C 12730

Human white preadipocytes (hPAd) derived from female abdominal subcutaneous adipose tissue of at least two different individuals were obtained from PromoCell (C-12730, Heidelberg, Germany). hPAd were cultivated in growth medium (PromoCell, C-27410), followed by switching to differentiation medium (PromoCell, C-27436) for 3 days, and then matured in nutrition medium (PromoCell, C-27438) for additional 15 days to become mature human white adipocytes (hAd). hAd-conditioned medium (hAd-CM) was collected every 3 days until day 45. Cellular debris was removed by centrifugation, and the supernatant was stored at −80 °C. Several collections of hAd-CM were pooled as one batch of hAd-CM and SERPINE1 concentration was measured by ELISA before use. The average SERPINE1 concentration was 16.44 ± 2.81 ng/ml in different batches of hAd-CM. For SERPINE1-depleted hAd-CM, hAd were transduced with lentivirus containing shScramble or shSERPINE1 24 h after maturation. For all the experiments, cancer cells were pre-treated with hAd-CM mixed with DMEM at 1:1 ratio for 3 days and were maintained in the same media throughout the experiments. Mature hAd culture medium (nutrition medium) mixed with DMEM (1:1) was used as a control.

IL-6, leptin and adiponectin ELISAs and human recombinant IL-6 were from R&D systems (Abingdon, UK). Anti-human antibodies (anti-CD31-FITC, anti-CD166-PerCP-efluor) were from eBioscience (Hatfield, UK), anti-CD105-APC, anti-CD45-Alexa fluor 700 and anti-CD11b-Brilliant Violet 421 were from Biolegend (Cambridge, UK) and anti-CD73-Brilliant Violet 605 was from BD Bioscience (Oxford, UK). Insulin ELISA was from Mercodia Diagnostics (Uppsala, Sweden). DAPI, LipidTOX Green Neutral Lipid, Inflammatory Cytokine Human Magnetic 5-Plex and Trizol were from Life Technologies (Warrington, UK). RT2 Profiler human adipogenesis PCR arrays and cDNA synthesis kits were from SABiosciences-Qiagen (Hilden, Germany). Human Oxidative Stress Magnetic Bead Panel was from Millipore (Watford, UK). Human white SC-pre-adipocytes from a lean individual (C-12730) were from PromoCell (Heidelberg, Germany). Other chemicals and reagents were from Sigma (Munich, Germany).

3T3-L1 fibroblasts (American Type Culture Collection) were maintained in culture and differentiated into adipocytes as previously described. Lipofectamin 2000 and RNAiMAX (Life Technology) were used for transfections of mammalian cells according to the manufacturer's protocol. Retroviral particles were produced in human embryonic kidney 293T cells after transfection with a standard combination of expression and packaging vectors (29 (link)). 3T3-L1 fibroblasts (30–40% confluent) were infected with respective virus particles for 48 h. After infection, stably expressing cells were selected in puromycin-containing culture media.
Primary human preadipocytes were purchased from Promo cell (C-12730) and cultured according to manufacturer's instructions. Primary human preadipocytes were differentiated using differentiation media (811D-250) according to their instruction. Experiments were performed after full differentiation protocol and 95% adipocyte differentiation.