Manumycin a

Rat INS-1 β cells were provided by the Institute of Basic Science, Medical University of Tianjin. The cells were cultured under standard cell culture conditions in Roswell Park Memorial Institute (RPMI)-1640 medium (Hyclone, Logan, UT), containing 11.1 mM glucose, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 2 mM l-glutamine, 10 mM hydroxyethyl piperazine ethanesulfonic acid (HEPES), 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Subcultures were established twice every week by using trypsin/ethylene diaminetetra-acetic acid (EDTA) treatment. Differentiated INS-1 cells were used for the experiment. Cells were then cultured in RPMI-1640 medium containing 5.6 or 25 mM glucose (5.6 and 25 G, respectively) with/without the intervention of atorvastatin for 2, 6, or 12 hours, and harvested for the assays described below. In some groups, subcultures were pretreated with manumycin A (Sigma, Ronkonkoma, NY) to inhibit GTPase activation.

The following stock solutions were used in the incubation experiments: manumycin A (MA; 10 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), MG132 (10 mM in DMSO; Selleck Chemicals, Houston, TX, USA), VER-155008 (VER; 20 mM in DMSO; Merck KGaA, Darmstadt, Germany), bortezomib (BTZ; 1.6 mM in DMSO; Selleckchem, Houston, TX, USA), bafilomycin A1 (BAF; 0.1 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), chloroquine (ChQ; 50 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Working solutions were freshly prepared from stock solutions prior to each experiment (in a culture medium or Optimem (Opti-MEM™ Reduced Serum Medium, GlutaMAX™ Supplement; Gibco, Thermo Fisher Scientific, Waltham, MA, USA)). Control cells were incubated with medium containing dimethyl sulfoxide (DMSO). All experiments were performed without the antibiotics addition.

Manumycin A was purchased from Sigma-Aldrich (St. Louis, MO, USA), manumycin B from Abcam (Cambridge, UK). Asukamycin A was isolated from the culture of Streptomyces nodosus ssp. asukaensis and colabomycin E from the culture of Streptomyces aureus SOK1/5-04 by a procedure described by Petrickova [34 (link)]. Strain SOK1/5-04 was isolated from colliery spoil heaps and is deposited in the Biology Centre Collection of Organisms (BCCO, No. BCCO 10_0005, www.actinomycetes.cz). The isolation quality check was performed using LC-MS described therein. All the compounds were dissolved in DMSO (Sigma-Aldrich (St. Louis, MO, USA), tissue culture grade) in 1 mM concentrations.

To inhibit exosome secretion, CD4⁺ T cells isolated from OT‐II mice were pre‐incubated with 2 μM manumycin A (Sigma) for 2 h before B‐cell co‐culture. Alternatively, isolated T cells were nucleofected with 250 nM of the ON‐TARGET plus mouse Rab27a siRNA‐SMART pool (Dharmacon), using the mouse CD4⁺ T‐cell nucleofector kit (Amaxa). Nucleofected cells were then resuspended in complete EXO‐free RPMI medium and incubated for 24 h before IS co‐culture.

Keratinocyte growth medium (KGM-SFM), pituitary gland extract and epidermal growth factor (EGF) etc. were from Gibco (Life Technologies, BRL, Grand Island, NY, USA). Catechol, arecoline, PD153035, manumycin A, pp2, curcumin, catalase, α-naphthoflavone, Zn-protoporphyrin dicoumarol and 3-(4,5-dimethyl-thiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from Sigma (Sigma Chemical Company, St. Louis, MO, USA). PGE₂ and PGF_2α ELISA kits were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Reagents for reverse transcription (RT) and polymerase chain reaction (PCR) were purchased from Invitrogen (Invitrogen Corporation, Carlsbad, CA, USA). Total RNA isolation kits were from Macherey-Nagel (Macherey-Nagel Inc., Easton, PA, USA). ANE and PBL extract were prepared and weighed as previously [12] (link), [15] (link). Hydroxychavicol (HC) was synthesized and characterized as before [16] . Specific PCR primer sets for COX-2, CYP450 isoforms, hemeoxygenase-1 (HO-1), cyclinB1, cdc25C, cdc2, keratin 5, keratin 14 and β-actin were synthesized by Genemed Biotechnologies, Inc. (San Francisco, CA, USA). Pathscan p-Src (Y416) and p-EGFR (Tyr845) ELISA kits were from Cell Signaling (Cell Signaling Technology Inc., MA, USA). EZ-Detect™ Ras activation assay kits were from Pierce (Rockford, IL, USA). Mouse anti-human COX-2 and anti-GAPDH antibody were from Santa Cruz.