Minocycline

Rotenone (#R8875) and Evans blue (#E2129) were provided by Sigma-Aldrich, Inc. (St. Louis, MO, USA). The AG RNAex Pro Reagent (#AG21102), Pro Taq HS qPCR Kit, and SYBR Green Premix (#AG11720) were provided by Accurate Biotechnology (Hunan, China). The PLX3397 (#S7818), minocycline (#S4226), and SB-3CT (#S7430) were provided by Selleck (Shanghai, China). Antibodies against tyrosine hydroxylase (TH, #AB152) and ionized calcium binding adaptor molecule-1 (Iba-1, #019-19741) antibodies were provided by EMD Millipore (Temecula, CA, USA) and Wako Chemicals (Richmond, VA, USA), respectively. Antibodies against fibrinogen (#ab34269), zonula occludens-1 (ZO-1, #ab96587), claudin-5 (#ab131259), occludin (#ab167161), MMP-2 (#ab92536), MMP-9 (#ab38898), GAPDH (#ab181602), and the MMP assay kit (#ab112146) were purchased from Abcam (Cambridge, MA, USA). The ECL reagents (#20-500-120) were provided by Biological Industries (Cromwell, CT, USA).

CEM (uninfected T-cell) and U1 (HIV-1-infected promonocytic) cells were cultured and maintained in complete RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine (RPMI) (Quality Biological) and incubated in 5% CO₂ at 37 °C. U1 cells were treated with varying concentrations of antiretroviral drugs, Indinavir (protease inhibitor) and Emtricitibine (nucleoside reverse transcriptase inhibitor), for 7 days. Both cell types and antiretroviral drugs were provided by the AIDS Reagent Program (National Institutes of Health). Additional drug treatments include Tetracycline class of antibiotics; OxyTetracycline (Selleckchem S1773), Tetracycline (Selleckchem S2574), Minocycline (Selleckchem S4226), Doxycycline (Selleckchem S4163), Demeclocycline (Selleckchem S4279), and Methacycline (Selleckchem S2527) were used to treat cells for 5 days. Interferon (IFNα-2a; PBL Assay Science 11100-1) was used at varying concentrations ranging from 0.5 K units to 100 K units for 5 days.

MPL is the product of Sigma (Saint Louis, MO, USA). Minocycline was purchased from Selleck (Shanghai, China). The MPL was dissolved in dimethyl sulfoxide (DMSO) as a stock solution, and was diluted to a final concentration 100 μg/mL using the Ringer’s solution. The Minocycline was dissolved in di-H₂O as a stock solution.

Drugs for this study were purchased from Selleck Chemicals (Minocycline, Vorinostat (SAHA), Donepezil, 4-aminobenzoic hydrazide (ABAH), Riluzole, Paclitaxel, Sulfasalazine, SnMP, P7C3, Etomoxir, BSO, and MK2206). AGI-25696 was purchased from Adooq Bioscience (A20250). AD drugs are chosen based on differential target function for Alzheimer’s disease or specific metabolic pathway (Table 1). Drugs were formulated with polyethylene glycol (PEG) 3,400 or 8,000 (Spectrum Chemical) as described in (Jonas et al., 2015 (link)). Briefly, compounds in the ratio of 20% drug were added to 80% PEG-3450 or PEG-8000 and vortexed for 5 min above its melting point (37 °C). For insoluble drugs, a mixture of drug, PEG, and an organic solvent (ethanol or acetone) was heated to ∼37°C until completely dissolved. The solution was placed on a rotary evaporator (Buchi) for ∼30–40 min at below respective vapor pressures to completely evaporate the solvent, leaving a homogeneous solid mixture of drug and PEG.
Drug/polymer mixtures were loaded into micro-reservoirs as described in (Jonas et al., 2015 (link)). Local drug concentration is measured using drug autofluorescence or Maldi imaging mass spectrometry as described in (Jonas et al., 2015 (link); Davidson et al., 2016 (link); Jonas et al., 2016 (link)), and calibrated using homogenized drug-tissue sections of identical thickness.