Figure 5.
CARD8 deletion in ECs and HCMs improves cell-intrinsic immunity against CVB3. (A) Viral genome (vg) copy numbers of CVB3 in control (Ctl) and CARD-KO HAECs and HCMs determined by qPCR. (B) Functional titers of CVB3 in Ctl and CARD8-KO HAECs and HCMs measured by plaque assays. P values indicated are from parametric, unpaired, two-tailed student’s t test. (C) Epifluorescence images of Ctl and CARD8-KO HAECs infected with Timer-CVB3 in the GFP channel, indicating early infection. Scale bars, 50 µm. (D) vg copy numbers of CVB3 in WT HAECs treated with specific inhibitors Belnacasan at 10 μM (caspase-1 inhibitor) and Disulfuram at 10 μM (GSDMD inhibitor) or both, determined by qPCR. CVB3 infection MOI = 0.006. (E) vg copy numbers of CVB3 in WT and CASP-1 KO HAECs, determined by qPCR. CVB3 infection MOI = 0.006. (F) Expression of ICAM-1 in CVB3-infected HAECs measured by flow cytometry. (G) Number of THP1 cells adhered to Ctl and CARD8 KO HAECs infected with CVB3 at the indicated time points. (H) mRNA levels of representative pro-inflammatory genes induced by CVB3 infection in HAECs, determined by qPCR. (I) mRNA of indicated interferon-response genes in Ctl and CARD8-KO HCMs measured by qPCR. (J) mRNA levels of representative pro-inflammatory and interferon response genes induced by CVB3 infection in HAECs in the presence and absence of specific inhibitors, Belnacasan at 10 μM (caspase-1 inhibitor), Disulfuram at 10 μM (GSDMD inhibitor), or both, determined by qPCR. CVB3 infection MOI = 0.006. (K) Schematic of co-culture between Ctl and CARD8-KO HAECs infected with the Timer-CVB3 and HCMs. (L) Epifluorescence images of HCMs co-cultured with Ctl and CARD8-KO HAECs and infected from the apical side of the endothelium with Timer-CVB3 in the DsRed channel indicating a later stage of infection. Scale bars, 50 µm. (M) CVB3 vg copy number in HCMs co-cultured with Ctl and CARD8 KO HAECs infected with the Timer-CVB3 measured by qPCR. P values indicated are from parametric, unpaired, two-tailed student’s t test. Data are represented by mean ± SEM, P values indicated are from two-way ANOVA test unless otherwise stated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. CVB3 infection MOI used is 0.1 unless indicated otherwise. Unless otherwise stated, each dot represents a biological replicate. All experiments were repeated twice.

CARD8 deletion in ECs and HCMs improves cell-intrinsic immunity against CVB3. (A) Viral genome (vg) copy numbers of CVB3 in control (Ctl) and CARD-KO HAECs and HCMs determined by qPCR. (B) Functional titers of CVB3 in Ctl and CARD8-KO HAECs and HCMs measured by plaque assays. P values indicated are from parametric, unpaired, two-tailed student’s t test. (C) Epifluorescence images of Ctl and CARD8-KO HAECs infected with Timer-CVB3 in the GFP channel, indicating early infection. Scale bars, 50 µm. (D) vg copy numbers of CVB3 in WT HAECs treated with specific inhibitors Belnacasan at 10 μM (caspase-1 inhibitor) and Disulfuram at 10 μM (GSDMD inhibitor) or both, determined by qPCR. CVB3 infection MOI = 0.006. (E) vg copy numbers of CVB3 in WT and CASP-1 KO HAECs, determined by qPCR. CVB3 infection MOI = 0.006. (F) Expression of ICAM-1 in CVB3-infected HAECs measured by flow cytometry. (G) Number of THP1 cells adhered to Ctl and CARD8 KO HAECs infected with CVB3 at the indicated time points. (H) mRNA levels of representative pro-inflammatory genes induced by CVB3 infection in HAECs, determined by qPCR. (I) mRNA of indicated interferon-response genes in Ctl and CARD8-KO HCMs measured by qPCR. (J) mRNA levels of representative pro-inflammatory and interferon response genes induced by CVB3 infection in HAECs in the presence and absence of specific inhibitors, Belnacasan at 10 μM (caspase-1 inhibitor), Disulfuram at 10 μM (GSDMD inhibitor), or both, determined by qPCR. CVB3 infection MOI = 0.006. (K) Schematic of co-culture between Ctl and CARD8-KO HAECs infected with the Timer-CVB3 and HCMs. (L) Epifluorescence images of HCMs co-cultured with Ctl and CARD8-KO HAECs and infected from the apical side of the endothelium with Timer-CVB3 in the DsRed channel indicating a later stage of infection. Scale bars, 50 µm. (M) CVB3 vg copy number in HCMs co-cultured with Ctl and CARD8 KO HAECs infected with the Timer-CVB3 measured by qPCR. P values indicated are from parametric, unpaired, two-tailed student’s t test. Data are represented by mean ± SEM, P values indicated are from two-way ANOVA test unless otherwise stated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. CVB3 infection MOI used is 0.1 unless indicated otherwise. Unless otherwise stated, each dot represents a biological replicate. All experiments were repeated twice.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal