IKKα controls canonical TGFβ–SMAD signaling to regulate genes expressing SNAIL and SLUG during EMT in Panc1 cells

Abstract

There was an error published in J. Cell Sci. 123, 4231-4239.In Fig. 4A, the cRel siRNA western blot panel was inadvertently constructed using the wrong images and all three western blots showed incorrect loading controls. The correct images and loading controls are shown in the figure below. The mistake in the figure did not affect the conclusions of the paper.We apologise for this mistake. . TGFb-dependent downregulation of E-cadherin is NFkB-independent. (A) Panc1 cells were transfected with a control or RelA/p65-, RelB-or c-Relspecific siRNA. At 24 hours after the transfection, cells were treated with 10 ng/ml TGFb or were left as an untreated control in DMEM without FCS. After an additional 48 hours, western blots detected RelA/p65, RelB or c-Rel and E-cadherin expression. The membrane was stripped and probed for a-tubulin or b-actin to ensure equal protein loading. (B) Panc1 (upper graph) and MDA-MB231 cells (lower graph) cells were co-transfected with a control or IKKa-specific siRNA and 500 ng of the pGL3control-, NFkB-or SMAD-luciferase reporter gene constructs as indicated. At 24 hours after the transfection, cells were treated with 10 ng/ml TGFb or were left as an untreated control. Luciferase activity was measured 6 hours after the TGFb treatment (Student's t-test: *P,0.05 versus control). (C) Panc1 (upper graph) and MDA-MB231 (lower graph) cells were transfected with a control or IKKa-specific siRNA. At 48 hours after the transfection, cells were stimulated with TGFb (10 ng/ml) for 20 minutes and binding of SMAD3 and SMAD4 to a SMAD consensus oligonucleotide was detected using ABCD assays. Input represents 5% of whole-cell extract of control siRNA-transfected cells. (D) Panc1 cells were treated as in C. Immunoprecipitation was performed with an IKKa-specific antibody or pre-immune serum as a control. Western blots of immunoprecipitates were probed with antibodies against IKKa and SMAD3. Input represents 5% of whole-cell extract of control siRNA-transfected Panc1 cells. (E) MDA-MB231 cells were transfected with a control or IKKa-specific siRNA. At 24 hours after the transfection, cells were treated with 10 ng/ml TGFb or were left as an untreated control in DMEM without FCS. After an additional 48 hours, western blots detected IKKa expression. The membrane was stripped and probed for b-actin to ensure equal protein loading.

Innate Antiviral Host Defense Attenuates TGF-β Function through IRF3-Mediated Suppression of Smad Signaling

“…HaCaT cells transition into a mesenchymal phenotype in response to TGF-β, with increased expression of Slug, a Snail-related transcription factor that drives EMT, dispersion of E-cadherin from junctions, decreased epithelial and increased mesenchymal gene expression, and changes in actin organization and cell shape (Lamouille and Derynck, 2007; Thuault et al, 2006). TGF-β-activated Smad3 directly controls Slug and several other EMT-regulated genes (Brandl et al, 2010; Xu et al, 2009). In contrast to many cell lines, HaCaT cells do not repress E-cadherin expression during EMT (data not shown).…”

Transforming growth factor-β2 promotes Snail-mediated endothelial–mesenchymal transition through convergence of Smad-dependent and Smad-independent signalling

“…Furthermore, all of these pathways are necessary for promoting increased expression of the EndMT-inducing transcription factor Snail, which suppresses cell adhesion and promotes EndMT [13]. Smad proteins have been shown to bind directly to the promoter of the Snail gene to regulate its transcription [31–34]. Transcription factors induced by Smad-independent signaling that regulate Snail transcription remain unclear, but we show that these pathways have a critical role in controlling Snail gene expression.…”

MYC and EGR1 synergize to trigger tumor cell death by controlling NOXA and BIM transcription upon treatment with the proteasome inhibitor bortezomib

“…Whole cell lysates and nuclear extracts were prepared and western blots were carried out as described (9,24). A formaldehyde-based cross-linking protocol was used for immunoprecipitations as described by Klockenbusch and Kast (25).…”

“…Antibodies are shown in Supplementary Table S1. Avidin-Biotin-Complex DNA-(ABCD) assay was done as described (24). Sequences of the 5′ biotinylated oligonucleotides are listed in Supplementary Table S1.…”