Article Figures & Data
Figures
- Figure 1. Arginine-rich domain is critical for the Tat-induced senescence-like phenotype in human astrocytes.
(A) Tat treatment for 48 h increased the percentage of SA-β-gal–positive cells in a concentration-dependent manner in human astrocytes (n = 3, scale bar = 40 μm). (B) Tat treatment for 48 h did not increase the release of LDH into the media of cultured astrocytes (n = 3). (C) Tat (100 nM) significantly increased the release of IL-6 at 48 h post-treatment (n = 6). (D) Tat (100 nM) significantly increased SA-β-gal–positive cells at 48 h and 72 h post-treatment (n = 3, scale bar = 40 μm). (E, F) Tat (100 nM for 48 h), but not mutant Tat (100 nM for 48 h), significantly increased SA-β-gal–positive cells ((E), n = 3, scale bar = 40 μm) and elevated SA-β-gal activity ((F), n = 3) in human astrocytes. (G, H) Tat (100 nM, 48 h), but not mutant Tat, significantly increased protein levels of the senescence marker p16Ink4a ((G), n = 4) and p21CIP1 ((H), n = 3) in human astrocytes. (I, J, K) Tat (100 nM, 48 h), but not mutant Tat, increased the release of IL-6 ((I), n = 6), IL-8 ((J), n = 5), and CCL2 ((K), n = 6) in the astrocyte culture media. Data information: Data were expressed as means ± SD. n = independent culture preparations. (C) Two-way ANOVA followed by Tukey’s post hoc test in (C) and one-way ANOVA followed by Tukey’s post hoc test for the rest of data.
- Figure 2. Arginine-rich domain is critical for Tat-induced endolysosomal dysfunction in human astrocytes.
(A) FITC-labeled Tat (Tat-FITC) colocalized with endolysosomes marked by LysoTracker (red) in human astrocytes at 1 h post-treatment, scale bar = 20 μm. (B) Mutant Tat labeled with Alexa 488 (mTat-Alexa 488) also colocalized with endolysosomes marked by LysoTracker (red) in astrocytes at 1 h post-treatment. Nuclear staining was performed using NucBlue (scale bar = 10 μm). (C) Tat (100 nM for 48 h), but not mutant Tat, led to endolysosome de-acidification, evident from the reduced Green/Deep Red fluorescence ratio (n = 3). (D) In astrocytes expressing EGFP-tagged galectin-3, Tat (100 nM), but not mutant Tat, significantly increased galectin-3 punctate formation after 2 and 24 h of treatment (n = 5). NucBlue was used for nuclear staining, scale bar = 20 μm. (E, F) Tat (100 nM for 48 h), but not mutant Tat, elevated galectin-3 levels ((E), n = 4) and cathepsin B levels ((F), n = 5) in the astrocyte culture media. Data information: Data were expressed as means ± SD. n = independent culture preparations. (C, D, E, F) One-way ANOVA followed by Tukey’s post hoc test in (C, D, E, F).
- Figure 3. Tat interacts with the endolysosome-resident arginine sensor SLC38A9.
(A) Biotin-labeled Tat was used as bait to pull down SLC38A9, but not TLR3, from U87MG cell lysates. (B) Biotinylated anti-SLC38A9 antibody was used to capture SLC38A9 from U87MG cell lysates, followed by incubation with Tat or mutant Tat. Tat, but not mutant Tat, was detected in the precipitates, whereas a biotinylated isotype IgG served as a negative control. (C) SLC38A9-RFP colocalized with Tat-FITC (green), yielding Pearson’s correlation coefficient of 0.37 (n = 3, scale bar = 10 μm). (D) Tat (100 nM, 2 h), but not mutant Tat, increased colocalization of ⍺-mTOR (red) with LAMP1-GFP in human astrocytes (n = 3, scale bar = 10 μm). (E) Tat (100 nM, 1.5 h) significantly increased phosphorylation of the mTORC1 downstream target 4E-BP1 at serine 65. Mutant Tat (100 nM, 1.5 h) also increased phosphorylation of 4E-BP1, but the extent is lower than that of Tat (n = 3). (F) Quantitative immunoblotting confirmed the knockdown of SLC38A9 in human astrocytes using specific siRNAs (n = 5). (G) SLC38A9 knockdown attenuated phospho-4E-BP1 and blocked Tat (100 nM, 1.5 h)-induced increases in phospho-4E-BP1 (n = 3). Data information: Data were expressed as means ± SD. n = independent culture preparations. (D, E, F, G) Two-tailed t test in (F), one-way ANOVA followed by Tukey’s post hoc test in (D, E), and two-way ANOVA followed by Tukey’s post hoc test in (G).
- Figure 4. SLC38A9 knockdown attenuates Tat-induced endolysosome dysfunction, LTR transactivation, and cellular senescence.
(A, B) Knockdown of SLC38A9 alone did not affect the release of endolysosome factors but significantly reduced Tat (100 nM, 48 h)-induced increases in galectin-3 ((A), n = 3) and cathepsin B ((B), n = 3) in the culture media of human astrocytes. (C) SLC38A9 knockdown significantly attenuated Tat-induced endolysosome membrane leakage, as indicated by the formation of endogenous galectin-3 puncta in LAMP-1–positive vesicles, at 24 h post-treatment (n = 3), scale = 15 μm. (D) SLC38A9 knockdown significantly decreased the cellular level of Tat at 48 h post-treatment (n = 3). (E) Quantitative immunoblotting confirmed the knockdown of SLC38A9 in U87MG cells using specific shRNAs (n = 3). (F) Knockdown of SLC38A9 reduced Tat-mediated HIV-1 LTR transactivation in U87MG cells stably transfected with HIV-1 LTR-luciferase reporter (n = 3). (G, H, I) SLC38A9 knockdown alone did not affect the release of inflammatory factors; however, SLC38A9 knockdown significantly attenuated Tat (100 nM, 48 h)-induced increases in IL-6 ((G), n = 3), IL-8 ((H), n = 5), and CCL2 ((I), n = 5) in media of cultured human astrocytes. (J) SLC38A9 knockdown significantly attenuated Tat (100 nM, 48 h)-induced increases in SA-β-gal activity in human astrocytes (n = 4). (K) SLC38A9 knockdown significantly attenuated Tat (100 nM, 48 h)-induced increases in p16Ink4a protein levels in human astrocytes (n = 6). Data information: Data were expressed as means ± SD. n = independent culture preparations. Two-tailed t test in (D, E, F) and two-way ANOVA followed by Tukey’s post hoc test in the rest of data.
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