Figure S6.

OTULIN Knockdown Studies, Related to Figure 7

(A) HEK 293ET (left) and U2OS (right) cell lines were transfected with NF-κB reporter plasmids, LUBAC and with siRNAs targeting OTULIN, HOIP or a nontargeting control siRNA. NF-κB reporter activity was measured as in Figure 4B. Error bars represent standard deviation from the mean of experiments performed in triplicate. p values are given to indicate significance and the asterisk indicate the mean value set to 1. Western blots with indicated antibodies show knockdown efficiency. Here, the asterisk indicates nonspecific bands.

(B) T-REx293 cells stably transfected with an inducible nontargeting control miRNA or with an inducible OTULIN-targeting miRNA are analyzed by western blotting with indicated antibodies. Doxycycline induces the OTULIN miRNA (together with GFP expression) from the GFP mRNA 3′UTR, and expression can be monitored by expressed GFP levels following doxycycline induction.

(C) LUBAC-induced NF-κB luciferase activity is increased in OTULIN-depleted cell lines. Error bars represent the standard deviation from the mean of experiments performed in duplicate. p values are given to indicate significance.

(D) Control or OTULIN-depleted cell lines were stimulated with TNFα (10 ng/ml) for the indicated times, and lysates were analyzed by western blotting with the indicated antibodies. ^*, nonspecific band.

(E) Cell viability counts of stable doxycycline-inducible control and OTULIN-miRNA expressing T-REx293 cells after treatment with and without doxycycline (+/− Dox, 1 μg/ml for 72 h) prior to TNFα treatment (+/− TNFα, 50 ng/ml for 24 hr) (see Extended Experimental Procedures). Error bars represent the standard deviation from the mean for experiments performed in triplicate.

(F) Clonogenic survival of cells treated as in (E).