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. 2018 Dec 11;7:e39944. doi: 10.7554/eLife.39944

Figure 1. Characterization of shMet B16-F10 cells and exosomes.

B16-F10 cells engineered to express shScr or shMet were used to purify exosomes. (A) Representative Western blots of exosomes and B16-F10 cells expressing the indicated shRNA were probed with antibodies specific for total Met (top panel) and Gapdh (bottom panel). Membranes were cut at ~75 kDa so that Met and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated exosome and cell lysate preparations from the same batch of infected cells. The fourth lane, labeled ‘Cells’ are lysate from B16-F10 cells expressing shScr. (B) Representative Western blots of exosomes and B16-F10 cells expressing the indicated shRNA were probed with antibodies specific for phosphorylated (Tyr 1234/1235) Met (pMet) (top panel) and Gapdh (bottom panel). Membranes were cut at ~75 kDa so that pMet and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated exosome preparations from the same batch of infected cells. (C) Western blot bands were quantified for cells. Met or pMet levels were normalized to Gapdh, and protein expression presented relative to shScr conditions. Expression level of shScr condition was assigned a value of 1. Means reported and error bars represent SD. Results are from 3 independent biological repeats for Met expression and 4 independent biological repeats for pMet expression. Exploratory analysis: one-sample t-test on Met levels (Met/Gapdh) in shMet cells compared to a constant of 1 (shScr cells): t(2) = 8.41, p = 0.014, Bonferroni corrected p = 0.028, Cohen’s d = 4.85, 95% CI [0.55, 9.43]; one-sample t-test on pMet levels (Met/Gapdh) in shMet cells compared to a constant of 1 (shScr cells): t(3) = 8.94, p = 0.003, Bonferroni corrected p = 0.006, Cohen’s d = 4.47, 95% CI [1.02, 8.01]. (D) Representative Western blot of exosomes isolated from cells expressing the indicated shRNA probed with exosome markers Hsc70, Tsg101, and Cd63 specific antibodies. Experiment performed on 3 independent biological repeats for each condition from the same batch of infected cells. Additional details for this experiment can be found at https://osf.io/aqm2m/.

Figure 1.

Figure 1—figure supplement 1. Multiplicity of infection (MOI) ratios tested for stable cell line generation.

Figure 1—figure supplement 1.

B16-F10 cells infected with various MOI ratios were characterized for shMet, Met, and Met expression. (A) Relative expression levels of shMet (normalized to U6) was determined by qRT-PCR for each cell line. For each MOI tested, fold change in shMet expression in shMet cells relative to shScr cells was determined. Expression level of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. (B) Relative expression levels of Met (normalized to Gapdh) was determined by qRT-PCR for each cell line. For each MOI tested, fold change in Met expression in shMet cells relative to shScr cells was determined. Expression levels of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. (C) Western blots using anti-Met (top panel) and anti-Gapdh (bottom panel) antibodies. Membranes were cut at ~75 kDa so that Met and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated cell lysate preparations from the same batch of infected cells. Additional details for this experiment can be found at https://osf.io/aqm2m/.