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. 2018 Jul 26;3(14):e121221. doi: 10.1172/jci.insight.121221

Figure 1. Biochemical characterization of lipolysis activity from C26c20 cell line medium.

Figure 1

(A and B) Characterization of cancer cell line medium-induced adipocyte lipolysis. Medium was collected, processed, and protein quantified from C26c20 or MC-38 cells as described in Methods. Differentiated adipocytes in a 12-well format were treated with 1.5 ml of medium E with the indicated amount of C26c20 or MC-38 medium (A) or 150 ng of recombinant IL-6, 150 ng of recombinant TNFα, or either 1.8 mg or 3.1 mg C26c20 medium in the absence or presence of 4.5 μg of the indicated antibody (B). After incubation for 20 hours at 37˚C, medium was collected and glycerol concentration was measured using the adipocyte lipolysis assay described in Methods. Data are shown as mean ± SEM (A) or dot plots with bars representing mean ± SEM (B) of 3 or 4 (A and B, respectively) experiments and represents the absolute increase of medium glycerol concentration over background (A) or as the relative change in medium glycerol concentration compared with conditions containing the indicated protein without antibody (B) (IL-6, 54 and 19 μM; TNFα, 25 and 36 μM; C26c20 medium, 37 and 20 μM). (C) Leukemia inhibitory factor (LIF) expression in medium of cancer cells. Medium (15 ml) from C26c20 and MC-38 was concentrated to a final volume of 150 μl using a 10 kDa MW cut-off Amicon Ultra centrifugal filter, and protein was quantified using a bicinchoninic acid kit. Protein (20 μg) was subjected to IB analysis with anti-LIF and Ponceau S stain described in Methods. (D) Immunodepletion of LIF from partially purified C26c20 medium. C26c20 medium was partially purified as described in Methods. Approximately 14 μg of the elution fractions containing lipolysis activity in Step 1 of the partial purification of C26c20 medium in 300 μl of buffer A with 0.2% BSA was subjected to immunodepletion described in Methods using 2 μg of the indicated antibody. The supernatant fraction from the immunodepletion was collected, and 30 μl was subjected to IB analysis with the indicated antibody and 20 μl subjected to the adipocyte lipolysis assay described in Methods. Data are shown as dot plots with bars representing mean ± SEM of 4 experiments and is represented as the relative change in medium glycerol concentration compared with conditions containing the indicated protein without antibody (33 and 64 μM). *P < 0.05 and ***P < 0.001 based on Student’s t test.