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. Author manuscript; available in PMC: 2018 Jun 1.
Published in final edited form as: J Immunol. 2017 May 1;198(11):4448–4457. doi: 10.4049/jimmunol.1601089

Figure 4. Effect of pharmacological PKD inhibitors on GBS-mediated activation of JNK and NF-κB and subsequent expression of proinflammatory cytokines in human and murine macrophages in vitro.

Figure 4

(A, B) THP1 cells were pretreated with vehicle (0.5% v/v DMSO), Gö6976 (1 μM) or Gö6983 (1 μM) or CRT0066101 (5 μM) for 1 hr and then stimulated with medium, GBS (1 × 108 cfu/ml) or TNFα (25 ng/ml) plus IFNγ (50 ng/ml) for 1 hr (A) or 4 hr (B). (A) Cell lysates were prepared. Phosphorylation status of PKD (pPKDs744/748, pPKDs916) and JNK (pJNK), and protein levels of IκBα were detected by Western blot assay. (B) Total RNA was isolated and mRNA levels of the indicated cytokines were analyzed by RT-PCR. Actin was used as a loading control. (C–F) RAW264.7 cells (1 × 106 cells/ml) were pretreated with vehicle (0.5% v/v DMSO), Gö6976 (0.66 μM) or Gö6983 (0.57 μM) for 30 min and then stimulated with medium or GBS (106 or 107 cfu/ml) for 24 hr in the complete DMEM supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. (D) RAW264.7 cells (1 × 106 cells/ml) were pretreated with vehicle (0.5% v/v DMSO) or CRT0066101 (1 μM) for 30 min and then stimulated with medium, GBS (5 × 106 cfu/ml) or IFNγ (10 ng/ml) for 24 hr in the complete DMEM supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. Levels of the selected cytokines in culture supernatants were analyzed by ELISA. Data represent the mean concentration (pg/ml) ± SD of quadruplicates. *, p < 0.05; **, p < 0.005; #, p < 0.0005. All experiments were repeated at least three times with similar results.