Fig 1: miR-7-5p downregulates NF-κB transcriptional activity and downstream NF-κB targets in melanoma cellsA. Luciferase reporter assay with WM266-4, A2058 and 1205Lu cells that were transfected with NF-κB cignal luciferase reporter or negative control luciferase reporter, and either miR-7-5p or miR-NC precursor molecules (10 nM), RelA siRNA (si-RelA7, positive control for assay) or negative control siRNA (si-NC) at 5 nM. *, p-value < 0.01. B. ELISA analysis of secreted and intracellular expression of IL-1β, IL-6 and IL-8 in 1205Lu cells 3 d post-transfection with either miR-7-5p or miR-NC precursor molecules, or si-RelA7 or si-NC. *, p-value < 0.01.
Fig 2: CyaA affects intracellular cAMP concentration and IL-6 secretion in human airway epithelial cells. (a) HNEC, HTEC and HBEC3-KT were incubated with CyaA or CyaA-AC– and intracellular cAMP concentration was analysed (n = 4). (b) Quantification of human IL-6 secreted by HNEC, HTEC and HBEC3-KT populations after treatment with CyaA or CyaA-AC– was assayed by ELISA of the supernatants (n = 3). (c) Quantification of human IL-6 secreted by human nasal mucosa tissue models after treatment with CyaA or CyaA-AC– (n=3). All data are presented as means (bars) + SEM (error bars). Differences after CyaA incubation compared to the control group (TUC) are indicated (*P ≤ 0.05, $P > 0.05 ≤ 0.1, ANOVA). Statistically significant differences of IL-6 basal levels are indicated (#P < 0.05, ANOVA). Significant cell type-specific differences are indicated (&P < 0.05, ANOVA).
Fig 3: 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.
Fig 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.
Fig 5: TLR7 knockdown attenuates Tat‐induced inflammatory response in human astrocytes. (A) Quantitative immunoblotting data showed that TLR7 was knocked down with specific siRNAs in human astrocytes (n = 3). (B‐F) TLR7 knockdown alone did not affect the release of these inflammatory factors; However, TLR7 knockdown significantly attenuated Tat‐induced increases in IL‐6 (B, n = 5), IL‐8 (C, n = 4), CCL2 (E, n = 5), galectin‐3 (E, n = 4), and cathepsin B (F, n = 3) in the media of cultured human astrocytes. Statistics: N = Independent replicates. Two‐way ANOVA with Tukey's post hoc test and two tailed student's t‐test.
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