Fig 5.

JINR1 inhibition attenuates DENV and WNV replication, neuronal apoptosis, and the expression of genes involved in ER stress and neuroinflammation. (A) JINR1 promotes DENV replication in SH-SY5Y cells transfected with either ASO-NS, ASO-JINR1-1, ASO-JINR1-2, empty vector, or JINR1-OE, and viral replication was determined by quantifying the intracellular levels of DENV RNA using qRT-PCR at 48 hpi. (B) JINR1 promotes WNV replication in SH-SY5Y cells transfected with either ASO-NS, ASO-JINR1-1, ASO-JINR1-2, empty vector, or JINR1-OE, and viral replication was determined by quantifying the intracellular levels of WNV RNA using qRT-PCR at 48 hpi. (C) JINR1 silencing reduces DENV titer. Quantification of viral titer upon JINR1 depletion during DENV infection is shown in Fig. S10A. (D) JINR1 silencing reduces WNV titer. Quantification of viral titer upon JINR1 depletion during WNV infection is shown in Fig. S10D. (E) JINR1 promotes apoptosis in DENV-infected SH-SY5Y cells. SH-SY5Y cells were transfected with either ASO-NS, ASO-JINR1-1, or ASO-JINR1-2, and the caspase-3/7 activity was determined at 60 hpi. (F) JINR1 promotes apoptosis in WNV-infected SH-SY5Y cells. SH-SY5Y cells were transfected with either ASO-NS, ASO-JINR1-1, or ASO-JINR1-2, and the caspase-3/7 activity was determined at 60 hpi. (G) JINR1 depletion reduces DENV-induced cleaved PARP protein expression. SH-SY5Y cells were transfected with ASO-NS or ASO-JINR1-1, and protein lysates were collected at 60 hpi. Cleaved PARP protein levels were analyzed by western blotting. A representative blot is shown from three independent experiments with similar results. Blots were reprobed for β-actin to establish equal loading. (H) JINR1 depletion reduces WNV-induced cleaved PARP protein expression. SH-SY5Y cells were transfected with ASO-NS or ASO-JINR1-1, and protein lysates were collected at 60 hpi. Cleaved PARP protein levels were analyzed by western blotting. A representative blot is shown from three independent experiments with similar results. Blots were reprobed for β-actin to establish equal loading. (I) JINR1 depletion attenuates DENV-induced ER stress genes. qRT-PCR analysis of indicated ER stress genes upon JINR1 depletion in SH-SY5Y cells at 48 hpi. (J) JINR1 depletion reduces DENV-induced neuroinflammatory genes. qRT-PCR analysis of indicated neuroinflammatory genes upon JINR1 depletion in SH-SY5Y cells at 48 hpi. (K) JINR1 depletion attenuates WNV-induced ER stress genes. qRT-PCR analysis of indicated ER stress genes upon JINR1 depletion in SH-SY5Y cells at 48 hpi. (L) JINR1 depletion reduces WNV-induced neuroinflammatory genes. qRT-PCR analysis of indicated neuroinflammatory genes upon JINR1 depletion in SH-SY5Y cells at 48 hpi. Error bars represent the mean ± SEM from three independent experiments. (A, B, I-L) RNA samples were analyzed by qRT-PCR. (A and B) *Significant change compared to DENV-ASO-NS/WNV-ASO-NS/ DENV-Con-Vec/WNV-Con-Vec. (C and D) *Significant change compared to DENV-ASO-NS/WNV-ASO-NS. (E, F, and I–L) *Significant change compared to MI-ASO-NS. #Significant change compared to DENV-ASO-NS/WNV-ASO-NS. Statistical comparison was made using the Student’s t-test.