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. Author manuscript; available in PMC: 2016 Aug 1.
Published in final edited form as: Eur J Immunol. 2015 May 18;45(8):2365–2376. doi: 10.1002/eji.201545462

Figure 5. HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition.

Figure 5

A. AICDA mRNA levels were measured by qRT-PCR in ZR75.1 cells expressing shRNA control (shScr) or shRNA against AID (AIDkd). TNF-α (50ng/ml) was used to induce AICDA. Bars show mean + SD out of 3-4 samples for each condition from ≥ 3 independent experiments.

B. Relative mRNA levels of various genes measured by qRT-PCR in ZR75.1 cells untreated or treated with 1 μM 17-AAG for 72 h. Expression of mesenchymal markers VIM and CDH2, and transcription factors SNAI2 (Slug), ZEB1 and ZEB2 (*: p<0.0005 VIM, CDH2 and ZEB2; p<0.005 SNAI2 and ZEB1) and, CDH1 (E-cadherin) and SNAI1 (Snail) were measured. Results are expressed as fold change relative to the levels seen in cells treated with DMSO. Bars show mean + SD out of 3-5 samples for each gene from ≥ 3 independent experiments. Expression was normalized to GAPDH and HPRT1.

C. Relative mRNA levels of EMT markers and transcription factors in ZR75.1 AIDkd cells, untreated or treated with 1 μM 17-AAG for 72 h were measured and plotted as in (B). Bars show mean + SD out of 3-5 samples for each gene from ≥3 independent experiments. Expression was normalized to GAPDH and HPRT1.

D. Bright field images (20X) of ZR75.1 cells treated with vehicle (DMSO) or 1 μM 17-AAG and/or TNF-α and TGF-β to induce the EMT (indicated as EMT). Treatment with 17-AAG decreases cell scattering at baseline and in EMT-inducing conditions, similarly to the effect we previously reported for AID knockdown in both conditions [30]. One representative image out of 3 independent experiments performed is shown.

E. Expression of Vimentin assessed by western blot in whole cell extracts of ZR75.1 cells, treated with DMSO, EMT inducers (TNF-α and TGF-β) and/or 17-AAG as indicated, or MDA-MB-231 breast cancer cells (as a positive control). β-actin was used as loading control. One representative blot out of 3 independent experiments performed is shown.

F. VIM, CDH2, CDH1 and SNAI1 mRNA levels measured by qRT-PCR in vehicle-treated ZR75.1 cells (DMSO) or EMT induced in the presence or absence of either shRNA targeting AID (AIDkd) or the HSP90 inhibitor 17-AAG. Bars show mean + SD out of 3-5 samples per gene from ≥ 3 independent experiments. Expression was normalized to GAPDH and HPRT1.

A-C, F. Differences in gene expression were compared by paired two-tailed t-test (assuming unequal variance, α=0.05).