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. 2017 Nov 24;8(8):1820–1832. doi: 10.1080/21505594.2017.1377881

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© 2017 Taylor & Francis

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — RIPK3 and mitochondrial ROS mediate M. tuberculosis-induced cell death both in the presence and absence of TNFα. (a) WT J774A.1 macrophages, RIP3K shRNA knockdown J774A.1 macrophages, and control shRNA J774A.1 macrophages were treated for 20 hours with DMSO control or 30 µM zVAD.fmk in the presence of 25ng/mL TNF, before measuring cell survival using a crystal violet assay. Results are mean +/- SEM of n = 10 samples and are expressed as a percentage of the TNFα+DMSO-treated control for each condition. Statistics are two way ANOVA with Sidak post-test. **p<0.01; ****p<0.0001. Results are representative of 3 independent experiments. (b) Knockdown of RIP3K mRNA was confirmed by RT-PCR, using murine RIP-3 and beta-actin primers (sc-61483-PR and sc-29192-PR, Santa Cruz). (c) RIP3K hRNA knockdown J774A.1 macrophages, and control shRNA J774A.1 macrophages were infected with M. tuberculosis for 3 hours, and subsequently incubated in the absence or presence of 25ng/ml TNF for 48 hours, before measuring cell survival using a crystal violet assay. Results are mean +/- SEM of n = 10 samples, and are expressed as a percentage of the uninfected control of each treatment. Statistics are two way ANOVA with Sidak post-test. *p>0.05; ***p<0.001; ****p<0.0001. Results are representative of 2 independent experiments (d) J774A.1 macrophages were infected with M. tuberculosis for 3 hours and incubated with Necrox-2 in the presence and absence of TNFα for 24 hours, before measuring cell survival using a crystal violet assay. Results are mean +/- SEM of n = 10 samples, and are expressed as a percentage of the uninfected control of each treatment. Statistics are one way ANOVA with Tukey's post-test. *p>0.05; ***p<0.001; ****p<0.0001.