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. 2021 Apr 29;118(3):772–784. doi: 10.1093/cvr/cvab156

Figure 4.

Figure 4

Drebrin inhibits SMC-to-foam cell transdifferentiation in a KLF4-dependent manner. (A) WT SMCs were treated with vehicle (control) or cholesterol-methyl-β-cyclodextrin (10 µg/mL) for 72 h and stained with anti-CD68 IgG. (Isotype control IgG yielded no colour.) Scale bar = 10 µm. Results represent three independent experiments with >100 SMCs evaluated per experiment. (B) Dbnflox/flox and SMC-Dbn−/− SMCs were treated ± cholesterol (as in A) for 24 h before mRNA isolation and qRT-PCR for CD68 and GAPDH. Threshold CD68 counts were normalized to cognate GAPDH values and plotted from four experiments (means ± SE) using three sets of independently isolated Dbnflox/flox and SMC-Dbn−/− SMC lines. Compared with cognate Dbnflox/flox: *P < 0.05 (two-way ANOVA with Sidak post hoc test). (C) SMCs from B were treated (Tx) as above with vehicle or cholesterol (‘Chol’) for 48 h; soluble SMC extracts were immunoblotted sequentially for Galectin-3 (Gal-3) and β-actin. (D) Band densities for Gal-3 were normalized to cognate β-actin band densities; ratios were plotted from three independent experiments (means ± SE) with distinct pairs of Dbnflox/flox and SMC-Dbn−/− SMC lines. Compared with vehicle control: *P < 0.01 (two-way ANOVA with Sidak post hoc test). (E) The mRNA of SMCs from B was subjected to qRT-PCR for SMMHC and GAPDH; data were obtained and processed as in B. Compared with vehicle-treated SMCs: *P < 0.001; compared with Dbnflox/flox: #, P < 0.05 (two-way ANOVA with Sidak post hoc test, n = 4/group). (F) Dbnflox/flox and SMC-Dbn−/− SMCs were grown to confluence, serum starved for 24 h, and then solubilized. SMC lysates were immunoblotted sequentially for KLF4 and β-actin. KLF migrates as a doublet due to its known post-translational modifications.24 (G) Band densities for KLF4 were normalized to cognate β-actin band densities; the ratios were plotted as arbitrary units (a. u.) from 3 independent experiments with three sets of independently isolated Dbnflox/flox and SMC-Dbn−/− SMC lines. Compared with Dbnflox/flox: *P < 0.05 (t test). (H) SMC-Dbn−/− SMCs were transfected with siRNAs targeting KLF4 or no known mRNA (‘control’). Forty-eight hours later, SMCs were treated for a further 48 h with cholesterol-methyl-β-cyclodextrin (10 µg/mL, ‘cholesterol’) or vehicle, and then solubilized. SMC lysates were immunoblotted sequentially for Galectin-3, KLF4 and β-actin. (I) Band densities for Gal-3 or KLF4 were normalized to cognate β-actin band densities; the ratios were plotted as means ± SE from three independent experiments with two independently isolated primary SMC-Dbn−/− SMC lines. Compared with cognate control siRNA-transfected SMCs: *P < 0.01 (Gal-3) or P < 0.05 (KLF4), two-way ANOVA with Sidak post hoc test.