Figure 6.
Knockdown of FHL2 prevents OGT-mediated mitochondrial arrest. (A and B) Mitochondrial motility in COS-7 cells expressing OGT or a control plasmid (GFP) along with shRNA to FHL2. (A) Representative images of mitochondria (upper panels) and heatmaps of the variance in Mito-DsRed over time (lower panels). (B) Quantification of mitochondrial motility as determined from the variance. The expression of OGT decreased mitochondrial motility in control cells, but not in those expressing shRNA to FHL2. n = 15–20 cells per condition from 3 independent transfections. (C and D) Knockdown of endogenous FHL2 in rat hippocampal neurons by lentiviral expression of shRNA. (C) Representative neuronal cell lysates on Western blots probed for FHL2 and GAPDH (as a loading control) are shown. Molecular weights (in kD) are indicated on the right. (D) Knockdown efficiency was quantified by measuring the ratio of the intensity of the FHL2 band to that of GAPDH, normalized to the control shRNA condition. n = 3–5 independent transductions per condition. P values were determined by ratio paired t tests. All data points are shown. Bars indicate mean ± SEM. (E and F) Effect of FHL2 knockdown in neurons on OGT-mediated mitochondrial arrest. Neurons were cotransfected with Mito-DsRed, GFP-2A-OGT or GFP, and shRNA against FHL2 or a control nontargeting shRNA. (E) Representative images of axons with GFP (top, green) and Mito-DsRed (middle, red) and kymographs (bottom) of mitochondrial motility. (F) Quantification of mitochondrial motility (percentage of time spent in motion for all mitochondria in the imaged regions) from kymographs. n = 10–12 axons per condition from 3 independent animals. Horizontal scale bars represent 20 µm, and vertical scale bars represent 30 s. All data except for bar graphs in D are represented as box-and-whisker plots. The line indicates the median, the box indicates the interquartile range, whiskers indicate the 10th and 90th percentiles. Outliers are represented as individual dots and are included in all statistical calculations. Significance testing was done using a two-tailed unpaired t test with Welch’s correction, and P values are indicated.

Knockdown of FHL2 prevents OGT-mediated mitochondrial arrest. (A and B) Mitochondrial motility in COS-7 cells expressing OGT or a control plasmid (GFP) along with shRNA to FHL2. (A) Representative images of mitochondria (upper panels) and heatmaps of the variance in Mito-DsRed over time (lower panels). (B) Quantification of mitochondrial motility as determined from the variance. The expression of OGT decreased mitochondrial motility in control cells, but not in those expressing shRNA to FHL2. n = 15–20 cells per condition from 3 independent transfections. (C and D) Knockdown of endogenous FHL2 in rat hippocampal neurons by lentiviral expression of shRNA. (C) Representative neuronal cell lysates on Western blots probed for FHL2 and GAPDH (as a loading control) are shown. Molecular weights (in kD) are indicated on the right. (D) Knockdown efficiency was quantified by measuring the ratio of the intensity of the FHL2 band to that of GAPDH, normalized to the control shRNA condition. n = 3–5 independent transductions per condition. P values were determined by ratio paired t tests. All data points are shown. Bars indicate mean ± SEM. (E and F) Effect of FHL2 knockdown in neurons on OGT-mediated mitochondrial arrest. Neurons were cotransfected with Mito-DsRed, GFP-2A-OGT or GFP, and shRNA against FHL2 or a control nontargeting shRNA. (E) Representative images of axons with GFP (top, green) and Mito-DsRed (middle, red) and kymographs (bottom) of mitochondrial motility. (F) Quantification of mitochondrial motility (percentage of time spent in motion for all mitochondria in the imaged regions) from kymographs. n = 10–12 axons per condition from 3 independent animals. Horizontal scale bars represent 20 µm, and vertical scale bars represent 30 s. All data except for bar graphs in D are represented as box-and-whisker plots. The line indicates the median, the box indicates the interquartile range, whiskers indicate the 10th and 90th percentiles. Outliers are represented as individual dots and are included in all statistical calculations. Significance testing was done using a two-tailed unpaired t test with Welch’s correction, and P values are indicated.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal