Confocal Immunofluorescent staining of nuclear HER3.

<p>C-TERM and N-TERM HER3 antibodies were used to visualize HER3 localization in H226^R, SKBr3, and BT549 cells. Alexa Fluor-546 secondary antibody was used to visualize HER3 (RED) and DAPI was used to visualize the nucleus (BLUE). Merged images were magnified to depict the nuclear localization of HER3 (see white arrows). Magnification 600X.</p

Nuclear HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain.

<p><b>A. HER3 knockdown decreases the activation of the cyclin D1 promoter.</b> H226^R, SKBr3, and MCF-7 cells were incubated with non-targeting (NT) or HER3 siRNA for 24 hr followed by transfection with the 122 bp cyclin D1 promoter-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 6). Percent decreases in cyclin D1 promoter-luciferase activity were normalized to NT transfected cells. The graph is representative of three independent experiments. Luciferase lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. <b>B. HER3 overexpression activates the cyclin D1 promoter.</b> SCC6, HCC1954, SKBr3, and BT474 cells were transfected with HER3WT or control vector, 122 bp cyclin-D1-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1-promoter luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Nuclear lysate was harvested from each cell line and fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for the Nuc fraction. <b>C. HER3ΔB₁ΔB₂ overexpression can prevent the activation of the cyclin D1 promoter while HER3DM remains functional.</b> SCC6, BT474 and HCC1954 cells were transfected with HER3WT, HER3DM, HER3ΔB₁ΔB₂ or control vector, the 122 bp cyclin-D1-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of seven independent experiments. <b>Inset 1:</b> CHOK1 cells were transfected with HER3WT, HER3ΔB₁ΔB₂ or control vector for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. <b>Inset 2:</b> CHOK1 cells were transfected with HER3WT, HER3DM, HER3ΔB₁ΔB₂ or control vector and stimulated for 40 min with 5 nM neuregulin-1. Whole cell lysate was fractionated on SDS-PAGE followed by immunoblotting for indicated proteins. <b>D. HER3 knockdown decreases cyclin D1 expression.</b> SCC6 and BT474 cells were incubated with NT or HER3 siRNA for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to NT transfected cells. The graph is representative of two independent experiments. <b>E. The overexpression of HER3WT enhances cyclin D1 expression while HER3ΔB₁ΔB₂ is hindered.</b> SCC6 and BT474 cells were transfected with HER3WT, HER3ΔB₁ΔB₂ or control vector for 72 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of four independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of <i>Renilla</i> luciferase. All data points are represented as mean +/− s.e.m. P<0.05.</p

HER3 can associate with the cyclin D1 promoter.

<p><b>A. Nuclear EGFR and HER2 associate with the cyclin D1 promoter.</b> ChIP using an anti-EGFR, anti-HER2, or normal rabbit IgG antibody was performed with SKBr3 cells and isolated DNA was subsequently used for qPCR with primers flanking the 122 bp cyclin D1 promoter region (n = 3). qPCR specificity for the 122 bp cyclin D1 promoter was confirmed by agarose gel electrophoresis of semi-qPCR products. DAPA analysis was performed using a biotinylated 122 bp cyclin D1 promoter probe incubated with 400 ug of nuclear lysate harvested from SKBr3 cells. Bound proteins were isolated with streptavidin agarose beads and subsequently fractionated on SDS-PAGE followed by immunoblotting for EGFR or HER2. Nuclear lysate incubated with beads only lacked association. <b>B. Nuclear HER3 can associate with the cyclin D1 promoter.</b> ChIP using a N-TERM anti-HER3 or a human IgG antibody was performed with H226^R, SKBr3, and BT549 cells. qPCR was performed as in 5A. <b>C. HER3 can associate with a cyclin D1 promoter probe.</b> DAPA analysis was performed using nuclear lysate harvested from H226^R, SKBr3, MCF-7, and BT549 cells as in 5A. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. <b>D. HER3 association with the cyclin D1 promoter probe is specific.</b> DAPA analysis was performed as in 5A using nuclear lysate harvested from H226^R, SKBr3, MCF-7, and BT549 cells transfected with either non-targeting (NT) or HER3 siRNA for 48 hr. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. All data points for ChIP are represented as mean +/− s.e.m and normalized to the IgG control. P<0.05.</p

The C-terminus of HER3 contains a strong transactivation domain.

<p><b>A. EGFR intracellular domain (ICD) map and plasmid validation.</b> The intracellular domain (ICD) and C-terminal domain (CTD) of EGFR were fused to the Gal4 DNA binding domain (Gal4DBD). CHOK1 cells were transfected with each construct for 48 hr prior to harvesting WC lysate and fractionation on SDS-PAGE followed by immunoblotting for EGFR and Gal4DBD. EGFRWT vector was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control<b>. B. EGFR-CTD contains strong transactivation potential.</b> CHOK1 cells were transfected with EGFR-ICD or EGFR-CTD constructs, UAS-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. <b>C. HER3-ICD map and plasmid validation.</b> The HER3- ICD, juxtamembrane and tyrosine kinase domain (JKD), and CTD were fused to the Gal4DBD. Transfection was performed the same as in 1A and immunoblot analysis was performed for HER3 and Gal4DBD. HER3WT was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control. <b>D. HER3-CTD contains strong transactivation potential.</b> CHOK1, H226^R, and SKBr3 cells were transfected with HER3-ICD, HER3-JKD, and HER3-CTD constructs, UAS-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. Luciferase activity was normalized to DNA content, protein content, and the expression of <i>Renilla</i> luciferase in all assays. Luciferase activity detected for each construct was normalized to the Gal4DBD vector control. Data points are represented as mean+/−s.e.m. p<0.05. TM (transmembrane); JM (juxtamembrane); KD (kinase domain).</p

The HER3 receptor is localized to the nucleus in its full-length form.

<p><b>A. HER3 is expressed in numerous cancer cell lines.</b> Whole cell protein lysates were isolated from various breast, lung, HNSCC, and colon cancer cell lines. Lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin was used as a loading control. <b>B. HER3 is localized to the nucleus in cancer cell lines.</b> H226^R, SKBr3, MCF-7, and BT549 cells were harvested for whole cell (WC), non-nuclear (NN) and nuclear (Nuc) protein, fractionated on SDS-PAGE followed by immunoblotting for HER3. Clathrin, dynamin, calnexin, α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. <b>C. Specificity of nuclear HER3 by siRNA.</b> H226^R and SKBr3 cells were harvested for NN and Nuc protein 48 hr post treatment with siHER3 or non-targeting (NT) siRNA. Experimental procedure as in 1B. α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. <b>D. Full-length HER3 is localized to the nucleus.</b> H226^R and SKBr3 cells were harvested for WC, NN, and Nuc lysate. WC lysates were harvested 48 hr post treatment with siHER3. 250 ug of cell lysate was immunoprecipitated with an N-TERM HER3 antibody or human IgG control. The immunoprecipitates were fractionated on SDS-PAGE followed by immunoblotting for HER3 with a C-TERM antibody.</p

An intracellular domain (ICD) mutant of HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain.

<p><b>A. HER3WT ICD (WT-ICD) can activate the cyclin D1 promoter while HER3ΔB₁ΔB₂-ICD (ΔB₁ΔB₂-ICD) is hindered.</b> SCC6, BT474, and HCC1954 cells were transfected with WT-ICD, ΔB₁ΔB₂-ICD or control vector, the 122 bp cyclin D1-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. <b>Inset 1:</b> Illustration of both the WT-ICD and ΔB₁ΔB₂-ICD constructs. <b>Inset 2:</b> CHOK1 cells were transfected with WT-ICD and ΔB₁ΔB₂-ICD for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. <b>B. The overexpression of WT-ICD can enhance cyclin D1 expression while ΔB₁ΔB₂-ICD is hindered.</b> SCC6 and BT474 cells were transfected with WT-ICD, ΔB₁ΔB₂-ICD or control vector for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of three independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of <i>Renilla</i> luciferase. All data points are represented as mean +/− s.e.m. P<0.05. ICD (intracellular domain).</p