Fig. 5: FABP5 depletion affects NF-κB activity. | Communications Biology

Fig. 5: FABP5 depletion affects NF-κB activity.

From: FABP5 is a key player in metabolic modulation and NF-κB dependent inflammation driving pleural mesothelioma

Fig. 5

A HMC35, IST-Mes-2, and MPP89 cells (3 × 106) were transfected with siRNA FABP5 or scrambled siRNA (200 pmoles) in presence of pSV-β-Gal (0.2 mg) and pκBluc, (0.2 mg). Forty-eight hours later, firefly luciferase activity and β-galactosidase activity were measured. The ratio of firefly luciferase activity to β-galactosidase activity was expressed as relative light units. Mean values ± SE of 3 independent experiments are shown. B HMC35, IST-Mes-2, and MPP89 cells (3 × 106) were transfected with siRNA FABP5 or scrambled siRNA (200 pmoles). The binding of p65, p50, p52, c-Rel, and RelB proteins to the NF-κB double-stranded oligonucleotide was measured in nuclear extracts using the NF-kB Combo Transcription Factor ELISA assay kit. Values are the mean ± SE of three independent experiments. C HMC35, IST-Mes-2, and MPP89 cells (3 × 106) were transfected with siRNA FABP5 or scrambled siRNA. IKK activity was measured in cytosolic cell extracts (30 μg) using the HTScan IKK kinase assay kit. Values are the mean ± SE of three independent experiments. The asterisks indicate statistically significant differences between stimulated and unstimulated cells, according to Student’s t-test (p < 0.05). D HMC35, IST-Mes-2, and MPP89 cells (3 × 106) were transfected with siRNA FABP5 or scrambled siRNA (200 pmoles). Total RNA of IST-Mes-2, MPP89, and HMC35 cells was analyzed by quantitative real-time PCR for the expression of NF-κB dependent genes IL-1β, IL6, CCL2, and IL8. E Schematic representation of CCTα, CHPT1, and CEPT1 promoters. Red arrows indicate the NF-κB enhancers with the related positions. F HMC35, IST-Mes-2, and MPP89 cells (3 x 106) were transfected with siRNA FABP5 or scrambled siRNA (200 pmoles). 48 h later, ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n  =  3) are shown. The asterisks indicate statistically significant differences compared to the control (siControl) according to the Student’s t-test (p <  0.05).

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