Supplementary Figure 2: A20 depletion does not affect tumor growth.

(a,e) MCF10CA1a (M4) (a) and 4T1-Luc (e) cells were infected with the indicated lentiviruses expressing shRNAs targeting A20 mRNA. Cell lysates were immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The data are representative of n = 3 independent experiments. (b,f) A20-knockdown MCF10CA1a (M4) (b) or A20-knockdown 4T1-Luc (f) cells were cultured in 6-well plates and harvested at the indicated time points. Cell proliferation was analyzed by counting cell numbers in each well, compared to shGFP-expressing control cells. The data were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. ^∗P < 0.05 compared to the shGFP control cells. n.s., not significant. (c,d) 5 × 10⁵ of A20-knockdown and shGFP-expressing MCF10CA1a (M4) cells were orthotopically injected into NOD/SCID mice (n = 6 mice per group). After the mice were sacrificed 5 weeks later, representative primary tumor images were shown in c and tumor volumes were measured (d). (g,h) 5 × 10⁴ of A20-knockdown and shGFP-expressing control 4T1-Luc cells were orthotopically injected into Balb/c mice (n = 6 mice per group) and the mice were sacrificed 5 weeks later. Representative images of primary tumors were shown in g and tumor volumes were measured (h). The data in d and h were statistically analyzed by a t-test and show the mean ± s.d. n = 6 mice per group per experiment. ^∗P < 0.05 compared to the shGFP control cells. n.s., not significant. Statistics source data for b,d,f and h are available in Supplementary Table 3. Unprocessed original scans of blots in a and e are in Supplementary Fig. 9.