Journal: bioRxiv

Article Title: Impaired peroxisomal import in Drosophila hepatocyte-like cells induces cardiac dysfunction through the pro-inflammatory cytokine Upd3

doi: 10.1101/659128

Figure Lengend Snippet: (a) Arrhythmia index of oenocyte-specific Cat and Sod1 knockdown flies (1-week-old). (b) Representative images of ROS levels (DHE staining) in dissected oenocytes from flies fed on normal diet or 10mM paraquat (for 24 h). All flies express mCD8::GFP under the oenocyte-specific driver ( PromE-Gal4 ). Sod1 was specifically over-expressed in the oenocytes ( Sod1 oe ). Scale bar: 20 µm. (c) Quantification of the percentage of DHE-positive staining in a selected region of interest (n ≥ 16). (d) Representative M-mode traces showing heart contraction in control and Sod1 over-expression flies fed on normal or 10mM paraquat diets. Sod1 was expressed using the oenocyte-specific GeneSwitch driver ( PromE GS -Gal4 ). RU: mifepristone (RU486). (e) Arrhythmia index of control and oenocyte-specific Sod1 over-expression flies fed on normal or 10mM paraquat diets. (f) Arrhythmia index of control and fat body/gut-specific Sod1 over-expression flies fed on normal or 10mM paraquat diets. over-expression specifically in fat body ( S106-Gal4 ). (g) Representative M-mode traces showing heart contraction in young (2 weeks) and old (6 weeks) flies with or without oenocyte-specific Sod1 over-expression. (h) Arrhythmia index of control and oenocyte-specific Sod1 oe flies at young and old ages. Data are represented as mean ± SEM. Panel e, f, h: N ≥ 16. Panel (a): N = 9. P values are calculated using unpaired t- test, ns: not significant.

Article Snippet: RU486 (mifepristone, Fisher Scientific) was dissolved in 95% ethanol, and added to standard food at a final concentration of 100 μM for all the experiments except for Pex5 over-expression, where 200 μM of RU486 was used.

Techniques: Knockdown, Staining, Control, Over Expression

Journal: bioRxiv

Article Title: Impaired peroxisomal import in Drosophila hepatocyte-like cells induces cardiac dysfunction through the pro-inflammatory cytokine Upd3

doi: 10.1101/659128

Figure Lengend Snippet: (a) Verification of oenocyte-specific GeneSwitch driver ( PromE GS -Gal4 ). RU: mifepristone (RU486). (b) Verification of gut/fat body-specific GeneSwitch driver (S106 GS -Gal4 ). Scale bar: 20 µm.

Article Snippet: RU486 (mifepristone, Fisher Scientific) was dissolved in 95% ethanol, and added to standard food at a final concentration of 100 μM for all the experiments except for Pex5 over-expression, where 200 μM of RU486 was used.

Techniques:

Journal: bioRxiv

Article Title: Impaired peroxisomal import in Drosophila hepatocyte-like cells induces cardiac dysfunction through the pro-inflammatory cytokine Upd3

doi: 10.1101/659128

Figure Lengend Snippet: RNAi knockdown verification by QRT-PCR for Upd3 (a) , ND75 (b) , Pex1 (c) , Pex5 (d) , Kay (e) . Oenocyte-specific GeneSwitch driver ( PromE GS -Gal4 ) was used. RU: mifepristone (RU486). Data are represented as mean ± SEM. P values are calculated using unpaired t- test, ns: not significant.

Article Snippet: RU486 (mifepristone, Fisher Scientific) was dissolved in 95% ethanol, and added to standard food at a final concentration of 100 μM for all the experiments except for Pex5 over-expression, where 200 μM of RU486 was used.

Techniques: Knockdown, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Impaired peroxisomal import in Drosophila hepatocyte-like cells induces cardiac dysfunction through the pro-inflammatory cytokine Upd3

doi: 10.1101/659128

Figure Lengend Snippet: (a) Schematic diagram showing the key genes involved in peroxisomal import and membrane assembly. (b) Arrhythmia index of oenocyte-specific knockdown of mitochondrial complex I subunit ND75 (two independent RNAi lines), mitochondrial Mn superoxide dismutase Sod2 , peroxisomal import factors ( Pex5, Pex1 , Pex14 ), and peroxisomal membrane assembly factor ( Pex19 ). N ≥ 15. Oenocyte-specific GeneSwitch driver ( PromE GS -Gal4 ) was used. RU: mifepristone (RU486). (c) Arrhythmia index of oenocyte-specific knockdown of peroxisomal matrix enzymes, Dhap-at , CG10268 , Prx5 . N ≥ 15. (d) QRT-PCR analysis showing relative mRNA levels of Upd3 from dissected oenocytes of Pex5 , Pex1 , Cat and ND75 KD flies. Mito: Mitochondrion. N = 4∼6. (e) QRT-PCR analysis showing relative mRNA levels of Upd3 , Upd1 , Upd2 from dissected oenocytes of oenocyte-specific Pex5 KD flies. N = 4∼6. (f) Representative images of STAT immunostaining of heart tissues dissected from flies with (+RU) or without (-RU) oenocyte-specific Pex5 KD. Arrows indicate cardiomyocyte nuclei. White boxes indicate the regions shown in the right insets. Scale bar: 20 µm (inset: 5 µm). (g) Quantification on the number of STAT punctae around cardiomyocyte nuclei of oenocyte-specific Pex5 KD flies. N = 6. Dot plot shows the quantifications of 6 biological replicates, 3-4 selected regions of interest (ROIs) per replicate. (h) Representative images of PMP70 immunostaining in three fly tissues. Arrows indicate cardiomyocyte and pericardial cell nuclei. Scale bar: 6.7 µm. (i) Quantification of PMP70-positive peroxisomes per region of interest. N = 6. Dot plot shows the quantifications of 6 biological replicates, 3-5 ROIs per replicate. (j) Arrhythmia index of flies with Pex5 KD in either oenocytes ( PromE GS -Gal4 ) or fat body/gut ( S106 GS -Gal4 ). N ≥ 15. Data are represented as mean ± SEM. P values are calculated using unpaired t- test, ns: not significant.

Article Snippet: RU486 (mifepristone, Fisher Scientific) was dissolved in 95% ethanol, and added to standard food at a final concentration of 100 μM for all the experiments except for Pex5 over-expression, where 200 μM of RU486 was used.

Techniques: Membrane, Knockdown, Quantitative RT-PCR, Immunostaining