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. 2019 Feb 4;216(2):450–465. doi: 10.1084/jem.20180742

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© 2019 Ruiz et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 6. — Inhibition of LUBAC or TAK1 sensitizes LSCC cells to cisplatin. (A) Both 5Z-7 and gliotoxin inhibit NF-κB signaling, as measured by phospho-p65 (Ser536). Blots represent two independent experiments. MW, molecular weight. (B) Expression of NF-κB–dependent target genes (CCL2 and TNFα) in murine LSCC cells is decreased in the presence of the TAK1 inhibitor 5Z-7 or the LUBAC inhibitor gliotoxin. Relative mRNA levels measured by real-time PCR. Data are mean + SD of three independent experiments performed in triplicate. (C) LSCC cells are highly sensitive to gliotoxin treatment, and gliotoxin sensitizes cells to cisplatin. Data are mean + SD of two independent experiments performed in duplicate. ***, P < 0.0001 versus vehicle, two-way ANOVA. (D) siRNA-mediated knockdown of the LUBAC components HOIP and HOIL-1 decreases LSCC cell resistance to cisplatin. Graph shows mean + SD of three independent experiments performed in triplicate. ***, P < 0.0001 versus vehicle, one-way ANOVA. (E) Validation of HOIL-1 knockdown in LSCC cells using two independent siRNAs. Graphs represent mean + SEM of three independent experiments. (F) Validation of HOIP knockdown in LSCC cells using three independent siRNAs. Graphs represent mean + SEM of three independent experiments. (G) In vitro LSCC and LADC cells are highly sensitive to cisplatin (CPPD) and 5Z-7-oxozeaenol combination treatment. Data are mean + SD of two independent experiments performed in duplicate. ***, P < 0.0001 versus vehicle, two-way ANOVA. (H) Validation of TAK1 knockdown in LSCC cells using three independent siRNAs. Graphs represent mean + SEM of three independent experiments. (I) siTAK1 LSCC cells are sensitive to cisplatin (CPPD), and addition of 5Z-7 does not affect CPPD sensitivity in the presence of TAK1 knockdown. P values calculated using one-way ANOVA test. Graph shows mean + SD of two independent experiments performed in quadruplicate. ***, P < 0.0001 versus vehicle, one-way ANOVA. See also Fig. S5.