Fig. 2: IL6 but not hypoxia is inducing JunB-dependent production and secretion of angiogenic factors.

A Impact of hypoxia on JunB versus Hif-1α expression. MM.1S cells were transiently transfected with non-targeting control (SCR) siRNA, siJunB, or siHif-1α and then treated with IL6 (25 ng/ml) and 1% O₂ (hypoxia) or left untreated. After 9 h, cell lysates were immunoblotted with antibodies against Hif-1α and JunB. ERK2 served as a loading control. B Doxycycline (Dox)-mediated JunB knockdown in TetR-shJunB/MM.1S inhibits IL6-induced production of AFs. TetR-shJunB/MM cells were cultured in RPMI-1640 medium with IL6 in the presence or absence of doxycycline (1 μg/ml). Expression profiles of VEGF, VEGFB, and IGF1 in TetR-shJunB/MM cells were determined using RT-qPCR. Data represent mean ± SD for triplicate samples of three independent experiments. *p < 0.05; **p < 0.01. C–E Doxycycline-mediated JunB knockdown in TetR-shJunB/MM.1S inhibits IL6-induced secretion of AFs. TetR-SCR/MM.1S or TetR-shJunB/MM.1S cells were cultured in RPMI-1640 medium with or without IL6 in the presence or absence of doxycycline (1 μg/ml). Supernatants from equal numbers of cells (1.2 × 10⁶) were collected after 18 h and analyzed for VEGF (C), VEGFB (D), and IGF1 (E) protein levels by ELISA. Supernatants from IL6-stimulated TetR-SCR/MM.1S cells served as a control. Data are expressed a mean ± SD of culture triplicates. F siRNA-mediated JunB knockdown in RPMI 8226, U266, KMS-11, and MR20 downregulates IL6-induced production of VEGF, VEGFB, and IGF1. MM.1S cells were transiently transfected with mock (200 nM) and JunB siRNA (200 nM). Expression profiles of VEGF, VEGFB, and IGF1 in indicated MM cell lines were determined using RT-qPCR. Data represent the decrease of VEGF, VEGFB, and IGF1 expression levels in MM cells transfected with siJunB versus mock control and represent mean ± SD for triplicate samples of three independent experiments.