Figure 1
From: A large-scale RNA interference screen identifies genes that regulate autophagy at different stages
Monodansylcadaverine (MDC) stains autophagic K562 cells with sensitivity equivalent to LC3B immunoblotting and GFP-LC3B fluorescence microscopy. (A) Fluorescence microscopy. K562 cells were treated with dimethyl sulfoxide (DMSO) or 1 μM imatinib (IM) overnight followed by MDC staining. Images of live cells were taken at different exposure times (milliseconds, ms) using an inverted fluorescence microscope. Scale bar: 25 μm. (B) MDC Fluorescence spectrophotometry. K562 cells treated with DMSO or IM were stained with MDC. MDC fluorescence of live K562 cells was quantified using a micro-plate reader at an excitation wavelength of 335 nm and an emission wavelength of 525 nm. The relative MDC levels were obtained by dividing MDC fluorescence of IM-treated cells to that of DMSO-treated cells. Error bars represent three independent experiments. (C) LC3B immunoblotting. Cropped images are shown and full images are included in supplemental materials. ACTB (β actin) is the loading control. The intensities of LC3B-II or ACTB were quantified using Image J. The fold changes of LC3B-II/ACTB were obtained by dividing the ratio of LC3B-II/ACTB in IM-treated cells with that of DMSO-treated cells. (D) MDC staining of K562 cells treated with IM and/or bafilomycin A1 (BFA1). K562 cells were treated with a combination of 1 μM IM (10 hours) and 5 nM of BFA1 (48 hours). Cells were imaged using a fluorescence confocal microscope. (E) Quantification of MDC intensities. Fluorescence intensities of MDC in 10 different cells from three images were quantified using Image J. (F) GFP-LC3B fluorescence microscopy. K562 cells were stably transfected with a construct encoding GFP-LC3B. Cells were treated with a combination of IM and BFA1 and then imaged. (G) Quantification of cells with GFP-LC3B puncta. Four to five images of more than thirty K562 cells from each treatment were randomly selected for the following quantification analyses. GFP-expressing K562 cells or K562 cells with GFP puncta (indicating autophagy) were counted by three persons. Percentages of K562 cells with puncta were obtained by dividing numbers of K562 cells with GFP puncta with those of GFP-expressing cells. Scale bar: 25 nm.
