Figure 6. TgNup503 depletion results in... | Rockefeller University Press

Figure 6.

$TgNup503 depletion results in a trend of mild enhanced mitochondrial potential and have no effect on other functions of the respiratory chain. (A) Quantification of mitochondrial tRNA levels in treated (24 h) relative to untreated parasites (three biological replicates all shown in I). Error bars show mean with SEM. Data were analyzed with one sample T test compared with untreated (NS). (B) Individual Northern blots were used for quantification in H. Top panels show Northern blots with probes against tRNAIle whereas the bottom panels show Northern blots with probes against a mitochondrial rRNA for loading control of mitochondrial fraction. (C) A representative image and graph of measurements from four biological replicates of in-gel assembly and activity assay for complex IV. Error bars display mean with SEM. Data were analyzed with one sample T test comparing to untreated. (D) Blue native page followed by Western blot to assess assembly of complex V (representative image of biological replicates). Error bars display mean with SEM. Two biological replicates, data were analyzed with one sample T test comparing to untreated (NS). (E) Membrane potential was assessed using JC-1 dye where membrane potential results in movement of the dye into the mitochondrion where it aggregates changing from green to red. Error bars show mean with SEM, three biological replicates, data were analyzed with two-tailed unpaired T test (NS). (F) Basal mitochondrial oxygen consumption rates (basal mOCR) and extracellular acidification rate (ECAR) of iHA-TgNup503 TgNup302-myc grown in the presence or absence of ATc for 24 h, three biological replicates. Error bars display mean with SEM. Data were analyzed with two-tailed unpaired T test (NS). (G) Representative images of mitochondria from 24 h ATc treated iHA-TgNup503 TgNup302-myc parasites showing their cristae. (H) Cristae count from EM of iHA-TgNup503 TgNup302-myc, untreated, and ATc treated for 16 and 24 h. Two biological replicates, 65–111 mitochondria analyzed per condition. Error bars display mean with SEM. Data were analyzed with one-way ANOVA (NS). (I) Level of mitochondrial ROS was assessed with MitoSOX dye and flow cytometry. Error bars show mean with SEM, four biological replicates. Data were analyzed with one sample T test compared with untreated (NS). Source data are available for this figure: SourceData F6.$

TgNup503 depletion results in a trend of mild enhanced mitochondrial potential and have no effect on other functions of the respiratory chain. (A) Quantification of mitochondrial tRNA levels in treated (24 h) relative to untreated parasites (three biological replicates all shown in I). Error bars show mean with SEM. Data were analyzed with one sample T test compared with untreated (NS). (B) Individual Northern blots were used for quantification in H. Top panels show Northern blots with probes against tRNA^Ile whereas the bottom panels show Northern blots with probes against a mitochondrial rRNA for loading control of mitochondrial fraction. (C) A representative image and graph of measurements from four biological replicates of in-gel assembly and activity assay for complex IV. Error bars display mean with SEM. Data were analyzed with one sample T test comparing to untreated. (D) Blue native page followed by Western blot to assess assembly of complex V (representative image of biological replicates). Error bars display mean with SEM. Two biological replicates, data were analyzed with one sample T test comparing to untreated (NS). (E) Membrane potential was assessed using JC-1 dye where membrane potential results in movement of the dye into the mitochondrion where it aggregates changing from green to red. Error bars show mean with SEM, three biological replicates, data were analyzed with two-tailed unpaired T test (NS). (F) Basal mitochondrial oxygen consumption rates (basal mOCR) and extracellular acidification rate (ECAR) of iHA-TgNup503 TgNup302-myc grown in the presence or absence of ATc for 24 h, three biological replicates. Error bars display mean with SEM. Data were analyzed with two-tailed unpaired T test (NS). (G) Representative images of mitochondria from 24 h ATc treated iHA-TgNup503 TgNup302-myc parasites showing their cristae. (H) Cristae count from EM of iHA-TgNup503 TgNup302-myc, untreated, and ATc treated for 16 and 24 h. Two biological replicates, 65–111 mitochondria analyzed per condition. Error bars display mean with SEM. Data were analyzed with one-way ANOVA (NS). (I) Level of mitochondrial ROS was assessed with MitoSOX dye and flow cytometry. Error bars show mean with SEM, four biological replicates. Data were analyzed with one sample T test compared with untreated (NS). Source data are available for this figure: SourceData F6.