Fig 2: LSS modulates NaV expression profiles in both TeloHAEC and HUVEC after 96 h of flow. mRNA expression profile of NaV channel subunits (SCN1A to SCN11A and SCN1B to SCN4B) in TeloHAEC and HUVEC (A, B). The mRNA relative expression of NaV channel subunits was determined by relative RT‐qPCR. The data are mean of mRNA relative expression ± SEM (n = 3) with the 2−ΔCq method. ND, not detected (Cq > 35). The protein expression of NaV channel subunits detected at transcript level was evaluated by western blot in comparison to control tissues as mouse heart (for NaV1.5 and NaVβ3), brain (for NaV1.6 and NaVβ1) or Dorsal Root Ganglion neurons (DRG) (for NaV1.7) (C, D). Fifty μg of cell protein extracts (TeloHAEC and HUVEC) and twenty μg of protein from control tissue (heart, brain and DRG) were loaded on 8% SDS‐PAGE. HSC70 protein was used as a loading control (C, D). In TeloHAEC or HUVEC, NaV1.5 and NaVβ3 share the same HSC70 loading control as well as NaV1.6 and NaVβ1 since the protein samples immunoblotted with these antibodies (NaV1.5/NaVβ3 and NaV1.6/NaVβ1) are the same after cutting the nitrocellulose membrane. SCN5A (E), SCN8A (F) and SCN9A (G), SCN1B (H), SCN3B (I) mRNA expression and NaVβ3 protein expression (J) in LSS (20 dyn/cm2 for 96 h) compared to static condition in TeloHAEC (left panel) and HUVEC (right panel). Values of fold change (mRNA and protein) are expressed as mean ± SEM for at least 3 until 6 independent experiments and compared to static condition, given the arbitrary value of 1 using the 2−ΔΔCq method for RT‐qPCR experiments or normalized to HSC70 used as loading control protein and compared to static condition, given the arbitrary value of 1 for the densitometry analysis. Immunoblot representative images of NaVβ3 and HSC70 protein expression in TeloHAEC and HUVEC (J) cultivated under static or LSS (20 dyn/cm2) condition for 96 h. Nonparametric Mann–Whitney tests were performed. ns, nonsignificant, *p < 0.05, **p < 0.01.

Fig 3: Identification of NaVβ3‐interacting partners in TeloHAEC and HUVEC by proteomic analysis. (A, B) Schematic representation of the NaVβ3 immunoprecipitation (IP) procedures. TeloHAEC (A) and COS‐7 (B) were transfected with pMyc‐SCN3B, and pCTL (negative control). (A) Myc tag‐NaVβ3 with its partners were coimmunoprecipitated using anti‐Myc tag magnetic beads from TeloHAEC protein extracts. (B) Myc tag‐NaVβ3 from COS‐7 protein extracts were first immunocaptured with anti‐Myc tag magnetic beads and subjected to pull‐down using HUVEC whole cell protein lysate. Below the schemes are shown representative control immunoblots (n = 3) with anti‐NaVβ3 (top blot) and anti‐myc tag (bottom blot). Input (Inp) was the whole protein extract after cell transfection and the flow throw (FT) corresponded to the supernatant recuperated after immunocapturing with the anti‐Myc tag magnetic beads. Wash and IP correspond to the washing step and a sample from the solution used to boil the beads (IP) to elute NaVβ3 and its partners. IP from TeloHAEC and HUVEC were subjected to MS/MS analysis. (C) Venn Diagram indicating the number of NaVβ3 interacting proteins identified in TeloHAEC and HUVEC after proteomic analysis. (D) Bubble plots of Gene ontology (GO)‐term enrichment analysis of the 76 putative NaVβ3 interactants. The four first GO‐terms belonging to the biological process and cellular component categories are indicated on the left. Below the bubble chart, is shown the protein ratio (number of proteins in our dataset compared to number of proteins in annotation). The scale of the bubble plot size (black, upper right) reflects the number of proteins. The significancy of the identification score is illustrated by a color intensity scale (Log10 Bonferroni, lower right). (E, F) Representative NaVβ3 and mTOR immunoblots (n = 3) of immunoprecipitated NaVβ3 with Myc tag antibody in TeloHAEC (E) and HUVEC (F).

Fig 4: SiRNA SCN3B decreased cell alignment and increased cell width after 24 h of LSS in TeloHAEC. (A) mRNA expression of SCN3B in TeloHAEC transfected with SiRNA Control (siRNA CTL) or SiRNA targeting SCN3B (SiRNA SCN3B) by ddPCR. Values of SCN3B mRNA fold change are expressed as mean of copy number of SCN3B per μL. (B) The histogram illustrates TeloHAEC amount between angle values of −5° and +5°, after transfection with SiRNA Control (siRNA CTL) or SiRNA targeting SCN3B (siRNA SCN3B). (C) Elongation of HUVEC was determined as the cell length along flow direction (left panel) divided by cell width (right panel). TeloHAEC were submitted to static or LSS (20 dyn/cm2) for 24 h. Values are mean ± SEM are shown (n = 5). Significance was analyzed using one‐way ANOVA statistical analysis, followed by Tukey multiple comparisons test or nonparametric Mann–Whitney tests. ns, nonsignificant, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 5: NaVβ3 partially colocalizes with LC3B, LAMP1, and mTOR in TeloHAEC under static or LSS conditions. (A) Representative images illustrating the immunostaining of LC3B, LAMP1 or mTOR (red) together with NaVβ3 (green) in TeloHAEC which were transfected with pmyc‐SCN3B and cultivated under static condition. Nuclei (blue) were stained with DAPI. (B) Quantification of the colocalization of NaVβ3 with LC3B, LAMP1 or mTOR was calculated using Pearson's correlation coefficient (PCC). Histogram bars represent the PCC value as mean ± SEM for each protein (LC3B, LAMP1 or mTOR) in TeloHAEC cultivated under static or LSS conditions for three independent experiments. Significance was analyzed using one‐way ANOVA statistical analysis, followed by Tukey multiple comparisons test. **p < 0.01, ***p < 0.001.