Supplementary Figure 2: Clinical and in vivo validation of MSK1 as regulator of homing, tumor mass dormancy and metastasis in ER+ breast cancer.
From: MSK1 regulates luminal cell differentiation and metastatic dormancy in ER+ breast cancer
(a) In vitro cell growth curve of shControl and shMSK1 DBM cells. Mixed-effect linear model in which the response was square-root transformed, n = 3 independent experiments were modeled as random effect and group, time point and their interaction were included as explanatory variables. (b) MSK1 KO generation by CRISPR/Cas9 genome editing. Five target sequences were located in exons 5, 6, 8, 9 or 13 (green), which overlap with residues critical for MSK1 catalytic and kinase activity (violet circles). The 20-nt target sequence in exon 5 (blue);the 3-nt PAM sequence(red); guide RNA is composed of complementary target sequence. The cleavage site is located between the 3rd and 4th nucleotides upstream from PAM (arrows). (c) Representative bioluminescence images showing bone metastasis progression of shControl and shMSK1 DBM cells. Experiment was repeated two times independently with similar results. (d) Bone metastasis incidence of ZR-75.1 cells infected with shControl (n=14 limbs) and shMSK1#1 (n=14 limbs) (left). Western blot analysis of MSK1, MSK2 and tubulin in ZR-75.1 cells (right). Western blot was repeated two times independently with similar results (e) Time-matched quantification of in vivo BLI signal in hind limbs during homing, latency and metastasis after injection of DBM shControl (n=4 limbs), shMSK1#1 (n=12 limbs) or shMSK1#2 (n=24 limbs). Two-tailed Wald tests from a linear model in which the response was log-transformed and group, status and their interaction were included as explanatory variables. (f) Time-matched quantification of apoptotic signal (Z-DEVD) normalized to lesion size (LUC) in hind limbs during homing, latency, metastasis after injection of DBM shControl (nh=6 limbs, nl=4 limbs, nm=3 limbs), shMSK1#1 (nh=6 limbs, nl=4 limbs, nm=4 limbs) or shMSK1#2 (nh=7 limbs, nl=4 limbs, nm=4 limbs). Two-tailed Wald test from a linear model in which the response was log-transformed and group, status and their interaction were included as explanatory variables. (g) Analysis of bone samples with detected metastatic lesions or single DTC by means of human ERα IHC (n=25 ROIs from 5 limbs). Panel (a) show data as mean ± SEM from three independent experiments (d,f) show data as whisker plots: mid-line, median; box, 25 – 75 percentile; whisker, min-max. ns (nonsignificant) P>0.05, * P≤0.05, ** P≤0.01, *** P≤0.001. Schaffer method was used for P-value correction when three or more groups are compared. Statistics source data P values are provided in Supplementary Table 9. Unprocessed original scans of blots are shown in Supplementary Fig. 8.
