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. Author manuscript; available in PMC: 2024 Jun 23.
Published in final edited form as: Circ Res. 2023 Jun 2;133(1):25–44. doi: 10.1161/CIRCRESAHA.122.322017

Figure 1: ERK5 S496 phosphorylation plays a crucial role in vulnerable plaque formation and SASP induction.

Figure 1:

WT BMDMs and ERK5 S496A KI BMDMs were treated with oxLDL (10 μg/ml) for 0-30 minutes (A, B), and an immunoblotting analysis was performed using antibodies against the indicated proteins in vitro. (B) The graphs represent densitometry data from 3 independent gels, one of which is shown in A. (C, D) 16 weeks after AAV-PCSK9 injection and fed an HFD, ERK5 S496A KI mice exhibited fewer oil-red O-stained atherosclerotic lesions in the en face whole aorta, scale bars=1 mm. (D) Quantified oil-red O-stained lesions are shown. (n=15, 21, all male). (E) The area occupied by the necrotic core (acellular lipid core) is shown as the percentage of the total lesion area (E, right). Scale bar=1 mm. (n=8, 10). (F) Sections of proximal aortas from each group were labeled using TUNEL reagents to detect apoptotic cells and counterstained with DAPI to detect nuclei. Only double-positive cells (TUNEL and DAPI) were counted. Scale bar=100 μm. (F, right) The graph shows the percentage of TUNEL-positive cells (TUNEL+ cells/total cells counted) in the lesion area. Over 200 cells were counted for each group. (n=8, 9). (G, H) BMDMs treated with vehicle or FMK-MEA (G) or WT BMDMs and ERK5 S496A KI BMDMs (H) were incubated with oxLDL (10 μg/mL) or vehicle, and ERK5 transcriptional activity was detected. (I, J) WT BMDMs and ERK5 S496A KI BMDMs were treated with oxLDL (10 μg/ml) for 24 hours, and immunoblotting analysis was performed using antibodies against the indicated proteins in vitro. (J) The graphs represent densitometry data from 5 independent gels, one of which is shown in I. (K) WT BMDMs and ERK5 S496A KI BMDMs were incubated with oxLDL as indicated. mtROS levels were detected by MitoNeoD as described in methods section in vitro. Cells treated with oxLDL were assayed 24 hours later. (L) The percentages of cells positive for SA-β-gal staining are shown in vitro. More than 200 cells/sample were counted. (M) WT BMDMs and ERK5 S496A KI BMDMs were transfected with the NF-κB luciferase reporter and the constitutively expressing Renilla luciferase vector for 16 hours and then incubated with oxLDL or vehicle. After 12 hours, NF-κB transcriptional activity was measured as described in Methods in vitro. (N, O) BMDMs treated with vehicle or FMK-MEA (10 μM) (N) or WT BMDMs and ERK5 S496A KI BMDMs (O) were incubated with oxLDL (10 μg/mL) or vehicle. After 24 hours, pHrodo-positive cells were quantified in vitro. The applied statistical tests, sample number, and results in all figures are summarized in Table S3. All data are expressed as mean±SD, **P<0.01, *P<0.05.