Correction: Smooth muscle 22 alpha protein inhibits VSMC foam cell formation by supporting normal LXRα signaling, ameliorating atherosclerosis

Correction to: Cell Death and Disease https://doi.org/10.1038/s41419-021-04239-w, published online 22 October 2021

The original version of this article unfortunately contained a mistake. Due to a typesetting error some of the figures were omitted and figure legends were misplaced. We sincerely apologize for the errors. The correct figures and legends can be found below.

Fig. 1: Impaired SM22α expression is associated with development of atherosclerosis.

a, b WT (n = 10) and Sm22α^−/− (n = 10) mice with or without Ldlr^−/− background (n = 10) fed Paigen diet for 8, 12, and 24 weeks, respectively. Representative images of en face ORO-stained aortas (a), aortic sinus, aortic cross sections (b), and quantification of lesion areas are shown. c M-mode and Doppler echocardiography images obtained from aortic arch and outflow tract of WT (n = 15) and Sm22α^−/− (n = 15) mice fed Paigen diet for 12 and 24 weeks. A_s: outflow tract and aortic diameter in systole; A_d: outflow tract and aortic diameter in diastole. d Identification of SMC-derived foam cells within atherosclerotic lesion of Sm22α^−/− mice (n = 6) by CD68 (blue), ACTA2 (red), and Bodipy (green). Scale bar, 20 µm. Arrows indicated foam cells, which were VSMCs-derived. e Representative immunofluorescence of LXRα (red) and quantification of cells with nuclear LXRα in the aortic sections from WT (n = 3) and Sm22α^−/− (n = 3) mice. Scale bar, 15 µm. Data and images are representative of at least three independent experiments. Data in a and b were analyzed by two-way and one-way ANOVA, respectively. Data in d and e were analyzed by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2: Expression and activity of LXRα is abnormal in Sm22α^−/− VSMCs.

a qRT-PCR and Western blot analysis of LXRα and LXRβ in WT and Sm22α^−/− VSMCs treated with LXRs agonist T090 for 0, 12, 24, 48, and 72 h, respectively, (n = 3). b qRT-PCR and western blot analysis of LXRα in WT VSMCs with or without cholesterol loading following knockdown of SM22α (n = 3). c qRT-PCR and western blot analysis of LXRα and SM22α expression in Sm22α^−/− VSMCs transducted with Ad-GFP and Ad-GFP-SM22α for 24 h (n = 3). d Confocal microscopy images of LXRα and LXRβ distribution in WT and Sm22α^−/− VSMCs. Scale bar, 10 µm. e Immunofluorescence staining for endogenous LXRα and LXRα-GFP in Sm22α^−/− VSMCs transducted with Ad-GFP and Ad-GFP-SM22α or not. Scale bar, 10 µm. f qRT-PCR analysis of cholesterol intake (LDLR, SR-BI), efflux genes (ABCA1, ABCG1) and sclerosis related genes (Col1α, Eln) in WT and Sm22α^−/− VSMCs incubated with or without cholesterol (n = 3). g The mRNA and protein levels of ABCA1 in WT and Sm22α^−/− VSMCs treated with cholesterol for 0, 12, 24, 48, and 72 h, respectively (n = 3). h ORO staining of WT and Sm22α^−/− VSMCs stimulated with cholesterol for 0, 24, 48, and 72 h, respectively, and quantification of positive ORO staining. Scale bar, 20 μm. i The binding activity of LXRα to the promoter of abca1 gene was decreased in Sm22α^−/− VSMCs (n = 4). j ChIP and RT-PCR detected LXRα binding to col1α promoter in WT and Sm22α^−/− VSMCs (n = 6). k The Young’s modulus of WT and Sm22α^−/− VSMCs treated with or without cholesterol (n = 120). Data and images are representative of at least three independent experiments. Data in a, g, and h were analyzed by Kruskal–Wallis rank sum test and two-way ANOVA. Data in b, c, f, i, and j were analyzed by unpaired t-test. N.S. not significantly different; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 3: Function of LXRα-ABCA1 axis is impaired in phenotypically switched VSMCs.

a Heatmap of proteomic analysis between synthetic and contractile VSMCs. b Analysis of KEGG pathway enriched by differentially expressed genes of proteomic analysis between synthetic and contractile VSMCs. c The mRNA expression of SM22α, LXRα, and ABCA1 in WT VSMCs treated with PDGF-BB for 0, 12, 24, and 48 h, respectively (n = 3). d Confocal microscopy images of LXRα distribution in WT VSMCs incubated with PDGF-BB for 0, 12, 24, and 48 h, respectively. Scale bar, 10 µm. e Confocal microscopy images of LXRα and ACTA2 in arterial walls of WT mice after ligation for 0, 7, 14, and 28 days. Scale bar, 20 µm. f Quantification of each lipid class in synthetic and contractile VSMCs. Lipid classes were expressed as μmol per g protein. g Heatmap of CEs between synthetic and contractile VSMCs. h ORO staining of WT VSMCs transducted with or without Ad-GFP-SM22α following with PDGF-BB and/or cholesterol treatment and quantification of positive ORO staining. Scale bar, 20 μm (n = 3). i M-mode and Doppler echocardiography images obtained from aortic arch and outflow tract of Sm22α^−/− mice transducted with AAV-GFP (n = 10) and AAV-SM22α (n = 10) fed Paigen diet for 12 weeks. A_s: outflow tract and aortic diameter in systole; A_d: outflow tract and aortic diameter in diastole. j Representative images of en face ORO-stained aortas and quantification of lesion areas (n = 6). k Representative immunofluorescence of LXRα (green) and quantification of cells with nuclear LXRα in the aortic sections from Sm22α^−/− mice transducted with AAV-GFP (n = 4) and AAV-SM22α (n = 4) fed Paigen diet for 24 weeks. Scale bar, 10 µm. Arrows indicated the distribution of LXRα. l Identification of SMC-derived foam cells within atherosclerotic lesion of Sm22α^−/− mice infected with AAV-GFP (n = 4) and AAV-SM22α (n = 4) fed Paigen diet for 24 weeks by CD68 (blue), ACTA2 (red), and Bodipy (green). Scale bar, 25 µm. Arrows indicated foam cells which were VSMCs-derived. Data and images are representative of at least three independent experiments. Data in c were analyzed by two-way ANOVA. Data in f, h, j, k, and l were analyzed by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 4: Nuclear import of LXRα is regulated by actin dynamics.

a Fluorescence recovery after photobleaching (FRAP) studies with LXRα-GFP to measure nuclear import. Cells were pretreated with LMB. Decreased accumulation of nuclear fluorescence indicates a lower rate of nuclear import of LXRα-GFP in Sm22α^−/− VSMCs relative to WT controls (n = 25). b Representative images of F-actin (phalloidin, red) and G-actin (DnaseI, green) in WT and Sm22α^−/− VSMCs. Scale bar, 10 μm. c, d Representative images for F-actin (phalloidin, red) and LXRα (green) in WT and Sm22α^−/− VSMCs with cholesterol loading or not (c) and in WT VSMCs treated with JPK, CytoB and after CytoB washout (d). Scale bars, 10 µm. e Western blot analysis of cytoplasmic and nuclear LXRα in WT VSMCs treated with CytoB at different time points (n = 6). Data and images represent at least three independent experiments. Statistical analyses, unpaired t-test and Kruskal–Wallis rank sum test. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5: G-actin interacts with and retains LXRα in the cytoplasm, blocking LXRα binding to Importin α.

a Double immunofluorescence staining for G-actin (DnaseI, red) and LXRα (green) in WT VSMCs accompanied with treatment of JPK or CytoB and also in Sm22α^−/− VSMCs. Scale bar, 10 μm. b, c Co-immunoprecipitation of ACTA2 and LXRα (b) and LXRβ (c), respectively, in F- and G-actin fractions of WT and Sm22α^−/− VSMCs (n = 3). d Double immunofluorescence staining of G-actin (Dnase1, red) and LXRα (green) or IgG in atherosclerotic lesion in aortic wall of Sm22α^−/− mice. Scale bar, 20 μm. e Representative immunofluorescence staining for endogenous LXRα (green) and LXRα-GFP (green) in WT VSMCs transfected with HA-ACTA2 (red, stained by anti-HA antibody) or not. Scale bar, 15 μm. f Representative immunofluorescence staining for LXRα-GFP (green) and HA-ACTA2 (red, stained by anti-HA antibody) in HEK-293A cells. Scale bar, 10 μm. g–j Two-color STORM images and quantification of the co-localization degree between LXRα and G-actin as well as Importin α in WT VSMCs with (h) or without (g) CytoB treatment and Sm22α^−/− VSMCs with (j) or without (i) Ad-GFP-SM22α infection (n > 10). k Co-immunoprecipitation of LXRα and Improtin α, Improtin β or ACTA2 in WT and Sm22α^−/− VSMCs with or without JPK, CytoB, PDGF-BB, and Ad-GFP-SM22α treatment (n = 3). l Double immunofluorescence staining for Importin α (red) and LXRα (green) in WT and Sm22α^−/− VSMCs as well as CytoB-treated WT VSMCs. Scale bar, 15 μm. m Co-immunoprecipitation of LXRα and Importin α, Improtin β or ACTA2 in WT VSMCs transfected with HA-ACTA2 of different concentration (n = 3). Data and images represent at least three independent experiments.

Fig. 6: The C-terminal domain mediates interaction between G-actin and LXRα.

a Representative immunofluorescence staining for LXRα-GFP (green) in WT VSMCs transfected with HA-ACTA2-CTD (red) or HA-ACTA2-NT (red). Scale bar, 15 μm. b Representative immunofluorescence staining for LXRα-GFP (green) and HA-ACTA2-CTD (red) or HA-ACTA2-NT (red) in HEK-293A cells. Scale bar, 15 μm. c LXRα-CTD-GFP (green) or LXRα-NT-GFP (green) was transfected into Sm22α^−/− VSMCs. Scale bar, 10 μm. d LXRα (-CTD, -NT)-GFP (green) and HA-ACTA2-CTD (red) were co-expressed in HEK-293A cells. Scale bar, 10 μm. e Interaction of HA-ACTA2 (-FL, -CTD, -NT) and GST-LXRα (-FL, -CTD, -NT) proteins analyzed by in vitro pull-down assay (n = 3). f Schematic representation of a working model which SM22α inhibits VSMC-derived foam cell formation by blocking actin-LXRα signaling ameliorating atherosclerosis. Data and images represent at least three independent experiments.