Journal: The FASEB Journal

Article Title: SARS ‐ CoV ‐2 Nucleocapsid Protein Does Not Induce Inflammation in Endothelial Cells or Monocytes

doi: 10.1096/fj.202501433R

Figure Lengend Snippet: Effects of recombinant N‐protein (endotoxin nondepleted, NPnon) on inflammatory marker mRNA levels in human microvascular endothelial cells. Cells were treated with vehicle, LPS (1 μg/mL), or NPnon (10 μg/mL, ThermoFisher) for 4 h. The total RNA was isolated, and qPCR analysis was performed to measure the mRNA levels of TNFα, IL‐6, E‐selectin, and ICAM‐1. Data are presented as mean ± SEM and analyzed using one‐way ANOVA followed by Bonferroni's multiple comparisons test. n = 4–5 independent cell cultures per group. **** p < 0.0001 vs. control.

Article Snippet: Human pulmonary artery microvascular endothelial cells (HMEC, American Type Culture Collection (ATCC)) were cultured in endothelial cell media as described above, in an incubator at 37°C with 5% CO 2 .

Techniques: Recombinant, Marker, Isolation, Control

Journal: The FASEB Journal

Article Title: SARS ‐ CoV ‐2 Nucleocapsid Protein Does Not Induce Inflammation in Endothelial Cells or Monocytes

doi: 10.1096/fj.202501433R

Figure Lengend Snippet: Effects of recombinant N‐protein (endotoxin nondepleted, NPnon) on inflammatory marker mRNA levels in mouse microvascular endothelial cells. Cells were treated with vehicle, LPS (1 μg/mL), NPnon (10 μg/mL, ThermoFisher), LPS + polymyxin B (PMB) (1.25 mg/mL), NPnon + PMB (250 μg/mL), or PMB (250 μg/mL) for 4 h. The total RNA was isolated and qPCR analysis was performed to measure the mRNA levels of TNFα, IL‐6, E‐selectin, and ICAM‐1. Data are presented as mean ± SEM and analyzed using one‐way ANOVA followed by Bonferroni's multiple comparisons test. n = 5 independent cell cultures per group. *** p < 0.001, ** p < 0.01 vs. control.

Article Snippet: Human pulmonary artery microvascular endothelial cells (HMEC, American Type Culture Collection (ATCC)) were cultured in endothelial cell media as described above, in an incubator at 37°C with 5% CO 2 .

Techniques: Recombinant, Marker, Isolation, Control

Journal: The FASEB Journal

Article Title: SARS ‐ CoV ‐2 Nucleocapsid Protein Does Not Induce Inflammation in Endothelial Cells or Monocytes

doi: 10.1096/fj.202501433R

Figure Lengend Snippet: Effects of endotoxin‐depleted N‐protein (NPd) on inflammatory marker mRNA levels in mouse microvascular endothelial cells. Cells were treated with vehicle, LPS (1 μg/mL), NPd (50 μg/mL), NPd + PMB (250 μg/mL), or PMB (250 μg/mL) alone (control) for 4 h. Total RNA was isolated and RT‐qPCR analysis was performed to measure mRNA levels of TNFα and E‐selectin. Data are presented as mean ± SEM and analyzed using one‐way ANOVA followed by Bonferroni's multiple comparisons test. n = 3 independent cell cultures per group. *** p < 0.001, ** p < 0.01 vs. control.

Article Snippet: Human pulmonary artery microvascular endothelial cells (HMEC, American Type Culture Collection (ATCC)) were cultured in endothelial cell media as described above, in an incubator at 37°C with 5% CO 2 .

Techniques: Marker, Control, Isolation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: ERM and Rac-1 activation are amplified by the CLIC5A/ezrin interaction. A , endogenous pERM abundance in lysates and detergent resistant pellets of COS-7 cells transfected with GFP-CLIC5A cDNA and increasing concentrations of GFP-ezrin 432-586 (T567D) cDNA . GFP-CLIC5A cDNA was kept constant and the GFP-CLIC5A: GFP-ezrin 432-586 (T567D) cDNA transfection ratio was 1:1, 1:2, 1:4, and 1:8. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± SD, n = 3 independent experiments, one-way ANOVA: F = 7.90; p = 0.0007. p values shown represent post hoc Dunnett’s multiple comparisons). B , pERM abundance in lysates and detergent resistant pellets of COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP- e zrin 432-570 cDNA. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± S.D., n = 3 biologically independent experiments, two-way ANOVA: interaction F = 12.25; p = 0.01; CLIC5A effect: F = 39.63, p = 0.0002; ezrin 432-570 effect F = 22.58. p = 0.0014; p values shown represent post hoc Tukey’s multiple comparisons). C , endogenous Rac1 WB for COS-7 cell lysates and endogenous Rac1-GTP captured by PAK-PBD pulldown (PD) from COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP-ezrin 432-586 (T567D) cDNA. Left panel, representative WB. Right panel: densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± SD, n = 3 independent experiments, two-way ANOVA: interaction F = 6.32, p = 0.036; CLIC5A effect F = 29.25, p = 0.001; ezrin 432–586 effect F = 5.88; p = 0.042, p values for post hoc Tukey’s multiple comparisons are shown). D , PAK-PBD pulldown (PD) of endogenous Rac1-GTP from lysates of COS-7 cells transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of transiently expressed GFP-ezrin 432 - 570 cDNA. Left panel, representative WB. Right panel, densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± S.D., n = 3 independent experiments, two-way ANOVA: interaction F = 0.029, p = 0.87; CLIC5A effect F = 62.17 p < 0.0001; ezrin 432–570 effect F = 0.012; p = 0.91, p values for post hoc Tukey’s multiple comparisons are shown). E , endogenous Rac1-GTP abundance in human glomerular endothelial cells (hGENs) determined by Rac1-GTP G-LISA. The cells were transduced with control adenoviral-vector (ad-Vector) or untagged CLIC5A cDNA in the same vector (ad- CLIC5A ) at an increasing multiplicity of infection (MOI) (mean ± SD, n = 3 independent experiments). F , representative WB of lysates from hGEN cells transduced with ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA. G , change relative to baseline of Rac1-GTP in hGEN cells transduced with 30 MOI ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA (mean ± SD, n = 4 independent experiments, two-way ANOVA: interaction F = 6.35, p = 0.027; CLIC5A effect F = 34.18, p < 0.0001; ezrin siRNA effect F = 6.86; p = 0.023, p values shown represent post hoc Tukey’s multiple comparisons). H , coimmunoprecipitation of endogenous Rho-GDI with endogenous ezrin in the presence and absence of transiently expressed GFP-CLIC5A. Left panel: representative α-Rho GDI, α-ezrin, and α-CLIC5A WB of lysates (input) and α-ezrin immunoprecipitates (IP). Right panel: Quantification of endogenous Rho-GDI and endogenous ezrin immunoprecipitated with α-ezrin antibodies (n = 3 biologically distinct experiments, mean ± SD, Student’s t test). CLIC, chloride intracellular channel; PBD, protein binding domain; pERM, phosphorylated ezrin, radixin, and moesin proteins; Rho-GDI, Rho guanine nucleotide dissociation inhibitor; WB, Western blot.

Article Snippet: Mycoplasma-free primary human glomerular endothelial cells (hGENs) were purchased from Angio-Proteomie (# cAP-0004) and maintained in EGM-2 MV Bulletkit growth media (# CC-3162, Lonza) containing 5% FBS and 1% penicillin/streptomycin at 37 °C in humidified air containing 5% CO 2 .

Techniques: Activation Assay, Amplification, Transfection, Transduction, Control, Plasmid Preparation, Infection, Immunoprecipitation, Protein Binding, Western Blot