Figure S4.
Cholesterol accumulation in the cells lacking FABP3 or FABP8. (A) Knockdown efficiency of human FABP7 family members determined by qPCR. Related to Fig. 7 A. The mRNA abundance of indicated cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM (from three biological replicates). (B–D) U251 cells were infected with lentivirus expressing the indicated shRNAs and harvested. (B) Knockdown efficiency of FABP3 and FABP8 determined by qPCR. The mRNA abundance of indicated cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM analysis (from three biological replicates). (C) U251 cells were fixed and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the bottom. Scale bars, 10 µm (main), 1 µm (inset). (D) Quantification of relative fluorescence intensity of filipin in C. The filipin fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 361 cells from three independent experiments). Mann–Whitney U test. (E–H) MA10 cells were infected with lentivirus expressing the indicated shRNAs and harvested. NC, negative control. (E and F) Immunoblotting analysis showing Fabp3 or Fabp8 knockdown MA10 cells. (G) MA10 cells were fixed and stained with filipin (magenta). Scale bars, 10 µm. (H) Quantification of relative fluorescence intensity of filipin in G. The filipin fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 749 cells from three independent experiments). Mann–Whitney U test. (I) SV589 cells were infected with lentivirus expressing the indicated shRNAs, washed with PBS and incubated in lipoprotein-deficient medium (5% lipoprotein-deficient serum) supplemented with 10 µg/ml Dil-labeled LDL for indicated time point. NC, negative control. Scale bars, 10 µm. (J) Quantification of relative fluorescence intensity of the internalized Dil-LDL in I. The Dil-LDL fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) and 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 = 828 cells from three independent experiments). Mann–Whitney U test. (K) SV589 cells were infected with lentivirus expressing the indicated shRNAs, and subjected to LysoSensor Green staining. NC, negative control. (L) Quantification of LysoSensor fluorescence intensity in K. The LysoSensor fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 413 cells from three independent experiments). Mann–Whitney U test. ns, no significance. (M) Sequence alignments of FABP7, 3, and 8. The residues conserved in all three FABPs are in shadow. The residues critical for cholesterol binding and transport are in yellow. The residues involved in cholesterol binding and transport but not conserved are circled in the blue box. h, human. Source data are available for this figure: SourceData FS4.

Cholesterol accumulation in the cells lacking FABP3 or FABP8. (A) Knockdown efficiency of human FABP7 family members determined by qPCR. Related to Fig. 7 A. The mRNA abundance of indicated cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM (from three biological replicates). (B–D) U251 cells were infected with lentivirus expressing the indicated shRNAs and harvested. (B) Knockdown efficiency of FABP3 and FABP8 determined by qPCR. The mRNA abundance of indicated cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM analysis (from three biological replicates). (C) U251 cells were fixed and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the bottom. Scale bars, 10 µm (main), 1 µm (inset). (D) Quantification of relative fluorescence intensity of filipin in C. The filipin fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 361 cells from three independent experiments). Mann–Whitney U test. (E–H) MA10 cells were infected with lentivirus expressing the indicated shRNAs and harvested. NC, negative control. (E and F) Immunoblotting analysis showing Fabp3 or Fabp8 knockdown MA10 cells. (G) MA10 cells were fixed and stained with filipin (magenta). Scale bars, 10 µm. (H) Quantification of relative fluorescence intensity of filipin in G. The filipin fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 749 cells from three independent experiments). Mann–Whitney U test. (I) SV589 cells were infected with lentivirus expressing the indicated shRNAs, washed with PBS and incubated in lipoprotein-deficient medium (5% lipoprotein-deficient serum) supplemented with 10 µg/ml Dil-labeled LDL for indicated time point. NC, negative control. Scale bars, 10 µm. (J) Quantification of relative fluorescence intensity of the internalized Dil-LDL in I. The Dil-LDL fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) and 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 = 828 cells from three independent experiments). Mann–Whitney U test. (K) SV589 cells were infected with lentivirus expressing the indicated shRNAs, and subjected to LysoSensor Green staining. NC, negative control. (L) Quantification of LysoSensor fluorescence intensity in K. The LysoSensor fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 413 cells from three independent experiments). Mann–Whitney U test. ns, no significance. (M) Sequence alignments of FABP7, 3, and 8. The residues conserved in all three FABPs are in shadow. The residues critical for cholesterol binding and transport are in yellow. The residues involved in cholesterol binding and transport but not conserved are circled in the blue box. h, human. Source data are available for this figure: SourceData FS4.

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