Figure S1.
Validation of APOE antibody via siRNA-mediated knockdown and measurement of intracellular and secreted APOE ± OA. (A) Western blot of lysates of TRAE3-H cells transfected with a non-targeting siRNA or one of two different siRNAs against APOE and treated ± OA for 5 h. The antibody used to probe for endogenous APOE is the same one used for both the immunofluorescence and immunogold experiments. (B) Quantification of Western blots of APOE knockdown from three independent biological replicates. APOE siRNA #2 demonstrated ∼94% knockdown of APOE in both − and + OA conditions, and was used for subsequent loss of function studies in Figs. 5 and 6. (C) Representative confocal slices of TRAE3-H cells transfected with NT siRNA or one of two APOE siRNAs and treated with 400 µM OA for 5 h. Cells were fixed and stained for endogenous APOE with an anti-APOE antibody and for LDs with BODIPY 493/503. Little to no endogenous APOE signal was observed by immunofluorescence upon APOE knockdown. Scale bar, 10 µm. (D) Normalized total APOE protein present in TRAE3-H and TRAE4-H lysates ± 5-h OA treatment. Cells were lysed in 100 µl of lysis buffer, and APOE protein concentrations in µg/ml were measured by ELISA. The APOE concentration in µg/ml was multiplied by the total lysate volume of 0.1 ml to derive the total amount of APOE protein in the sample. These values were then normalized by dividing the corresponding total lysate protein concentration of each sample measured via Bradford assay to account for differences in cell number. N = 3 independent biological replicates. Data are expressed in bar graphs as means, and error bars represent ± standard deviation. P value calculated via Tukey’s HSD. All pairwise comparisons were statistically insignificant. (E) Normalized total APOE protein present in TRAE3-H and TRAE4-H media ± 5-h OA treatment. Cells were grown in 1 ml of media, and APOE protein concentrations in µg/ml were measured by ELISA. The APOE concentration in µg/ml was multiplied by the total media volume of 1 ml to derive the total amount of APOE protein in the sample. These values were then normalized by dividing the total lysate protein concentration of the corresponding lysate sample measured via Bradford assay to account for variations in total material. N = 3 independent biological replicates. Data are expressed in bar graphs as means and error bars represent ± standard deviation. P value calculated via Tukey’s HSD. All pairwise comparisons were statistically insignificant. (F) The percentage of APOE present in the media out of the total APOE protein present in the lysate + media. Around 5% of total APOE protein was secreted into the media after 5 h ± OA treatment in all conditions. N = 3 independent biological replicates. Data are expressed in bar graphs as means, and error bars represent ± standard deviation. P value calculated via Tukey’s HSD. All pairwise comparisons were statistically insignificant. Source data are available for this figure: SourceData FS1.

Validation of APOE antibody via siRNA-mediated knockdown and measurement of intracellular and secreted APOE ± OA. (A) Western blot of lysates of TRAE3-H cells transfected with a non-targeting siRNA or one of two different siRNAs against APOE and treated ± OA for 5 h. The antibody used to probe for endogenous APOE is the same one used for both the immunofluorescence and immunogold experiments. (B) Quantification of Western blots of APOE knockdown from three independent biological replicates. APOE siRNA #2 demonstrated ∼94% knockdown of APOE in both − and + OA conditions, and was used for subsequent loss of function studies in Figs. 5 and 6. (C) Representative confocal slices of TRAE3-H cells transfected with NT siRNA or one of two APOE siRNAs and treated with 400 µM OA for 5 h. Cells were fixed and stained for endogenous APOE with an anti-APOE antibody and for LDs with BODIPY 493/503. Little to no endogenous APOE signal was observed by immunofluorescence upon APOE knockdown. Scale bar, 10 µm. (D) Normalized total APOE protein present in TRAE3-H and TRAE4-H lysates ± 5-h OA treatment. Cells were lysed in 100 µl of lysis buffer, and APOE protein concentrations in µg/ml were measured by ELISA. The APOE concentration in µg/ml was multiplied by the total lysate volume of 0.1 ml to derive the total amount of APOE protein in the sample. These values were then normalized by dividing the corresponding total lysate protein concentration of each sample measured via Bradford assay to account for differences in cell number. N = 3 independent biological replicates. Data are expressed in bar graphs as means, and error bars represent ± standard deviation. P value calculated via Tukey’s HSD. All pairwise comparisons were statistically insignificant. (E) Normalized total APOE protein present in TRAE3-H and TRAE4-H media ± 5-h OA treatment. Cells were grown in 1 ml of media, and APOE protein concentrations in µg/ml were measured by ELISA. The APOE concentration in µg/ml was multiplied by the total media volume of 1 ml to derive the total amount of APOE protein in the sample. These values were then normalized by dividing the total lysate protein concentration of the corresponding lysate sample measured via Bradford assay to account for variations in total material. N = 3 independent biological replicates. Data are expressed in bar graphs as means and error bars represent ± standard deviation. P value calculated via Tukey’s HSD. All pairwise comparisons were statistically insignificant. (F) The percentage of APOE present in the media out of the total APOE protein present in the lysate + media. Around 5% of total APOE protein was secreted into the media after 5 h ± OA treatment in all conditions. N = 3 independent biological replicates. Data are expressed in bar graphs as means, and error bars represent ± standard deviation. P value calculated via Tukey’s HSD. All pairwise comparisons were statistically insignificant. Source data are available for this figure: SourceData FS1.

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