Figure S2.
LDLR-mediated LDL uptake provides cholesterol for FABP7-mediated transport. (A and B) Two lines of FABP7 KO cells (FABP7 KO1 and FABP7 KO2) were transfected with the indicated siRNAs for 48 h and harvested for qPCR (A) and immunoblotting (B) to determine the knockdown efficiency of LDLR gene and the resultant reduction in LDLR protein, respectively. 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). (C and D) Two lines of FABP7 KO cells (FABP7 KO1 and FABP7 KO2) were transfected with the indicated siRNAs for 48 h transfection, fixed, and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the right. Scale bars, 10 µm (main), 1 µm (inset). NC, negative control. Relative fluorescence intensity of filipin in C was quantified by ImageJ and shown in D. The filipin fluorescence intensity 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 median with interquartile range (n = 575 cells from three independent experiments). Mann–Whitney U test. (E) SV589 fibroblasts were infected with lentivirus expressing indicated shRNAs and harvested for surface biotinylation assay. Ponceau S staining indicates equal amounts of samples were loaded. (F) WT and FABP7 KO2 cells were washed with PBS and incubated in lipoprotein-deficient medium (5% lipoprotein-deficient serum) supplemented with 10 µg/ml Dil-labeled LDL for the indicated time point. Scale bars, 10 µm. (G) Quantification of relative fluorescence intensity of the internalized Dil-LDL in F. The Dil-LDL fluorescence intensity of wild-type (WT) cells incubated with LDL for 0.5 h is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 497 cells from three independent experiments). Mann–Whitney U test. (H) WT and FABP7 KO2 cells were subjected to LysoSensor Green staining to assess lysosomal acidity. Scale bars, 10 µm. (I) Quantification of LysoSensor fluorescence intensity in H. The LysoSensor fluorescence intensity of wild-type (WT) cells is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 366 cells from three independent experiments). Mann–Whitney U test. Source data are available for this figure: SourceData FS2.

LDLR-mediated LDL uptake provides cholesterol for FABP7-mediated transport. (A and B) Two lines of FABP7 KO cells (FABP7 KO1 and FABP7 KO2) were transfected with the indicated siRNAs for 48 h and harvested for qPCR (A) and immunoblotting (B) to determine the knockdown efficiency of LDLR gene and the resultant reduction in LDLR protein, respectively. 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). (C and D) Two lines of FABP7 KO cells (FABP7 KO1 and FABP7 KO2) were transfected with the indicated siRNAs for 48 h transfection, fixed, and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the right. Scale bars, 10 µm (main), 1 µm (inset). NC, negative control. Relative fluorescence intensity of filipin in C was quantified by ImageJ and shown in D. The filipin fluorescence intensity 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 median with interquartile range (n = 575 cells from three independent experiments). Mann–Whitney U test. (E) SV589 fibroblasts were infected with lentivirus expressing indicated shRNAs and harvested for surface biotinylation assay. Ponceau S staining indicates equal amounts of samples were loaded. (F) WT and FABP7 KO2 cells were washed with PBS and incubated in lipoprotein-deficient medium (5% lipoprotein-deficient serum) supplemented with 10 µg/ml Dil-labeled LDL for the indicated time point. Scale bars, 10 µm. (G) Quantification of relative fluorescence intensity of the internalized Dil-LDL in F. The Dil-LDL fluorescence intensity of wild-type (WT) cells incubated with LDL for 0.5 h is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 497 cells from three independent experiments). Mann–Whitney U test. (H) WT and FABP7 KO2 cells were subjected to LysoSensor Green staining to assess lysosomal acidity. Scale bars, 10 µm. (I) Quantification of LysoSensor fluorescence intensity in H. The LysoSensor fluorescence intensity of wild-type (WT) cells is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 366 cells from three independent experiments). Mann–Whitney U test. Source data are available for this figure: SourceData FS2.

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