The subcellular localization of FABP7 and characterization of FABP7 knockdown and knockout in cultured cells. (A) SV589 cells infected with lentivirus expressing FABP7-Flag were permeabilized without (−) or with (+) 0.005% digitonin (Dig) diluted in DMEM containing 0.3 M sucrose for 5 min before fixation with 4% PFA for 30 min at room temperature. Boxed areas are shown at a higher magnification on the right. Scale bars, 10 µm (main), 1 µm (inset). (B) Cells were permeabilized in a liquid nitrogen bath (N₂) for 30 s before fixation and immunostained with anti-Flag and anti-EEA1 antibodies, or permeabilized with digitonin (Dig) as described above and immunostained with anti-Flag antibody and other organelle markers. Boxed areas are shown at a higher magnification on the right. Scale bars, 10 µm (main), 1 µm (inset). (C) Quantification of FABP7 colocalization with organelle-specific markers. Data are presented as mean ± SEM (n = 80 cells from three independent experiments). (Dand E) Knockdown efficiency of FABP7 in SV589 fibroblasts (D) and U251 cells (E). Indicated cells were transfected with the indicated siRNAs for 48 h and harvested for quantitative PCR (qPCR). The mRNA abundance of indicated cells transfected with scrambled control siRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM (from three biological replicates). (F) Cartoon showing that the exon (E) 2 of the human FABP7 gene was edited by Cas9/sgRNA. Two targeting sequences are in magenta and the protospacer-adjacent motifs are in green. (G) Sanger sequencing of two lines of FABP7 KO cells. (H) WT and FABP7 KO2 cells were harvested, and lysosomes were purified and subjected to immunoblotting analysis. Related to Fig. 1 G. Source data are available for this figure: SourceData FS1.