Fig 1: DCex and miR-203-3p contribute to alleviate AS in vivo. The HFD-fed ApoE-/- mice did not receive any treatment as controls, or were treated with DCex alone, DCex + antagomir-NC, or miR-203-3p antagomir (n = 8 for each group). (A, B) Oil red O staining for atherosclerotic plaque in ApoE-/- mice. (C–E) HE staining (200 ×) and Masson staining (200 ×) for atherosclerotic plaque in ApoE-/- mice (In panel C, red arrows show plaque location). (F) Serum LDL, HDL, TC, and TG levels in ApoE-/- mice determined by ELISA. (G) miR-203-3p expression and mRNA expression of Ctss in vascular tissues in ApoE-/- mice evaluated by RT-qPCR. (H, I) The protein expression of Ctss in vascular tissues in ApoE-/- mice evaluated by Western blot analysis. (J) Immunofluorescent double staining for the expression of F4/80 and Ctss (400 ×). * p < 0.05 vs. mice without treatment; # p < 0.05 vs. mice treated with DCex. Statistical data were measurement data, and described as mean ± standard deviation. The one-way analysis of variance was adopted for comparisons among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated 3 times independently.
Fig 2: The elevation of Ctss and decline of miR-203-3p are identified in AS. (A) The volcanic map of DEGs between from GSE56143 with -log10 logarithm of p value as the abscissa and logFC multiple as the ordinate, and |logFC| > 2 as threshold, where 19 genes are up-regulated and indicated as red, and 7 genes are down-regulated and indicated as green. (B) Protein interaction map of DEGs in which the brighter color indicates the higher core level. (C) Online prediction of miRNAs that target Ctss. (D) miR-203-3p expression in DCexs determined by RT-qPCR. * p < 0.05 vs. the healthy volunteers. Statistical data were measurement data, and described as mean ± standard deviation. The paired t test was used for comparison between two groups. n = 76 for AS patients, while n = 49 for healthy volunteers.
Fig 3: Ctss affects BMDM expression of atherosclerotic phenotype. (A) The mRNA expression of Ctss in BMDMs after 24-h treatment with ox-LDL determined by RT-qPCR. (B) The protein expression of Ctss in BMDMs after 24-h treatment with ox-LDL determined by Western blot analysis. (C) Silencing efficiency of Ctss after 24-h treatment with ox-LDL and different siRNA detected by RT-qPCR. (D–E) The proportion of foam cells in BMDMs treated with si-Ctss by oil red O staining (200 ×) (Five visual fields were randomly read and photographed, and the mean value was obtained. Each experiment was repeated three times). (F) Serum TC, FC and CE levels in BMDMs treated with si-Ctss determined by ELISA. (G, H) BMDM migration in response to the treatment of si-Ctss evaluated by Transwell assay (200 ×) (Five visual fields were randomly read and photographed, and the mean value was obtained. Each experiment was repeated three times). (I) Ctss protein expression in BMDMs in response to the treatment of si-Ctss evaluated by western blot analysis. ** p < 0.01 vs. the BMDMs treated with NC mimic, si-NC or without treatment. Statistical data were measurement data, and described as mean ± standard deviation. The paired t test was used for comparisons between two groups. The one-way analysis of variance was adopted for comparisons among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated 3 times independently.
Fig 4: Validation of the relationship between miR-203-3p and Ctss. (A) TargetScan prediction of miR-203-3p-Ctss binding sites. (B) The binding of miR-203-3p to Ctss confirmed by dual-luciferase reporter gene assay. (C) mRNA expression of Ctss in BMDMs treated with miR-203-3p mimic determined by RT-qPCR. (D) The protein expression of Ctss in BMDMs treated with miR-203-3p mimic determined by Western blot analysis (50 μg protein was loaded). (E) mRNA stability of Ctss following miR-203-3p mimic and miR-203-3p inhibitor evaluated using RNA degradation experiment. (F) The binding of miR-203-3p and Ctss detected using RIP assay. Ago2 antibody was used for immunoprecipitation of the RNA-induced silencing complex (Ago2-RISC) in BMDMs (Input: total sample before co-immunoprecipitation treatment). IgG was employed as a negative control and β-actin was used as an internal control. (G) Relative mRNA expression of serum Ctss detected by RT-qPCR (Relative quantification based on the expression of healthy population). (H) Protein expression of serum Ctss detected by ELISA. * p < 0.05 vs. healthy persons. ** p < 0.01. Healthy persons = 50; AS patients = 76. Statistical data were measurement data, and described as mean ± standard deviation. The paired t test was used for comparisons between two groups. The one-way analysis of variance was adopted for comparisons among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated 3 times independently.
Fig 5: The miR-203-3p/Ctss/p38/MAPK axis mediates the progression of AS in vitro. BMDMs were treated with NC mimic or miR-203-3p in the presence of green-fluorescent protein (GFP) or rAd-Ctss. (A, B) The proportion of foam cells in BMDMs by oil red O staining (200 ×). (C) Serum TC, FC and CE levels determined by ELISA. (D, E) BMDM migration evaluated by Transwell assay (200 ×). BMDMs were treated with PBS or DCexs in the presence of GFP or rAd-Ctss. (F, G), The proportion of foam cells in BMDMs by oil red O staining (200 ×). (H) Serum TC, FC and CE levels determined by ELISA. (I, J) BMDM migration evaluated by Transwell assay (200 ×). (K, L) Protein expression of genes related to the p38/MAPK signaling pathway tested by Western blot analysis in response to the treatment of NC mimic or miR-203-3p in the presence of green-fluorescent protein (GFP) or rAd-Ctss. (M, N) Protein expression of genes related to the p38/MAPK signaling pathway tested by Western blot analysis in response to the treatment of PBS or DCexs in the presence of GFP or rAd-Ctss. The results for the inflammation-related proteins IL-1ß and caspase 1 were derived from additional independent experiments. * p < 0.05 vs. the BMDMs treated with NC mimic or PBS plus GFP. # p < 0.05 vs. the BMDMs treated with miR-203-3p mimic or DCex plus GFP, or NC mimic or PBS plus rAd-Ctss. Statistical data were measurement data, and described as mean ± standard deviation. The one-way analysis of variance was adopted for comparisons among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated 3 times independently.
Supplier Page from Abcam for Anti-Cathepsin S antibody