Figure 6. KLF4-dependent activation of Lgals3, MSC markers, and phagocytotic activity in cholesterol loaded cultured SMC.

(a-c) Aortic SMCs were isolated from SMC YFP^+/+ Klf4 ^WT/WT and SMC YFP^+/+ Klf4^Δ/Δ mice and sorted using a FACSVantage SE DIVA to ensure a pure SMC (YFP+) cell population. (a) Induction of Lgals3 mRNA expression following cholesterol loading (80 μg/mL cholesterol for 72 hours) was decreased in cells derived from SMC YFP^+/+ Klf4^Δ/Δ as compared to cells derived from SMC YFP^+/+ Klf4 ^WT/WT mice. P-values based on two-way ANOVA with Tukey post-test. *P < 0.05. Data normalized to Klf4 ^WT/WT 0 ug/mL cholesterol. Error bars based on S.E.M. n = 3 independent experiments. (b-c) Klf4^WT/WT and Klf4^Δ/Δ cells were incubated with 0.8 μm polysterene beads for 1.5 hours after 72 hours of cholesterol loading to induce a Mϕ-like state. Cells were then analyzed on an Amnis ImagestreamX Mark II to assess expression of YFP, LGALS3, and to identify bead uptake. (b) Quantification of bead uptake was performed using Amnis IDEAS software. After normalization to the total population, values were subjected to Fisher's exact test, which showed that the Klf4^WT/WT YFP⁺LGALS3⁺ cells contained significantly (P < 0.05) more beads than the Klf4^Δ/Δ YFP⁺LGALS3⁺ cells (representative experiment from n = 2). (c) Representative images from the Amnis IDEAS software, YFP (green), LGALS3 (yellow), beads (red), Dark Field (magenta). Scale bar = 5 μm.