64 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-B-dependent resistance to apoptosis in 3D mammary acini

JCS03484 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Pharmacological inhibition of Rac perturbs tissue polarity and sensitizes mammary acini to death induction. (A) Representative confocal immunofluorescence images of β4 integrin (green) in control and toxin-A-treated MEC acini showing that inhibiting Rho GTPase activity disrupts the polarity of rBM-differentiated mammary tissue structures. Bar, 50 μm. (B) Dose-response curves of the percentage death, calculated using the live/dead assay, induced following treatment with increasing doses of the chemotherapeutic agents etoposide and taxol. The results demonstrate how inhibiting Rho GTPase activity compromised both acini polarity and apoptosis resistance. Similar data were obtained following treatment with doxorubicin and camptothecin (data not shown). Results are the mean ± s.e.m. of at least three separate experiments of duplicates. *, P≤0.05.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Rac-dependent Pak activation regulates phosphorylation of Bad. (A) Representative immunoblots of phospho-Bad, total Bad and E-cadherin in 3D control, N17Rac and N17Rac/wild-type-Pak1-expressing 3D mammary acini. The data indicate that Rac-dependent Pak1 activity regulates phospho-Bad levels. (B) Quantification of experimental data shown in A of phosphorylation-mediated Bad activity inhibition, calculated by densitometric analysis of immunoblots of phospho-(inactivated)-Bad divided by total Bad after normalization to E-cadherin.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Transgene manipulation of NF-κB in 3D mammary acini. (A) Confocal immunofluorescence images of elevated nuclear (DAPI) p65 (green) in 3D control mammary acini and N17-expressing acini ectopically expressing p65. The data indicate that elevated expression of p65 increases nuclear p65 in 3D mammary acini even in the absence of Rac activity. (B) Representative immunoblots of total cellular NF-κB p65 and E-cadherin in control and N17Rac-infected mammary acini and acini ectopically expressing p65 or IκBαM. (C) Quantification of the immunoblots shown in B depicting the relative p65 levels normalized to total cellular E-cadherin expressed in control and N17Rac-infected mammary acini and acini expressing p65 or IκBαM.