Figure 2.
HSP47 is required for BrM
(A) 4T1 and LLC1 cells expressing HSP47 were analyzed by immunoblotting (top) and RT-qPCR (bottom), respectively. The RT-qPCR data were normalized to GAPDH (mean ± SEM of n = 3 independent experiments, two-tailed Student’s t test). ∗∗∗p < 0.001.
(B) 4T1 and LLC1 cells (5 × 104 cells/mouse) expressing HSP47 were intracardially injected into mice, respectively. Representative BLI images of two mice for each group are shown. BLI photon fluxes in the brain for each mouse were quantified (mean ± SD, n = 8 mice for each group, two-sided Mann-Whitney test). ∗∗p < 0.01.
(C) The percentages of mice forming brain and bone metastases after intracardial cell implantation.
(D) The BMFS of mice was evaluated (n = 8 mice for each group, Kaplan-Meier model with two-sided log-rank test). ∗∗p < 0.01.
(E and F) H&E staining showing the BrMs in (B). Representative images are shown (E). Scale bars, 200 μm. Insets: high-magnification images corresponding to the areas marked by dotted red lines. The number of BrMs were quantified (F). Large metastases are >300 μm on the longest axis (mean ± SD, n = 8 mice, two-sided Mann-Whitney test). ∗∗∗p < 0.001.
(G) 4T1-BMT5 and LLC1-BMT5 cells expressing HSP47 shRNAs were analyzed by immunoblotting (top) and RT-qPCR (bottom). For RT-qPCR, GAPDH was used as an internal control. Data are expressed as mean ± SEM, n = 3 independent experiments, two-tailed Student’s t test. ∗∗∗p < 0.001.
(H) 4T1-BMT5 and LLC1-BMT5 cells expressing HSP47 shRNAs were intracardially injected into mice, respectively. Representative BLI images of mice are shown. BLI photon fluxes in the brain for each mouse were quantified (mean ± SD, n = 8 mice for each group, two-sided Mann-Whitney test). ∗∗∗p < 0.001.
(I) The BMFS of mice was evaluated (n = 8 mice for each group, Kaplan-Meier model with two-sided log rank test). ∗∗∗p < 0.001.
(J) H&E staining of the BrMs in (H). Representative images are shown. Scale bars, 200 μm. Insets: high-magnification images corresponding to the areas marked by dotted red lines. The number of BrMs were quantified (mean ± SD, n = 8 mice, two-sided Mann-Whitney test). ∗∗∗p < 0.001.