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. Author manuscript; available in PMC: 2022 Feb 23.
Published in final edited form as: Cell Rep. 2022 Feb 1;38(5):110309. doi: 10.1016/j.celrep.2022.110309

Figure 3. Macrophage IL-1β drives nascent VEGF-A mRNA transcription, nuclear-localized NF-κB and STAT3 activity but not HIF-1a activity, and gain-of-function mutations for STAT3 or IKK2 increase pro-angiogenic VEGF-A165a isoform expression.

Figure 3.

(A) BMDMs from control or myeloid IL-1β-deleted mice (mIL-1β KO) were treated with either LPS+IFN-γ or IL-4+IL-13 for 12 h followed by 20-min 5-ethynyl Uridine pulse, Click-iT Nascent RNA purification, and quantitative RT-PCR for relative nascent VEGF-A mRNA (**, p ≤ 0.0002 compared to all others by ANOVA; n = 4 biological replicates from two mice, one male and one female).

(B) BMDMs from control or mIL-1β KO mice were treated with either LPS+IFN-γ or IL-4+IL-13 for 24 h followed by a modified ELISA for relative HIF-1α activity on nuclear fraction lysates (n = 4 mice total, two males and two females).

(C) BMDMs treated as in (B) followed by a modified ELISA for relative STAT3 activity on nuclear fraction lysates (***, p < 0.0001 compared to all others by ANOVA; n = 4 mice total, two males and two females).

(D) BMDMs treated as in (B) followed by a modified ELISA for relative NF-κB activity on nuclear fraction lysates (***, p < 0.0001 compared to all others by ANOVA; n = 4 mice total, two males and two females).

(E) BMDMs from control or mIL-1β KO mice were transfected with plasmid for constitutively active mutations of STAT3 or IKK2 (NF-κB activator) alone or in combination followed by ELISA on culture supernatants for secreted VEGF-A protein (**, p ≤ 0.0075 compared to wild-type control transfected with vector control by ANOVA; n = 4 mice total, two males and two females).

(F) BMDMs treated as in (E) followed by quantitative RT-PCR for relative VEGF-A165a mRNA expression (*, p < 0.05; **, p < 0.005 compared to wild-type control transfected with vector control by ANOVA; n = 4 mice total, two males and two females).

(G) BMDMs treated as in (E) followed by quantitative RT-PCR for relative VEGF-A165b mRNA expression (***, p < 0.0001 compared to wild-type control transfected with vector control by ANOVA; n = 4 mice total, two males and two females). Data, mean ± SD.