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. 2017 Aug 21;13(10):1648–1663. doi: 10.1080/15548627.2017.1343768

Figure 2.

Figure 2.

Increased SQSTM1 levels and decreased aggregate clearance in BORC-KO cells. (A) Confocal micrographs of WT, BORCS5-KO, BORCS5-rescue, BORCS6-KO, BORCS7-KO, and BORCS8-KO cells immunostained for SQSTM1 and LAMP1. Images of SQSTM1 staining are in negative grayscale for easier visualization. Nuclei were stained with DAPI. Scale bar: 10 μm. (B) Quantification of SQSTM1 puncta from experiment in A. Bars represent the mean ± SEM of SQSTM1 puncta per cell from 30 cells. ***P < 0.0001, one-way ANOVA, followed by multiple comparisons using the Dunnett test. (C) Immunoblotting of extracts from WT, BORCS5-KO, BORCS5-rescue, BORCS6-KO, BORCS7-KO and BORCS8-KO cells with antibodies to SQSTM1 and tubulin (control). The positions of molecular mass markers (in kDa) are indicated at left. (D) Quantification of SQSTM1 normalized to tubulin from experiments as in (C). Bars represent the mean ± SEM from 3 independent experiments. *P < 0.05, **P < 0.001, ***P < 0.0001, one-way ANOVA, followed by multiple comparisons using the Dunnett test. (E) Confocal images of WT, BORCS5-KO and BORCS5-rescue cells transfected with a plasmid encoding the aggregation-prone HTT103Q-EGFP for 48 h. Arrowheads indicate intracellular aggregates of HTT103Q-EGFP. Nuclei were stained with DAPI. Scale bar: 50 μm. (F) Percentage of cells with EGFP-positive aggregates. Over 600 GFP-positive cells from 3 independent experiments were analyzed. Bars represent the mean ± SEM of the percentage of cells with EGFP-positive aggregates. ***P < 0.0001, one-way ANOVA, followed by multiple comparisons using the Dunnett test.