rd cells  (Vector Laboratories)

Journal: Frontiers in Pharmacology

Article Title: Genistein inhibits the replication of enterovirus A71

doi: 10.3389/fphar.2026.1787050

Figure Lengend Snippet: Effect of the genistein on EV71 replication in HeLa cell. (A) Cytotoxicity of genistein on HeLa cells. HeLa cells were treated with serially diluted genistein for 24 h, after which the cell viabilities were measured by cell counting kit-8 assay (CCK8). N = 3 replicates in each concentration. (B) HeLa cells were infected with EV71 at a MOI of 5 for 2 h. At 2 h post-infection, cells were treated with 10% DMEM or genistein (GET, 75 μM) for 22 h. Morphology was recorded using an inverted microscope. N = 3 independent experiments. (C) At 2 h post-infection, cells were treated with genistein (0, 50, 75, 100 μM) for 22 h. Western blot analysis of VP1 was performed. Tubulin was used a loading control. (D) Corresponding to (C) the gray value ratio of the VP1 protein band to the Tubulin protein band was shown. Data were presented as the mean ± SD (N = 3 independent experiments); statistical analysis was performed by one-way ANOVA test with Tukey’s multiple comparisons test. (*** P < 0.001). (E) At 2 h post-infection, cells were treated with genistein (75 μM) for 22 h. Relative mRNA levels of viral genome were measured using VP1 primers. GAPDH was used as a housekeeping gene. Data were presented as the mean ± SD (N = 3 independent experiments); statistical analysis was performed using T-test. (*** P < 0.001). (F) At 2 h post-infection, cells were treated with genistein (0, 50, 75, 100 μM) for 22 h. Virus from the supernatant was collected for plaque assay analysis. Plaques in RD cells were recorded after diluting the supernatant 5000-fold. (G) Corresponding to (F) data were presented as the mean ± SD (N = 3 independent experiments); statistical analysis was performed using one-way ANOVA test with Tukey’s multiple comparisons test. (** P < 0.01, *** P < 0.001). (H) At 2 h post-infection, cells were treated with genistein (75 μM) for 22 h. Intracellular and supernatant progeny virions were titrated using RD cells to determine TCID50/ml. Data were presented as the mean ± SD (N = 3 independent experiments); statistical analysis was performed using a T-test (*** P < 0.001).

Article Snippet: Human rhabdomyosarcoma RD cells (CCL-136) and human cervical cancer HeLa cells (CCL-2TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Counting, Concentration Assay, Infection, Inverted Microscopy, Western Blot, Control, Virus, Plaque Assay

Journal: Frontiers in Pharmacology

Article Title: Genistein inhibits the replication of enterovirus A71

doi: 10.3389/fphar.2026.1787050

Figure Lengend Snippet: Genistein inhibited autophagy. (A) Cherry-GFP-LC3 plasmid was transfected into HeLa cells, at 24 h post transfection, HeLa cells treated with rapamycin (Rapa; 8 nM), genistein (GET; 75 μM), or both rapamycin (8 nM) and genistein (75 μM) for 24 h. Immunofluorescence analysis was recorded. Scale bars = 10 μm. (B) Corresponding to (A) , the GFP-LC3 positive dots was counted by ImageJ (N = 3, *** P < 0.001, ns: no significant difference). (C) RD cells were treated with rapamycin (8 nM) or together with doses of genistein (GET, 0, 50, 75, 100 μM) for 24 h. Then cells were collected for Western blotting analysis. (D,E) is calculated according to (C) . Data were presented as the mean ± SD (N = 3 independent experiments); Statistical analysis of was performed using one-way ANOVA with Tukey’s multiple comparisons test. (ns: No significant difference, * P < 0.05, ** P < 0.01,*** P < 0.001). (F) RD cells were infected with EV71 at a MOI of 1 for 2 h, at 2 h post infection, cells were treated with genistein (GET, 0, 50, 75, 100 μM) for 22 h. At 24 h post infection, cells were collected for Western blotting analysis. (G,H) is calculated according to (F) . The quantification of relative VP1 and LC3II/Tubulin were shown. Data were presented as the mean ± SD (N = 3 independent experiments); Statistical analysis of was performed using one-way ANOVA with Tukey’s multiple comparisons test. (* P < 0.05, ** P < 0.01,*** P < 0.001). (I) RD cells were infected with EV71 at a MOI of 1 for 2 h, at 2 h post infection, cells were treated with 3-MA (2, 4, 6 mM) for 22 h. At 24 h post infection, cells were collected for Western blotting analysis. (J,K) is calculated according to (I) . The quantification of relative VP1 and LC3II/Tubulin were shown. Data were presented as the mean ± SD (n = 3 independent experiments); Statistical analysis of was performed using one-way ANOVA with Tukey’s multiple comparisons test (* P < 0.05, ** P < 0.01,*** P < 0.001).

Article Snippet: Human rhabdomyosarcoma RD cells (CCL-136) and human cervical cancer HeLa cells (CCL-2TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Plasmid Preparation, Transfection, Immunofluorescence, Western Blot, Infection

Journal: Frontiers in Pharmacology

Article Title: Genistein inhibits the replication of enterovirus A71

doi: 10.3389/fphar.2026.1787050

Figure Lengend Snippet: Genistein induces G2/M arrest, which inhibits viral replication. (A,B) HeLa cells were treated with genistein (GET, 75 μM) or 10% DMEM for 24 h. Flow cytometry was used to analyze the proportion of cells in G0/G1, G2/M, and S phases of the cell cycle. (G2/M of Con vs. GET, ### P < 0.001). (C,D) HeLa cells were infected with EV71 at a MOI of 5 for 2 h, then treated with genistein (75 μM) for 22 h. Flow cytometry analysis was performed to determine the distribution of cells in G0/G1, G2/M, and S phases. (S of Mock + Con vs. EV + Con, ** P < 0.01; G2/M of Mock + Con vs. Mock + GET, ### P < 0.001; G2/M of EV + Con vs. EV + GET, ### P < 0.001). (E,F) RD cells were infected with EV71 at a MOI of 1 for 2 h, then treated with genistein (75 μM) for 22 h. Flow cytometry analysis was performed to determine the distribution of cells in G0/G1, G2/M, and S phases. (S of Mock + Con vs. EV + Con, *** P < 0.001; G2/M of Mock + Con vs. Mock + GET, ### P < 0.001; G2/M of EV + Con vs. EV + GET, ### P < 0.001) (G–M) HeLa cells were treated with genistein (0, 50, 75, 100 μM) for 24 h. Western blotting was used to analyze the relative protein levels of CDK4, CyclinB1, CDK1, CyclinE1, CDK2, and CyclinD1. Data were presented as mean ± SD (N = 3 independent experiments); statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons test. (ns: no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001). (N,O) HeLa cells were treated with nocodazole (Noco; 20 nM) or 10% DMEM for 24 h. Flow cytometry was used to analyze the proportion of cells in G0/G1, G2/M, and S phases. (S of Mock + Con vs. EV + Con, ** P < 0.01; G2/M of Mock + Con vs. Mock + Noco, ### P < 0.001; G2/M of EV + Con vs. EV + Noco, ### P < 0.001). (P,Q) HeLa cells were infected with EV71 at a MOI of 5 for 2 h; at 2 h post-infection, nocodazole (20 nM) was administered, and at 24 h post-infection, cells were collected for Western blotting. Tubulin served as the loading control. Data were presented as mean ± SEM (N = 3 independent experiments); statistical analysis was performed using a T-test. (*** P < 0.001).

Article Snippet: Human rhabdomyosarcoma RD cells (CCL-136) and human cervical cancer HeLa cells (CCL-2TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Infection, Western Blot, Control