Fig 2: HTRA2 overexpression or knockdown affects CCR2-H SUM225 and CCR2-KO DCIS.com breast cancer cell growth and invasion. (A–D) CCR2-H SUM225 cells expressing vehicle control (pHAGE) or HTRA2 (HTRA2-OE) were analyzed for HTRA2 expression by immunoblot (A), spheroid size in 3D cultures (B), PCNA expression (C) or invasion index (D). Mean number of spheroids analyzed per group±s.e.m.: pHAGE, 269±63; HTRA2-OE, 227±28. (E–H) CCR2-KO DCIS.com cells were transduced with lentivirus expressing Con or HTRA2 shRNAs (HTRA2-KD#2, HTRA2-KD#3) and analyzed for HTRA2 expression by immunoblot (E), spheroid size in Matrigel: Collagen (F), PCNA expression (G) or transwell invasion (H). Spheroid size and invasion index were normalized to spheroid number. Experimental groups were plated in triplicate. Experiments were repeated three times. Two slides containing three sections each were subject to immunostaining. Mean number of spheroids analyzed/group±s.e.m.: Con, 196±80; HTRA2-KD#2, 219±99; HTRA2-KD#2, 161±40. Statistical analysis was performed using two-tailed t-test (A–D) or one-way ANOVA with Bonferroni post-hoc comparison (E–H). *P<0.05, **P<0.01, ***P<0.001; n.s, not significant. Mean±s.e.m. is shown.

Fig 3: Overexpression of HSF1 participates in mitochondrial apoptosis by regulating Omi/HtrA2 expression in H9C2 cells. HSF1 expression increased after transfection of plasmid pcDNA3.1, which was not affected by RNAi Omi/HtrA2 (A, B). Omi/HtrA2 protein expression increased after transfection of plasmid pcDNA3.1, which decreased after RNAi Omi/HtrA2 (A, C). Caspase -9 activity was induced by HSF1 overexpression, which was reversed after transient transfection of si-Omi/HtrA2 (D). Caspase-3 activity was suppressed by HSF1 overexpression, but similar change was detected after RNAi using Omi/HtrA2 (E). Data are represented as mean +/- SEM. *P<0.05, **P<0.01 vs. pcDNA3.1, #P<0.05, ##P<0.01 vs. HSF1+scramble, n = 4.

Fig 4: HTRA2 overexpression inhibits DCIS.com breast cancer cell growth and invasion. (A–F) DCIS.com cells expressing pHAGE control or HTRA2 were analyzed for (A) ALDH1A1 and HTRA2 expression by immunoblot, (B) spheroid size in 3D cultures, (C) PCNA expression, (D) transwell invasion, (E) E-cadherin or (F) TWIST1 expression. Mean number of spheroids analyzed per group±s.e.m.: pHAGE, 325±25; pHAGE±CCL2, 358±33; HTRA2-OE, 343±17; HTRA2-OE±CCL2, 316±44. Protein levels in immunoblots were measured by densitometry. Expression levels were normalized to control group. Representative blots of three experiments are shown. Spheroid size and invasion index were normalized to spheroid number. Experimental groups were plated in triplicate. Experiments were performed three times. Two slides containing three sections each were subject to immunostaining. Statistical analysis was performed using two-tailed t-test (A) or one-way ANOVA with Bonferroni post-hoc comparison (B–F). *P<0.05, **P<0.01, ***P<0.001; n.s, not significant. Mean±s.e.m. is shown.

Fig 5: Cardiac dysfunction in transgenic mice with cardiac-specific overexpression of Omi/HtrA2.(a) Echocardiography of WT and transgenic mice overexpressing cardiac-specific Omi/HtrA2 (TG-Omi). (b) EF (ejection fraction), %. (c) FS (fractional shortening), %. (d) Doppler imaging of the mitral annulus. (e) E/A ratio, the ratio of the early (E) to late (A) ventricular filling velocities. (f) IVRT (isovolumetric relaxation time). All indices were recorded at 9 months age. n = 3–5 /genotype. **p < 0.01 versus WT.