Fig 2: HTLV-1 co-culture and expression of cellular markers in HIV-1 and HTLV-1 infected neurospheres. (a) Differentiated neurospheres were exposed to dual-tropic HIV-1 89.6 with or without cART (lamivudine, tenofovir disoproxil fumarate, emtricitabine, indinavir), or were co-cultured with irradiated HTLV-1 infected cells with or without IFN/AZT for a period of fourteen days. Western blot was performed to examine the relative expression of various CNS-relevant cellular markers including those associated with microglia-like cells, astrocytes, neurons (dopaminergic, GABAergic, glutamatergic), and NSCs. (b) Western blot was performed to examine the relative expression of Tax and p19 in neurospheres co-cultured with HTLV-1 infected cells with or without IFN/AZT treatment. (c) RT-qPCR was performed to quantify the copy numbers of tax, env, and hbz RNA in neurospheres co-cultured with HTLV-1 infected cells with or without IFN/AZT treatment. n = 3. ** p < 0.005, *** p < 0.0001.

Fig 3: HTLV-1 proteins are differentially packaged after Viral Activation. HUT102 cells were treated with IR (10 Gy) and cultured in Exo-Free media for 5 days. Supernatants were collected from control and IR treated cells for precipitation by ExoMAX and separation into 11 fractions by OptiPrep/ultracentrifugation. Each resulting fraction was concentrated with NT080/082/086 particle mixture overnight. (A) WB analysis was conducted for each fraction from control (lanes 1–11) and IR treated cells (lanes 12–22), and probed for p19, gp61, gp46, HBZ, Actin. Each figure represents two blots with identical exposure combined into one for control and one for IR. (B) Densitometry analysis was performed on Tax protein bands from three independent Iodixanol gradients, averaged, and normalized to Actin. EV RNA was analyzed by RT-qPCR for (C) env RNA, (D) tax RNA, (E) and hbz RNA. Error bars represent one standard deviation above and below the averaged RNA copies/mL of technical triplicates.

Fig 4: Treatment with HTLV-1 EVs does not generate HTLV-1 viral proteins and RNA in Recipient cells. CEM and Jurkat cells were treated with density fractions (6 to 18) of HUT102 EVs for 5 days. WB analysis was conducted on CEM (A) and Jurkat (B) cells treated with HUT102 control EV fractions (lanes 1–11) and HUT102 IR-treated EV fractions (lanes 12–22) for p19, Tax, and Actin. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. (C) RT-qPCR was conducted on WCE from Jurkat, CEM, and HUT102 cells (controls; lanes 1, 2, and 3, respectively). Jurkat cells (±IR) treated with fractions 6 (lanes 4 and 5), 12 (lanes 6 and 7), and 18 (lanes 8 and 9), and CEM cells (±IR) treated with fractions 6 (lanes 10 and 11), 12 (lanes 12 and 13), and 18 (lanes 14 and 15) for the presence of env RNA. Negative controls levels were denoted by a solid line (-) and positive control levels for infection by a dashed line (—). We used fraction 6 as negative control for presence of virus; fraction 18 as a potential source of virus; and fraction 12 as a control for fraction 18, which contained EVs with Tax/gp61.

Fig 5: Sirt1 overexpression inhibits cell senescence and TIME signaling, and deacetylates H3K9/14ac in pulmonary fibroblasts from VD‐deficient mice. Pulmonary fibroblasts were isolated from 9‐week‐old WT, Sirt1 Tg , Cyp27b1 −/− and Sirt1 Tg Cyp27b1 −/− mice. (a) Representative micrographs of cells stained cytochemically for SA‐β‐gal. (b) Percentage of SA‐β‐gal‐positive areas relative to the total areas. (c) Second‐passage fibroblast proliferation was determined by CCK‐8 assays and spectrophotometry at 450 nm. Cell number was determined at the indicated times relative to the cell number at hour 0. (d) Western blotting for Sirt1, VDR, TGF‐β1, TGF‐β RII, Smad2, p‐Smad2(Ser465/467), p‐Smad2/3(Ser423/425), Snail, Collagen 1, IL‐11, IL‐11Rα1, MEK1/2, p‐MEK1/2(Ser217/221), ERK1/2, p‐ERK1/2 (Thr202/Tyr204), eIF4E, p‐elF4E(Ser209), p16, p19, p21, p53, acetyl‐histone H3(Lys9/Lys14), and α‐SMA. β‐actin was used as the loading control. (e) Protein expression relative to β‐actin was assessed by densitometric analysis. (f) Representative micrographs of cells immunofluorescently stained for p16 and IL‐11, with DAPI staining the nucleus. (g) The percentage of p16‐positive cells or IL‐11‐positive areas. Three biological replicates were used per experiment. Values are means ± SEM of six determinations. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the WT group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the Sirt1 Tg group; & p < 0.05, && p < 0.01, &&& p < 0.001 compared with the Cyp27b1 −/− group