Fig 2: Coronin 1A downregulation attenuates expression of HIV-1 Tat-induced GFAP, IL-6, and pro-inflammatory genes in reactive astrocytes. Human astrocytes downregulated for coronin 1A using siRNA for 48 h followed by transfection with HIV-1 Tat for 24 h, were harvested and analyzed for (A) Coronin 1A and GFAP protein expression, using western blotting. (A) representative blots and (B) the densitometric analysis of the GFAP expression. (C) Released IL-6 levels were measured in the conditioned media collected from the above experimental groups, using Flow cytometry and (D) pro-inflammatory genes viz PTGS2 and NOS2, assessed through real-time PCR assay. GAPDH was used as a normalization control for all the mRNA and protein analyses. Data represents mean ± SD of three to four independent experiments. *p < 0.05, **p < 0.005, ***p < 0.0005. SCR, scrambled; CORO1A, coronin 1A

Fig 3: Coronin 1A knockdown does not affect the resting-state human astrocytes. Astrocytes transfected with CORO1A siRNA for 48 h, were harvested, and transcript and protein levels were analyzed using real-time qPCR and western blotting, respectively. (A) The bar graph represents the relative expression of coronin 1A mRNA and (B) protein, compared to the control group. Cells downregulated for coronin 1A were assessed for the expression of GFAP (C) mRNA and (D) protein. Vimentin (E) mRNA and (F) protein expression was also assessed in the same experimental group. Blots are representative and the bar graph shows the densitometric analysis of the blots. GAPDH was used as a normalization control for all the analyses. Data represent mean ± SD of three to four independent experiments. * represents p < 0.05; *** p < 0.0005. SCR, scrambled; CORO1A, coronin 1A; GFAP, glial fibrillary acidic protein

Fig 4: Downregulation of Coronin 1A attenuates HIV-1 Tat-induced indirect-neuronal death. Human primary neurons were treated for 24 h with (A) ACM collected from astrocytes transfected with HIV-1 Tat expression vector for 24 h after which neuronal death was assessed by analyzing the TUNEL-positive neurons, (B) ACM collected from astrocytes transfected with CORO1A siRNA for 48 h followed by transfection with HIV-1 Tat expression vector for 24 h and assessed for neuronal death by analyzing the TUNEL-positive neuronal cells, (C) ACM collected from astrocytes transfected with mimic-92b-5p for 48 h followed by transfection with HIV-1 Tat expression vector for 24 h and neurons were then assessed for neuronal death. Data represent the mean ± SD of four independent experiments. *p < 0.05, **p < 0.005. SCR, scrambled; CORO1A, coronin 1A; ACM, astrocyte conditioned media

Fig 5: MiR-92b-5p is downregulated in response to HIV-1 Tat and targets 3’ UTR of CORO1A. Astrocytes were transfected with HIV-1 Tat expression vector for 24 h and were assessed for the expression of miRNAs. (A) Heatmap was prepared using fold-change derived from absolute values of sequencing data, showing miRNAs which are downregulated by -1.3 to -2.5-fold in the presence of HIV-1 Tat, compared to the vector control group, and predicted to target CORO1A. (B) The bar graph represents miRNA levels compared to the vector control group, analyzed using real-time PCR assay; U6 was used for normalizing the data. (C) CORO1A 3’ UTR was cloned downstream to the luciferase gene in the pMIR-Report vector and was co-transfected along with mimic control or mimic-92b-5p into Hela cells. Quantitative analysis shows the normalized relative luciferase activity in the indicated groups. The data shown represent three to four independent experiments (mean ± SD). * represents p < 0.05