Fig 2: The roles of ETS family transcription factors in the regulation of neuroendocrine differentiation in small cell lung cancer cell lines.(a) Upregulated genes by KRASG12V overexpression for 1 day and 7 days in comparison with a GFP overexpression control in H2107 and H82 cells. The numbers of genes upregulated (>1.5 fold) are indicated. ETV1, ETV4, and ETV5 are shown in red. (b) Heat map of the PEA3 family ETS transcription factors (ETV1, ETV4, and ETV5) and neuroendocrine transcription factors (ASCL1, NEUROD1, INSM1, and POU3F2 [BRN2]) in small cell lung cancer and lung adenocarcinoma cell lines. Red and blue denote high and low expression, respectively. (c) Western blot showing the effects of ERK inhibition using 1 µM SCH772984 on the expression of ETV1, ETV4, and ETV5 with or without KRASG12V transduction for 72 hr. Lysates from HA-tagged ETV1-overexpressing H524 cells were used as a positive control for ETV1. GAPDH was used as a loading control. (d) Effects of HA-tagged ETV1 induction as assessed by western blot in H2107, H82, and H524 cells, upon treatment with 100 ng/mL doxycycline (dox) for 3 and 7 days. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated control values (set to two in each panel) after normalization to GAPDH. (e) Effects of HA-tagged ETV5 induction as assessed by western blot in H82 and H524 cells, upon treatment with 100 ng/mL dox for 3 and 7 days. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated control values (set to one in each cell line panel) after normalization to GAPDH. (f) Western blot showing the effects of KRASG12V induction and treatment with siRNA pools targeting ETV4, ETV5, or both on expression of neuroendocrine transcription factors in H82 cells. Cells were treated with 100 ng/mL dox and indicated siRNAs for 72 hr. Scrambled siRNA (siScr) was used as a negative control. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated and siScr-treated control values (set to 1) after normalization to GAPDH. (g) Western blot showing the effects of MSK/RSK and/or CBP/p300 inhibition on KRASG12V-mediated expression of ETV4 and ETV5 in H82 cells. Cells were treated with 5 µM SB-747651A (a MSK-RSK inhibitor) and/or 400 nM A-485 (a CBP/p300 inhibitor) as well as 100 ng/mL dox for 72 hr. GAPDH was used as a loading control. Numbers below the ETV5 blots indicate the amounts of ETV5 relative to the dox-treated and non-drug-treated (DMSO-treated) control values (set to lane 5) after normalization to GAPDH. Immunoblots are representative of at least two biological replicates.

Fig 3: RSK1 functions upstream of GSK3β to mediate Tau phosphorylation induced by HIV exposure and cocaine.(A & B) H80 cells were cultured in twelve independent dishes (three biological replicates per condition) under each identical condition and whole-cell lysates were collected 48 h after cocaine, HIV exposure and cocaine plus HIV exposure. Protein lysates were prepared separately from each dish and quantified. Equal amounts of protein were loaded and analyzed by immunoblotting. Immunoblot analysis revealed a significant increase in Tau phosphorylation at Ser396 (p-Tau-S396), a marker of pathological Tau, and RSK-1 expression under all conditions (cocaine, HIV exposure and both) compared with untreated controls. Concurrent analysis demonstrated enhanced phosphorylation of GSK3β at Ser9 (p-GSK3β-Ser9), indicating functional inactivation of GSK3β under all conditions, while GSK3β and Tau levels remained unchanged. Densitometric analysis of immunoblots was performed by normalizing band intensities to β actin or total protein, with values expressed relative to the control (Ctrl/No HIV). (C & D) To delineate the relative contributions of RSK1 and GSK3β, H80 cells were pretreated for 24 h with selective inhibitors BI-D1870 (RSK1 inhibitor) or CHIR-99021 (GSK3β inhibitor) prior to HIV and/or cocaine exposure. Immunoblotting and densitometry revealed that BI-D1870 effectively suppressed RSK1 activation and significantly reduced Tau phosphorylation, whereas CHIR-99021 failed to alter Tau phosphorylation induced by HIV or cocaine, confirming that Tau modification is primarily mediated through RSK1-dependent signaling. Inhibition of RSK1 also reversed GSK3β inactivation, as evidenced by reduced Ser9 phosphorylation, suggesting a hierarchical relationship in which RSK1 lies upstream of GSK3β. Densitometric analysis of immunoblots was performed by normalizing band intensities to GAPDH, with values expressed relative to the control (Ctrl/No HIV). (E) An acute time point study was conducted for 1 h, 3 h and 6 h with cocaine and cocaine along with HIV, Immunoblot results show enhanced phosphorylation of GSK3β at Ser9 (p-GSK3β-Ser9), indicating functional inactivation of GSK3β at 6h while simultaneously seen the enhanced tau phosphorylation at S396. Immunoblots are representative of at least three independent biological replicates. Data are presented as mean ± S.D. Statistical significance was assessed using one way ANOVA with Dunnett’s multiple comparisons test. Significance is indicated as P < 0.05 (*) and P < 0.01 (**).

Fig 4: Model summarizing HIV- and cocaine-induced Tau phosphorylation in neuronal cells.Proposed schematic illustrating distinct yet convergent signaling mechanisms by which HIV exposure and cocaine promote Tau phosphorylation in H80 neuronal cells. HIV exposure induces a robust and sustained activation and upregulation of RSK1, which drives Tau phosphorylation through a pathway that is independent of AKT signaling while concurrently promoting inhibitory phosphorylation of GSK3β at Ser9. In contrast, cocaine exposure engages a partially overlapping but mechanistically distinct pathway, characterized by modest RSK1 induction and strong activation of AKT, as evidenced by phosphorylation at Thr308 and Ser473. Activated AKT subsequently catalyzes inhibitory phosphorylation of GSK3β at Ser9, leading to its functional inactivation. Despite GSK3β inactivation under both conditions, Tau phosphorylation persists, indicating the existence of a GSK3β-independent mechanism regulated by RSK1. Collectively, these findings identify RSK1 as a central signaling hub that integrates viral and substance-induced signaling to drive Tau dysregulation, highlighting its critical role in neurodegenerative processes relevant to HIV-associated neurocognitive disorders.

Fig 5: RSK1 overexpression modulates AKT and GSK3β signaling in H80 cells. / RSK1 as a central upstream modulator of AKT and GSK3β signaling(A–B) Immunoblot analysis confirmed robust overexpression of RSK1 following transient transfection with a CMV-driven RSK1 construct, as evidenced by a marked increase in total RSK1 protein and elevated levels of RSK isoforms (RSK1/2/3). Overexpression also resulted in a significant increase in phosphorylation of RSK1 at Ser380, an autophosphorylation site associated with partial catalytic activation, while phosphorylation at Thr348 decreased relative to empty vector transfected control. (C–D) RSK1 overexpression induced a robust increase in AKT phosphorylation at both Thr308 and Ser473, two canonical regulatory sites required for full AKT activation, without altering total AKT protein levels. These findings indicate that RSK1 positively regulates AKT activity predominantly through post-translational mechanisms. (E–F) Overexpression of RSK1 also enhanced phosphorylation of GSK3β at Ser9, a canonical inhibitory site that suppresses GSK3β kinase activity, while total GSK3β levels remained unchanged. Densitometric analysis of immunoblots was performed by normalizing band intensities to β actin or total protein, with values expressed relative to the empty vector transfected control. Immunoblots are representative of at least three independent biological replicates. Data are presented as mean ± S.D. Statistical significance was assessed using an unpaired, two tailed Student’s t test. Significance is indicated as P < 0.01 (**) and ns denotes not significant.