Figure 3

Assessment of CFC frequencies in BC and LP according to their autophagy activity. (A) Frequencies of CFCs from the G⁺ and R⁺ cells isolated from four-day cultures of BCs and LPs. Values shown are the mean ± SEM from paired analyses of cells from three normal donors. (B) Frequencies of CFCs in the R⁺ and G⁺ fractions of BCs and LPs isolated from collagen gels initially containing 10⁵ RFP-GFP-LC3B-transduced BCs and transplanted subcutaneously four weeks previously into female NRG mice. Results are from cells originally isolated from two different normal donors. (C) CFC frequencies in FACS-purified BCs and LPs from three different normal donors after incubation at 37 °C for two hours in SF7 medium containing either 200 nM BafA₁, 10 μM CQ, or an equivalent concentration of DMSO (0.5%). CFC frequencies are expressed as a percent of matched control values. (D) Effect of inhibiting ATG7 in BCs and LPs on their CFC activities. Normal BCs and LPs isolated by FACS from three different donors were transduced with pTRIPZ-shScr or shATG7-1 or shATG7-2 vectors, selected for a day with puromycin, and then plated in CFC assays in the presence of doxycycline. CFC activity expressed as a percent of matched control values. P-values were calculated using the Student t-test. *P < 0.05.