Aicar

AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) (Sigma-Aldrich), an analog of adenosine monophosphate (AMP), was used to stimulate AMP-dependent protein kinase (AMPK) activity. TBP/Q₇₉-GFP SH-SY5Y cells were plated on 6-well or 24-well plates in the presence of retinoic acid as described. On day 2, the cells were pretreated with LM-031 or licochalcone A (100 nM) for 4 h; AICAR (0.1 mM) treatment and TBP/Q₇₉-GFP induction followed. On day 8, the cells were collected for AMPKα and pAMPKα protein analysis or stained with Hoechst 33342 and analyzed for aggregation and neurite outgrowth as described.

Immortalized human keratinocytes (NTERTs) are maintained in KSFM media (Gibco) supplemented with 0.4 mM calcium chloride^{48 (link)}. Immortalized dermal papilla cells^{49 (link)} are maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin, PAN-Biotech GmbH, Germany). Immortalized dermal papilla cells are maintained in 10% CO₂ at 37 °C. 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, Sigma) is administered for 24 hours at 0.5 mM. Dermal papilla cells are cultured in activated charcoal stripped FBS supplemented DMEM one day prior to treatment.

Mice were deeply anesthetized with a ketamine/xylazine mixture and mounted in a stereotaxic apparatus (8003, RWD Life Science). The eyes of the mice were protected by ophthalmic gel. The 26-gauge guide cannulas (Plastics One) were bilaterally implanted into the DH. The coordinates (in reference to bregma) were as follows: lateral (L), ±1.1 mm; AP, −1.80 mm; dorsoventral (V), and −1.0 mm. The guide cannulas were fixed by dental cement. Then miR-181a agomir, antagomir or their control (20 nM, 0.5 μl/per site respectively, RiboBio.Co. Guangzhou) were injected using a 33-gauge infusion needle (1.0 mm longer than the guide cannulas, Plastics One) connected to a 10 μl Hamilton microsyringe at a rate of 0.1 μl /min. After the injection, the needle was left for an additional 5 min before withdrawal. After surgery, the animals were allowed to recover for 1 week prior to behavior tests. The 5′-prime-AMP-activated protein kinase (AMPK) specific activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, 0.01, 0.1 or 1 mM, Sigma) and inhibitor compound C (CC, 0.1, 1 or 10 mM, Merck) were injected 30 min before the behavior test via the same infusion needle as above.

PF-06409577 was purchased from MedKoo Bioscience (Shanghai, China). Antibodies were purchased from Cell Signaling Technologies (Beverly, MA, USA). AICAR, metformin, puromycin, and agents for cell culture were purchased from Sigma-Aldrich (St Louis, MO, USA).

[γ-³²P]ATP was purchased from PerkinElmer. HG-10-102-01, MLi-2, GSK2578215A, LRRK2-IN1, MRT67307, GDC0941, AG1478, BID1870, H-89, PD0325901, UO1216, Rapamycin and Phos-tag acrylamide were all synthesized by Natalia Shpiro (University of Dundee). Phos-tag acrylamide stocks are stored in aliquots at −80°C and working stocks are stored for a few weeks at 5 mM aqueous solution at 4°C in opaque tubes given the photo-sensitive nature of this reagent. iN04 was purchased from ChemBridge Corporation (#7989904). Full-length recombinant human LRRK2[G2019S] was purchased from Invitrogen (#A15202). GZD-824 was purchased from Cayman Chemical (#21508) and calyculin A (#ab141784) was purchased from Abcam, Inc. Phorbol 12-myristate 13-acetate (PMA) (#P8139), interleukin-1A (#I2778), oligomycin (#75351), antimycin A (#A8674), AICAR (#A9978) and forskolin (#F6886) were all purchased from Sigma–Aldrich. Human EGF (#8916), and human IGF-1 (#3093) were purchased from cell signaling technology (CST). PKC-specific inhibitor GO6983 was purchased from Selleckchem (#S2911). ON-TARGETplus SMARTpool human LRRK1 (#L-005320-00-0005), human LRRK2 (#L-049666-00-0005) and non-targeting control pool (#D-001810-10-20) siRNA were purchased from Horizon Discovery.