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TGFBR3 and JUND are functionally important for 3D morphogenesis. ( a ) Time-dependent expression of TGFBR3 during 3D morphogenesis . ( b ) Knockdown of TGFBR3 and inducible addback of murine RNAi-resistant Tgfbr3. TGFBR3/Tgfbr3 levels for cells cultured in the absence (Lane 1 and 2) or presence (Lane 3) of 1 µg/ml DOX for 24 hours were analyzed by immunoblotting. Hsp90 was used as a loading control. Densitometry of TGFBR3/Tgfbr3 abundance is shown normalized to the shGFP control. ( c and d ) Blocking TGFBR3 induction specifically elicits a ductal-branching phenotype. The MCF10A-5E lines described in ( b ) were placed in morphogenesis in the absence (control and <t>shTGFBR3)</t> or presence (Tgfbr3 addback) of 1 µg/ml DOX from day 4–10. Acini were fixed at day 10 of 3D culture, stained for E-cadherin (green) and HA-tagged Tgfbr3 (red), and analyzed by confocal immunofluorescence. Cells were counterstained with DRAQ5 (blue) to label nuclei. ( e ) Constitutive expression of HA-tagged JUND analyzed by immunoblotting. Densitometry of JUND abundance is shown normalized to pBabe vector control. ( f and g ) Constitutive JUND expression causes stable cribiform-like acinar structures. Acini from the MCF10A-5E lines described in ( e ) were placed in morphogenesis, fixed at day 28, stained for E-cadherin (green) and HA-tagged JUND (red), and analyzed by confocal immunofluorescence. Cells were counterstained with DRAQ5 (blue) to label nuclei. ( h ) Homogenization of JUND expression by knockdown of JUND and addback with murine RNAi-resistant JunD to near-endogenous expression levels. JUND/JunD levels were determined by immunoblotting. Densitometry of JUND/JunD abundance is shown normalized to the shGFP control. ( i ) Quantification of the cribiform-like phenotype at day 28 of 3D culture for the cells in ( h ). For ( a ), ( c ), ( g ), and ( i ), data are shown as the mean ± s.e.m. of n=3 ( a ) or n=4 ( c , g , i ) independent experiments. For ( d ) and ( f ), scale bar is 20 µm. For ( e ) and ( h ), tubulin was used as a loading control and n.s. denotes a non-specific band. For source data, see .
Plko 1 Shtgfbr3 Puro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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TGFBR3 and JUND are functionally important for 3D morphogenesis. ( a ) Time-dependent expression of TGFBR3 during 3D morphogenesis . ( b ) Knockdown of TGFBR3 and inducible addback of murine RNAi-resistant Tgfbr3. TGFBR3/Tgfbr3 levels for cells cultured in the absence (Lane 1 and 2) or presence (Lane 3) of 1 µg/ml DOX for 24 hours were analyzed by immunoblotting. Hsp90 was used as a loading control. Densitometry of TGFBR3/Tgfbr3 abundance is shown normalized to the shGFP control. ( c and d ) Blocking TGFBR3 induction specifically elicits a ductal-branching phenotype. The MCF10A-5E lines described in ( b ) were placed in morphogenesis in the absence (control and shTGFBR3) or presence (Tgfbr3 addback) of 1 µg/ml DOX from day 4–10. Acini were fixed at day 10 of 3D culture, stained for E-cadherin (green) and HA-tagged Tgfbr3 (red), and analyzed by confocal immunofluorescence. Cells were counterstained with DRAQ5 (blue) to label nuclei. ( e ) Constitutive expression of HA-tagged JUND analyzed by immunoblotting. Densitometry of JUND abundance is shown normalized to pBabe vector control. ( f and g ) Constitutive JUND expression causes stable cribiform-like acinar structures. Acini from the MCF10A-5E lines described in ( e ) were placed in morphogenesis, fixed at day 28, stained for E-cadherin (green) and HA-tagged JUND (red), and analyzed by confocal immunofluorescence. Cells were counterstained with DRAQ5 (blue) to label nuclei. ( h ) Homogenization of JUND expression by knockdown of JUND and addback with murine RNAi-resistant JunD to near-endogenous expression levels. JUND/JunD levels were determined by immunoblotting. Densitometry of JUND/JunD abundance is shown normalized to the shGFP control. ( i ) Quantification of the cribiform-like phenotype at day 28 of 3D culture for the cells in ( h ). For ( a ), ( c ), ( g ), and ( i ), data are shown as the mean ± s.e.m. of n=3 ( a ) or n=4 ( c , g , i ) independent experiments. For ( d ) and ( f ), scale bar is 20 µm. For ( e ) and ( h ), tubulin was used as a loading control and n.s. denotes a non-specific band. For source data, see .

Journal: Nature cell biology

Article Title: A time- and matrix-dependent TGFBR3–JUND–KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies

doi: 10.1038/ncb2930

Figure Lengend Snippet: TGFBR3 and JUND are functionally important for 3D morphogenesis. ( a ) Time-dependent expression of TGFBR3 during 3D morphogenesis . ( b ) Knockdown of TGFBR3 and inducible addback of murine RNAi-resistant Tgfbr3. TGFBR3/Tgfbr3 levels for cells cultured in the absence (Lane 1 and 2) or presence (Lane 3) of 1 µg/ml DOX for 24 hours were analyzed by immunoblotting. Hsp90 was used as a loading control. Densitometry of TGFBR3/Tgfbr3 abundance is shown normalized to the shGFP control. ( c and d ) Blocking TGFBR3 induction specifically elicits a ductal-branching phenotype. The MCF10A-5E lines described in ( b ) were placed in morphogenesis in the absence (control and shTGFBR3) or presence (Tgfbr3 addback) of 1 µg/ml DOX from day 4–10. Acini were fixed at day 10 of 3D culture, stained for E-cadherin (green) and HA-tagged Tgfbr3 (red), and analyzed by confocal immunofluorescence. Cells were counterstained with DRAQ5 (blue) to label nuclei. ( e ) Constitutive expression of HA-tagged JUND analyzed by immunoblotting. Densitometry of JUND abundance is shown normalized to pBabe vector control. ( f and g ) Constitutive JUND expression causes stable cribiform-like acinar structures. Acini from the MCF10A-5E lines described in ( e ) were placed in morphogenesis, fixed at day 28, stained for E-cadherin (green) and HA-tagged JUND (red), and analyzed by confocal immunofluorescence. Cells were counterstained with DRAQ5 (blue) to label nuclei. ( h ) Homogenization of JUND expression by knockdown of JUND and addback with murine RNAi-resistant JunD to near-endogenous expression levels. JUND/JunD levels were determined by immunoblotting. Densitometry of JUND/JunD abundance is shown normalized to the shGFP control. ( i ) Quantification of the cribiform-like phenotype at day 28 of 3D culture for the cells in ( h ). For ( a ), ( c ), ( g ), and ( i ), data are shown as the mean ± s.e.m. of n=3 ( a ) or n=4 ( c , g , i ) independent experiments. For ( d ) and ( f ), scale bar is 20 µm. For ( e ) and ( h ), tubulin was used as a loading control and n.s. denotes a non-specific band. For source data, see .

Article Snippet: pLKO.1 shGFP puro (Addgene #12273), pLKO.1 shTGFBR3 puro (TRCN0000033430), and pLKO.1 shJUND puro (TRCN0000014974) were obtained from The RNAi Consortium or Addgene. pBabe JunD-HA neo, pBabe JUND-HA neo, pBabe RFP1-Smad2 neo, pBabe JunD-Venus puro, and pBabe RFP1-KRT5 hygro were constructed by PCR cloning from plasmid templates (Open Biosystems) into the retroviral vector pBabe neo, pBabe puro, or pBabe hygro.

Techniques: Expressing, Knockdown, Cell Culture, Western Blot, Control, Blocking Assay, Staining, Immunofluorescence, Plasmid Preparation, Homogenization