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. 2021 Jun 16;43(6):488–500. doi: 10.1093/eurheartj/ehab222

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© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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TBCE knockdown led to a proliferating phenotype in both primary murine VSMCs and in TBCE-silenced HAoSMCs. Confirmation of ER stress induction in vitro. Representative immunofluorescence images of (A) primary VSMCs isolated from murine aortas, stained for DAPI (blue), TBCE (green) and α-tubulin (magenta) (upper panel) or phalloidin (green, F-actin; n = 3 representative images per group), α-tubulin (magenta) and DAPI (blue) (lower panel) as cellular morphology markers; and (B) primary VSMCs isolated from murine aortas (±AngII treatment) were stained for DAPI (blue), α-tubulin (magenta) and calnexin (cyan). Graphs of integrated fluorescence intensity of (C) TBCE/DAPI (n = 3–4 per group) (D) α-tubulin/DAPI (n = 7–8 per group) (E) Calnexin/DAPI (n = 5–9 per group) (F) MTT proliferation assay of primary VSMCs (±AngII treatment) (n = 3–7 per group). Representative immunofluorescence images of (G) human aortic VSMCs (HAoSMCs) transfected with negative control siRNA (NgCTL) or with TBCE siRNA stained for DAPI (blue), TBCE (green), and α-tubulin (magenta) (upper panel) or phalloidin (green, F-actin; n = 3 representative images per group), α-tubulin (magenta) and DAPI (blue) (lower panel) as cellular morphology markers; and of (H) transfected HAoSMCs (± AngII treatment) stained for DAPI (blue), α-tubulin (magenta), and calnexin (cyan). Graphs of integrated fluorescence intensity of (I) TBCE/DAPI (n = 4 per group) (J) α-tubulin/DAPI (n = 6 per group) (K) Calnexin/DAPI (n = 4–3 per group) (L) MTT proliferation assay of primary VSMCs (±AngII treatment) (n = 4 per group) (Tukey’s multiple comparison test; one-way ANOVA; different numbers in the in vitro experiments refer to individual experiments).