flag or myc antibodies. (C) N-TAD domain of AR interacted with SMAD3. Myc-SMAD3 was co-expressed with the Flag-tagged AR fragments (N-terminal transactivation domain, N-TAD; DNA binding domain, DBD; Ligand-binding domain, LBD) in 293T cells, and analyzed as described in (B). (D) Heatmap showing two replicates of AR ChIP-seq peaks relative to IgG negative control antibody. (E) AR ChIP-seq peak annotation. (F) The Homer motif analysis showing the significant enrichment of ARE or AR half-site motifs on AR peaks. (G) Venn diagram showing the overlap of AR peaks and SMAD3 peaks in the ChIP-seq analysis. Cut&Run ChIP-seq studies were performed on Rv1 cells using AR or SMAD3 antibodies. Peak calling identified 12745 AR peaks and 11779 SMAD3 peaks. The overlapping of AR and SMAD3 peaks was determined by the ChIPpeakAnno package in R. (H) Heatmap showing the AR enriched peaks, common peaks and SMAD3 enriched peaks. (I) GO analysis of the genes associated with the common peaks between AR and SMAD3. (J) Distribution of AR (blue) and SMAD3 (green) ChIP-seq peak signal near the common peak center. (K) Example signal track image showing the SMAD3 peaks and AR peaks at the KLK3 enhancer or KLK2 promoter, which is indicated with an arrow. (L and M) ChIP-PCR showing the enrichment of AR and SMAD3 at KLK3 enhancer (L) or KLK2 promoter (M) of C4-2 cells after androgen treatment. ChIP assays were performed with control, AR or SMAD3 antibodies. Precipitated chromatin was analyzed by qPCR for KLK3 enhancer or KLK2 promoter. % of input was calculated and presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; ***P < 0.001).

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: ChIP-sequencing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Binding Assay, Ligand Binding Assay, Negative Control, Control

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 6. SMAD3 KD decreases the global ChIP-seq signal of AR. (A) Alteration of AR ChIP-seq peaks after SMAD3 KD in Rv1 cells. Cut&Run ChIP-seq studies were performed on Rv1 cells (control and SMAD3 KD) using AR antibodies. The alteration of AR peaks is shown in the Venn diagram including the control-enriched peaks, common peaks and SMAD3-KD-enriched peaks. (B) The genome distribution of AR peaks in control (n = 13073) and SMAD3-KD (n = 3997) Rv1 cells. (C) Heatmap showing the AR ChIP-seq peaks in control and SMAD3-KD Rv1 cells including the control-enriched peaks, common peaks and SMAD3-KD-enriched peaks. (D) Example signal track image showing the AR peak at KLK3 enhancer, KLK2 promoter or NKX3-1 promoter (indicated with an arrow) in control and SMAD3-KD Rv1 cells.

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: ChIP-sequencing, Control

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 7. Re-expression of AR partly rescues the AR target expression and growth of SMAD3-KD PCa cells. (A) Western blot showing re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells. Optimal amounts of lentivirus were used to restore the expression of AR and AR-V7 in the SMAD3- KD cells to the levels seen in control cells. (B) Real-time RT-PCR results showing that re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued the expression of example AR targets. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (C and D) Re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued colony formation in cell-culture plates. The indicated cells were seeded at low density and maintained for 2 weeks. Colony numbers were scored in the 12 high-power fields. The example image represents 4 high-power fields. Colony number per field was presented as mean ± SD (n = 12), and ANOVA was used for statistical analysis (***P < 0.001). (E and F) Re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued the colony formation in soft agar. The indicated cells were grown in soft agar for 3 weeks and colony numbers were scored in 12 high-power fields. The example image represents 1 high-power field. Colony number per field was presented as mean ± SD (n = 12), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (G) Western blot showing re-expression of AR in the SMAD3-KD C4-2 cells. (H) Real-time RT-PCR results showing that re-expression of AR in the SMAD3-KD C4-2 cells partly rescued the expression of example AR targets. Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (I) Re-expression of AR in the SMAD3-KD C4-2 cells partly rescued the colony formation in cell-culture plates. The procedure is as described in C and D. Colony numbers were scored in 8 high-power fields. Colony number per field was presented as mean ± SD (n = 8), and ANOVA was used for statistical analysis (***P < 0.001). (J) Re-expression of AR in the SMAD3-KD C4-2 cells partly rescued the colony formation in soft agar. The procedure is as described in E and F. Colony number per field was presented as mean ± SD (n = 12), and ANOVA was used for statistical analysis (***P < 0.001). (K and L) Re-expression of AR and AR-V7 in the SMAD3-KD Rv1 cells partly rescued the xenograft tumor formation. The indicated cells (1 × 106) were subcutaneously injected into athymic nude mice (n = 10 per group). Xenograft tumors were collected at 5 weeks post injection. The image (K) and weight (L) of xenograft tumors are shown. Tumor weight was presented as mean ± SD (n = 10), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001).

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Cell Culture, Injection

Journal: Nucleic acids research

Article Title: SMAD3 promotes expression and activity of the androgen receptor in prostate cancer.

doi: 10.1093/nar/gkad043

Figure Lengend Snippet: Figure 8. SMAD3 promotes the expression of AR mRNA in human PCa and can be targeted with PROTAC inhibitor. (A) The SMAD3 peak in AR intron 3 overlapped with the H3K27ac peak in human PCa cells. The H3K27ac peaks in AR upstream enhancer are also shown. The indicated datasets of H3K27ac ChIP-seq were download from GEO database and visualized by IGV software. (B) H3K27ac peaks in AR upstream enhancer and AR intron 3 enhancer in a published dataset of 8 CRPC PDX samples. (C) ATAC-seq peaks in AR upstream enhancer and AR intron 3 enhancer in a published dataset of 6 PCa PDX samples. (D) Positive correlation (Pearson correlation, R = 0.29, P-value = 2.9e-11) between AR mRNA and SMAD3 mRNA in the TCGA PCa dataset (n = 492). The correlation analysis was performed using the web server of GEPIA2. (E and F) Upregulation of AR mRNA and SMAD3 mRNA in metastatic PCa (E) or CRPC (F) relative to the primary PCa. The indicated datasets of profiling array studies on human PCa tissues were downloaded from the GEO database. Cai dataset includes 22 primary PCa and 29 metastatic PCa. Chandra dataset includes 207 primary PCa and 13 CRPC. The intensity values of AR and SMAD3 were Log2 transformed, and the mean values of the indicated PCa groups were compared. Quantification was presented as mean ± SD, and t test was used for statistical analysis (**P < 0.01; ***P < 0.001). (G and H) Partial knockdown of SMAD3 reduced the levels of AR mRNA (G) and protein (H) in the enzalutamide-resistant C4-2B cells (EnzR). Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (I and J) A SMAD3 PROTAC inhibitor decreased levels of AR, AR-V7 and AR targets. Rv1 cells were treated with the indicated concentration of SMAD3 PROTAC inhibitors for 24 hours before western blot (I) or real-time RT-PCR analysis (J). Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: SMAD3 truncation mutants ( N, lacking the MH1 domain (133–425) or C, lacking the MH2 domain (1–225)) were generated by PCR and subcloned into FlagpcDNA3. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2aPuro plasmid was a gift of Dr Charles Gersbach (Addgene plasmid #71236). shRNAs in pLKO.1 vector targeting SMAD3 (TRCN0000330127, TRCN0000330128), SMAD2 (TRCN0000010477) and SMAD4 (TRCN0000040031) were from Sigma-Aldrich (St. Louis, MO).

Techniques: Expressing, ChIP-sequencing, Software, Transformation Assay, Knockdown, Concentration Assay, Western Blot, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor Smad3 Is Required for the Inhibition of Adipogenesis by Retinoic Acid *

doi: 10.1074/jbc.M109.054536

Figure Lengend Snippet: Retinoic acid treatment triggers up-regulation of Smad3 protein expression. A, semi-quantitative RT-PCR analysis of Smad3 mRNA expression in C3H10T1/2 cells treated with retinoic acid or vehicle for 48 h. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a loading control. B, semi-quantitative RT-PCR analysis of Smad3 mRNA expression in C3H10T1/2 cells treated with RA for the indicated times. Amplification of glyceraldehyde-3-phosphate dehydrogenase is used as a loading control. C, Western analysis of Smad3 expression in C3H10T1/2 cells treated with vehicle or RA for the indicated time period in days. Actin serves as a loading control. D, Western analysis of Smad expression in 2-day post-confluent 3T3L1 cells induced to differentiate with the cAMP phosphodiesterase inhibitor MIX and insulin (+MI) in the presence or absence of the synthetic glucocorticoid dexamethasone (DEX) and RA as indicated for 48 h. Actin serves as a loading control. E, C3H10T1/2 cells treated with RA and cycloheximide (CHX) as indicated for 48 h were harvested for RNA isolation and semi-quantitative RT-PCR for Smad3 and glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The retroviral vector pLPCX-Smad3 (Addgene plasmid 12638), pGEX-Smad3 (Addgene plasmid 12630), and pEXL-FLAG-Smad3 (Addgene plasmid 10920) were purchased through Addgene and were kindly deposited by Dr. Rik Derynck and Dr. Bob Weinberg, respectively ( 25 , – 27 ). pLXSN, pLXSN-C/EBPβ, RSV-βgal, and the wild type and mutant Cebpa -reporter construct have been described previously ( 8 ).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Western Blot, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor Smad3 Is Required for the Inhibition of Adipogenesis by Retinoic Acid *

doi: 10.1074/jbc.M109.054536

Figure Lengend Snippet: Smad3 interferes with C/EBPβ occupancy of target promoters. A, interaction of in vitro translated full-length Smad3 and a truncated Smad3 lacking the MH1 domain (ΔMH1) with GST and GST-C/EBPβ. Following binding, precipitated Smad3 was revealed by Western blotting. B, avidin biotin-conjugated DNA assay evaluating the interaction of endogenous C/EBPβ from 3T3-L1 cells induced to differentiate for 24 h, with a double-stranded oligonucleotide coding for four repeats of a C/EBP consensus motif in the presence or absence of recombinant Smad3. Input represents 10% of the material used in the binding reaction. C, quantification of the interaction of C/EBPβ with a consensus DNA motif as in A by phosphorimager analysis. Data represent results from three experiments. Error bars represent the mean ± S.E. D, Western analysis of Smad and C/EBPβ expression in 3T3-L1 cells retrovirally transduced to express Smad3 or with empty virus and induced to differentiate into adipocytes with standard mixture for 48 h in the presence or absence of RA as indicated. Note that retrovirally expressed Smad3 is FLAG-tagged and thus migrates higher than endogenous Smad3. Endogenous Smad3 is indicated as the lower band observed in RA-treated empty virus control cells. The larger Smad2 is indicated by an arrowhead. Actin is used as a loading control. E, ChIP analysis of C/EBPβ occupancy of the Cebpa (C/EBPα) promoter and the Retn (resistin) promoter in 3T3-L1 cells retrovirally transduced to express Smad3 or with empty virus and induced to differentiate for 48 h. Chromatin was immunoprecipitated using anti-C/EBPβ antibody (β) or a type-matched nonspecific antibody (NS). Inputs represent 25% of the material used for precipitation. F, transient transcription assay in C3H10T1/2 cells measuring activation of the mouse Cebpa promoter by C/EBPβ and Smad3. Both the wild type (WT) promoter and a mutant reporter (MT) with the C/EBP-response element abolished were used. Data are reported as fold induction over the activity of the respective promoters in the absence of C/EBPβ and Smad3. Luciferase activity was corrected with β-galactosidase activity from a cotransfected reporter to correct for transfection efficiency. Data represent the means of two independent experiments performed in duplicate. Error bars represent the mean ± S.E.

Article Snippet: The retroviral vector pLPCX-Smad3 (Addgene plasmid 12638), pGEX-Smad3 (Addgene plasmid 12630), and pEXL-FLAG-Smad3 (Addgene plasmid 10920) were purchased through Addgene and were kindly deposited by Dr. Rik Derynck and Dr. Bob Weinberg, respectively ( 25 , – 27 ). pLXSN, pLXSN-C/EBPβ, RSV-βgal, and the wild type and mutant Cebpa -reporter construct have been described previously ( 8 ).

Techniques: In Vitro, Binding Assay, Western Blot, Avidin-Biotin Assay, Recombinant, Expressing, Immunoprecipitation, Transcription Assay, Activation Assay, Mutagenesis, Activity Assay, Luciferase, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor Smad3 Is Required for the Inhibition of Adipogenesis by Retinoic Acid *

doi: 10.1074/jbc.M109.054536

Figure Lengend Snippet: RA treatment increases nuclear Smad3. A, time course of Western analysis of nuclear Smad3 in 3T3-L1 cells induced to differentiate with MIX, insulin, and dexamethasone (DEX) (+MID) and treated with vehicle, RA, or TGFβ for the times indicated. B, quantification of nuclear Smad3 following RA or TGFβ treatment as in A. C, nuclear (N) and cytoplasmic (C) localization of Smad3 and Smad4 in 3T3-L1 cells induced to differentiate for 48 h in the presence or absence of RA. Tubulin is a cytoplasmic marker, whereas C/EBPβ is used to evaluate the integrity of the nuclear compartment. D, transient transcription assay measuring the activation of a synthetic Smad-responsive reporter construct by a 48-h RA treatment in NIH 3T3 cells. Relative light units were corrected for β-galactosidase activity from a cotransfected constitutively active reporter construct to correct for transfection efficiency. Data represent three independent experiments, and error bars represent the mean ± S.E. (*, p < 0.05). Veh, vehicle.

Article Snippet: The retroviral vector pLPCX-Smad3 (Addgene plasmid 12638), pGEX-Smad3 (Addgene plasmid 12630), and pEXL-FLAG-Smad3 (Addgene plasmid 10920) were purchased through Addgene and were kindly deposited by Dr. Rik Derynck and Dr. Bob Weinberg, respectively ( 25 , – 27 ). pLXSN, pLXSN-C/EBPβ, RSV-βgal, and the wild type and mutant Cebpa -reporter construct have been described previously ( 8 ).

Techniques: Western Blot, Marker, Transcription Assay, Activation Assay, Construct, Activity Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor Smad3 Is Required for the Inhibition of Adipogenesis by Retinoic Acid *

doi: 10.1074/jbc.M109.054536

Figure Lengend Snippet: Smad3 overexpression is not sufficient to inhibit adipocyte differentiation. A, Oil red O micrographs of 3T3-L1 cells retrovirally infected to express Smad3 or with empty vector (pLP) and induced to differentiate into adipocytes with standard mixture for 8 days in the presence or absence of RA. B, Western analysis of adipocyte marker expression in 3T3-L1 cells transduced and induced to differentiate as in A. Actin expression is shown as a loading control. MID, MIX, insulin, and dexamethasone.

Article Snippet: The retroviral vector pLPCX-Smad3 (Addgene plasmid 12638), pGEX-Smad3 (Addgene plasmid 12630), and pEXL-FLAG-Smad3 (Addgene plasmid 10920) were purchased through Addgene and were kindly deposited by Dr. Rik Derynck and Dr. Bob Weinberg, respectively ( 25 , – 27 ). pLXSN, pLXSN-C/EBPβ, RSV-βgal, and the wild type and mutant Cebpa -reporter construct have been described previously ( 8 ).

Techniques: Over Expression, Infection, Plasmid Preparation, Western Blot, Marker, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor Smad3 Is Required for the Inhibition of Adipogenesis by Retinoic Acid *

doi: 10.1074/jbc.M109.054536

Figure Lengend Snippet: Inhibition of adipogenesis by RA is abrogated by loss of Smad3 expression. A, Western analysis of Smad and C/EBPβ expression in 3T3-L1 cells retrovirally transduced to express a small hairpin RNA directed against Smad3 (shSmad3) or with empty vector (pMKO) and induced to differentiate into adipocytes with standard mixture for 48 h in the presence or absence of RA. B, Oil red O micrographs of 3T3-L1 cells retrovirally transduced as in A and induced to differentiate into adipocytes with induction mixture for 8 days. MID, MIX, insulin, and dexamethasone. C, Western analysis of adipocyte marker expression in 3T3-L1 cells transduced and induced to differentiate as in B. D, ChIP analysis of C/EBPβ occupancy of the Cebpa promoter in cells retrovirally transduced and induced to differentiate as in A. Chromatin was immunoprecipitated with anti-C/EBPβ antibody (β) or a type-matched nonspecific antibody (NS) as indicated. Input represents 25% of the material used for immunoprecipitation.

Article Snippet: The retroviral vector pLPCX-Smad3 (Addgene plasmid 12638), pGEX-Smad3 (Addgene plasmid 12630), and pEXL-FLAG-Smad3 (Addgene plasmid 10920) were purchased through Addgene and were kindly deposited by Dr. Rik Derynck and Dr. Bob Weinberg, respectively ( 25 , – 27 ). pLXSN, pLXSN-C/EBPβ, RSV-βgal, and the wild type and mutant Cebpa -reporter construct have been described previously ( 8 ).

Techniques: Inhibition, Expressing, Western Blot, Plasmid Preparation, Marker, Immunoprecipitation

Journal: PLoS ONE

Article Title: Sox17 Promotes Cell Cycle Progression and Inhibits TGF-β/Smad3 Signaling to Initiate Progenitor Cell Behavior in the Respiratory Epithelium

doi: 10.1371/journal.pone.0005711

Figure Lengend Snippet: (A) MLE-15 cells were transfected with the TGF-β/Smad-responsive reporter 3TP-luciferase (3TP-Luc) and increasing amounts of vector, t-Sox17, or full length Sox17 in the presence or absence 2 ng/ml TGF-β1. Sox17 inhibited TGF-β1-mediated activation of 3TP-Luc. The graph represents average fold activity±standard deviation. Asterisks indicate statistical significance determined by Student's t-test (p<0.05). (B–C) Lysates from MLE-15 cells expressing full length or mutant FLAG-Smad3 or Sox17-V5 (schematic representations) were incubated with GST only and GST-Sox17 (B) or GST-Smad3 (C), respectively. GST-pulldowns revealed an interaction between amino acids 129–359 of Sox17 and the linker region and MH2 domain of Smad3. MH1 and MH2, MAD homology domains; HMG, High mobility group. (D) MLE-15 cells were transfected with 3TP-Luc in the presence or absence Smad3 and wild type or mutant Sox17. Sox17 full length and C-terminal deletion inhibited Smad3-dependent transcriptional activity. Representative results are shown±standard deviation of the mean. Asterisks indicate statistical significance determined by Student's t-test (p<0.05). (E) Sox17 antagonizes TGF-β1/Smad3-mediated repression of cyclin D1 promoter activity. MLE-15 cells were co-transfected with −944/+187 cyclin D1-luciferase and Smad3 or Sox17 in the presence or absence of 5 ng/ml TGF-β1. The graph represents average fold activity±standard deviation. Pound signs and asterisk indicate statistical significance determined by Student's t-test (p<0.05). (F) Sox17 blocks Smad3 DNA binding. MLE-15 cell were transfected with FLAG-Smad3 in the presence or absence of Sox17. After 24 h, cells were incubated with 5 ng/ml TGF-β1 for 8 h before harvesting. Binding of Smad3 to the p15 promoter was assessed by chromatin immunoprecipitation and quantified by real time PCR. Graph represents average fold enrichment of FLAG immunoprecipitated samples relative to IgG negative control samples. Asterisk indicates statistical significance determined by Student's t-test (p<0.05). Expression of Smad3 and Sox17 inputs were assessed by immunoblot.

Article Snippet: The pGEX-human Smad3 construct (Addgene plasmid 12630) encoding for GST-Smad3 was generated in the lab of Dr. Rik Derynck (UCSF) , and the FLAG-tagged Smad3 expression vectors were generated by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center) (FLAG-Smad3 (1–425), Addgene plasmid 14052; FLAG-Smad3 (1–145), Addgene plasmid 14965; FLAG-Smad3 (146–425), Addgene plasmid 14966; FLAG-Smad3 (220–425), Addgene plasmid 14967) , .

Techniques: Transfection, Luciferase, Plasmid Preparation, Activation Assay, Activity Assay, Standard Deviation, Expressing, Mutagenesis, Incubation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Western Blot