Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients

Figures

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  Figure 1. Expression of the importin family genes in COVID-19 and non-COVID-19 patients. (A) Experimental design of the GSE152075 dataset, composed of transcriptome data from nasopharyngeal swabs collected from 430 COVID-19 and 54 non-COVID-19 patients. (B–I) Gene expression analysis for importin genes (B) KPNA1, (C) KPNA2, (D) KPNA3, (E) KPNA4, (F) KPNA5, (G) KPNA6, (H) KPNA7, and (I) KPNB1. Each gene is divided in two panels: (I) COVID-19 versus non-COVID-19 patients (p-values correspond to Wilcoxon rank-sum test, except when the assumption normality was maintained, then two-tail Welch’s t test was used). (II) COVID-19 and non-COVID-19 patients categorized by age groups (p-values correspond to increasing and decreasing Jonckheere–Terpstra trend tests). Blue boxes indicate genes with significant or marginal significant p-value when comparing gene expression between COVID-19 and non-COVID-19 patients.

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  Figure 2. Expression of the Rho GTPase family genes in COVID-19 and non-COVID-19, and correlation analysis. (A–C) Gene expression analysis for Rho GTPase genes (A) RHOA, (B) CDC42, and (C) RAC1. Each gene is divided in two panels: (I) COVID-19 versus non-COVID-19 patients (p-values correspond to Wilcoxon rank-sum test, except when the assumption normality was maintained, then two-tail Welch’s t test was used). (II) COVID-19 and non-COVID-19 patients categorized by age groups (p-values correspond to increasing and decreasing Jonckheere–Terpstra trend tests). Light green boxes indicate genes with significant or marginal significant p-value when comparing gene expression between COVID-19 and non-COVID-19 patients. (D) Summary of importin and Rho GTPase genes selected for correlation analysis. Red color represents genes that are downregulated and light green represents genes that are upregulated in COVID-19 and non-COVID-19 patients. (E) Pairwise Spearman correlation matrix analysis between age, KPNA3, KNA5, KPNA7, KPNB1, RHOA, and CDC42. The upper half displays the Spearman coefficients (r) considering all patients (Corr.), non-COVID-19 patients (Neg.), or COVID-19 patients (Pos.). Black…

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  Figure 3. Dysregulation of importin and Rho GTPases gene expression in A549 cells transfected with Poly(I:C) and treated with IVM and ATV. (A) Experimental design of IVM and ATV treatment in a viral infection simulation context. (B) Heatmap depicting NFKB, KPNA7, KPNA5, KPNA2, RHOA, RAC1, and CDC42 fold changes in mRNA levels assessed by real time PCR (RT-qPCR) in A549 cells transfected with Poly(I:C) (10 µg/mL) and treated with PBS as the control, IVM (2.5 µM), ATV (10 µM), or the combination of both drugs (IVM + ATV). Values were normalized using PPIA as a reference gene. Gene expression for control Poly(I:C) transfected cells was relativized to mock condition. Gene expression for IVM, ATV, and IVM + ATV treated cells was relativized to the control Poly(I:C) condition. Red, white, and blue represent a fold change >1, fold change = 1 or fold change <1, respectively. * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 4. Effect of IVM and ATV on importin α nuclear accumulation. (A–D). Importin α nuclear to cytoplasmic distribution was analyzed by confocal microscopy in Hela (A,C) and Vero (B,D) cells treated with 2.5 μM IVM for 1 h (A,B) or 10 μM ATV for 24 h (C,D). Each data point represents Fn/Fc from a single cell; data were normalized to control cells and median ± quartiles are indicated. ** p < 0.01; **** p < 0.0001; Mann–Whitney test. (E,F) Representative confocal images of Hela cells exposed to IVM (E) or ATV (F) showing the effect of the drugs on importin α cellular distribution after treatment. Scale bar = 20 μm.

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  Figure 5. Evaluation of importin α nuclear accumulation after combination treatment using IVM plus ATV. (A) Hela and (B) Vero cells were treated with 2.5 μM IVM, 10 μM ATV, or IVM + ATV and importin α cellular distribution was evaluated by confocal microscopy. Each data point represents Fn/Fc from a single cell; data were normalized to control cells and median ± quartiles are indicated. ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control group; ### p < 0.001 vs. IVM and ATV monotherapies; Kruskal–Wallis followed by Dunn’s multiple comparisons test. (C) Representative confocal images of Hela cells treated with IVM, ATV, and IVM + ATV showing a combination treatment effect on importin α nuclear localization. Scale bar = 20 μm.

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  Figure 6. Effect of IVM at low concentration on importin α nuclear accumulation. (A,B) Importin α nuclear to cytoplasmic distribution evaluated in Hela (A) and Vero (B) cells treated with 0.2 μM IVM for 24 h. Each data point represents Fn/Fc from a single cell; data were normalized to control cells and median ± quartiles are indicated. * p < 0.05; **** p < 0.0001; Mann–Whitney test. (C) Representative confocal images of Hela cells treated with 0.2 μM IVM. Scale bar = 20 μm.

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  Figure 7. Evaluation of importin α nuclear accumulation after combination treatment using IVM at low concentration plus ATV. (A) Hela and (B) Vero cells were treated with 0.2 μM IVM, 10 μM ATV, or IVM + ATV and importin α cellular distribution was evaluated by confocal microscopy. Each data point represents Fn/Fc from a single cell; data were normalized to control cells and median ± quartiles are indicated. * p < 0.05; *** p < 0.001, **** p < 0.0001 vs. control group; Kruskal–Wallis followed by Dunn’s multiple comparisons test. (C) Representative confocal images of Hela cells treated with IVM, ATV, or IVM + ATV showing a combination treatment effect on importin α nuclear localization when low concentration IVM was used. Scale bar = 20 μm.