α linolenic acid

Tween 80, Tween 20, Transcutol P, Cremophor EL, PEG 400 and α-linolenic acid were purchased from Sigma (Sigma Aldrich, Dorset, UK). The derivatisation reagent, BCl₃-methanol 12% w/w (12% boron trichloride in methanol), n-hexane (HPLC grade, purity, ≥99%) were also purchased from Sigma (Sigma Aldrich, Dorset, UK). Labrasol, Capryol 90 and Labrafil M2125 were kindly gifted from Gattefosse Company (Bracknell, UK).

The PnLOXA coding sequence (Vv06s0004g01510) without the plastid targeting sequence was amplified from Pinot Noir berry cDNA using Phusion DNA polymerase (Finnzymes) and the primers LOXfw5′BamHI (5′-GGATCCGTTGGCTACGTCCCTG-3′) and LOXrev3′HindIII (5′-AAGCTTTCAAATGGAGATACTGTATGGAA-3′) and inserted into the pGEM-T vector for sequencing. PnLOXA was then transferred to the expression vector pQE30 using the BamHI/HindIII restriction sites, thus adding an N-terminal His₆ tag. Escherichia coli M15 [pRep4] cells transformed with pQE30:PnLOXA were induced with 1 mM IPTG and 2% ethanol for 16 h at 20°C. A 1-L bacterial culture pellet was resuspended in 40 ml of 50 mM HEPES/NaOH buffer (pH 7.5) containing 150 mM NaCl, 5 mM DTT and protease inhibitors (Sigma). After sonication and lysozyme treatment (0.2 mg/ml), the cleared bacterial lysate was adjusted to 0.5M NaCl and loaded onto a 5-ml HisTrap™ FF crude Column (GE Healthcare, AKTA Purifier system) pre-equilibrated with binding buffer (20 mM HEPES-NaOH pH 7.5, 0.5 M NaCl). The column was washed with binding buffer and 50 mM imidazole (Merck), and the His₆-PnLOXA protein was eluted using 250 mM imidazole. Protein yield was 2 mg of pure protein per liter of bacterial culture. Recombinant PnLOXA activity was studied using 0.1 mM α-linolenic acid (Sigma) at pH 6.5 and 25°C.

DMSO, α-linolenic acid, and BSA were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). Antibodies against FASN (C2065), GAPDH (#5174), Phosoho-Akt (Ser473#4060), and Akt (#4691) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against Bax (ab32503), Bcl-2 (ab32124), IRE1 (ab124945), PERK (ab229912), and ATF6 (ab227830) were purchased from Abcam (Boston, MA, USA).

An overview of the tested plant extracts and compounds is provided in Table 1. Levamisole, albendazole, trans-cinnamaldehyde, dimethyl sulfoxide (DMSO), RPMI-1640 media and α-linolenic acid were obtained from Sigma-Aldrich (Stellenborsch, Germany). Grape seed extract, consisting of >95% condensed tannins [35 (link)], was purchased from Bulk Powders (Colchester, UK). A chicory (Cichorium intybus) extract (cv. Spadona) enriched in sesquiterpene lactones was prepared as previously described [36 (link)]. Four different seaweed extracts were produced as described by Bonde et al. [24 (link)]. Briefly, Saccharina latissma was sourced from either Grenå, Denmark, or the Faroe Islands. From each source location, extracts were prepared using water and methanol (polar extracts; SW1, SW2), or dichrolormethane and methanol (non-polar extracts; SW3, SW4). The chemical composition of the extracts has previously been reported [24 (link)].

Oleic acid (18∶1, n-9; OA), linOleic acid (18∶2, n-6; LA), α-linolenic acid (18∶3, n-3; LNA), arachidonic acid (20∶4, n-6; AA), eicosapentenoic acid (20∶5, n-3; EPA), docosahexaenoic acid (22∶6, n-3; DHA), bovine serum albumin fraction V (BSA, fatty acid free), and 5-aza-2′-deoxy-cytidine (5-aza-dC) were from Sigma.