Authorship Contributions
K.H.C. and R.G.S were involved in the conception, design and interpretation of experiments and wrote the manuscript.
K.H.C. performed experiments and analyzed data.
Figure 1. Formoterol restores RhoA and Drp1 activity in glucose treated RPTC. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were treated with either vehicle (0.1% DMSO) or formoterol (30nM) for 24 hr. Cells were harvested and proteins were subjected to immunoblot analysis of (A) GTP-bound RhoA and total RhoA, and (B) GTP-bound Drp1 and total Drp1. Data represent mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 2. Formoterol, CCG-1423 and Y-27632 restore RhoA and Drp1 activity and mitochondrial function in RPTC. CCG-1423 (300nM) and Y-27632 (100nM) are RhoA and ROCK1 inhibitors, respectively. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were treated with either vehicle (0.1% DMSO), formoterol, CCG-1423 or Y-27632 for 24 hr. Cells were harvested and proteins were subjected to immunoblot analysis of (A) GTP-bound RhoA and total RhoA and (B) and (C) GTP-bound Drp1 and total Drp1. Seahorse XF96 Analyzer was used to measure oxygen consumption under the same conditions and co-treated with either CCG-1423 (D) basal-OCR measurements and (E) FCCP-OCR, or Y-27632 (F) basal-OCR and (G) FCCP-OCR. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 3. Formoterol and Mdivi-1 restore mitochondrial function in glucose treated RPTC. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were co-treated with either vehicle (0.1% DMSO), formoterol (30nM) or Mdivi-1 (100nM) for 24 hr. Seahorse XF96 Analyzer was used to measure (A) basal-OCR or (B) FCCP-OCR. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 4. Carvedilol blocks the effect of formoterol on RhoA and Drp1 activity. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were co-treated with either vehicle (0.1% DMSO), formoterol (30nM), carvedilol (10nM) or carvedilol+formoterol for 24 hr. Cells were harvested and proteins were subjected to immunoblot analysis of (A) GTP-bound RhoA and total RhoA and (B) GTP-bound Drp1 and total Drp1. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 5. Gallein blocks the effect of formoterol on RhoA and Drp1 activity. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were co-treated with either vehicle (0.1% DMSO), formoterol (30nM), gallein (100nM) or gallein+formoterol. Cells were harvested and proteins were subjected to immunoblot analysis of (A) GTP-bound RhoA and total RhoA and (B) GTP-bound Drp1 and total Drp1. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 6. Formoterol blocks the interaction between p114RhoGEF and RhoA. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were co-treated with either vehicle (0.1% DMSO), formoterol (30nM), gallein (100nM) or gallein+formoterol. p114RhoGEF and RhoA were measured by immunoblot after immunoprecipitation of RhoA. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 7. Formoterol, PLX4032 and GSK11202 restore Mfn1 activity in glucose treated RPTC. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were co-treated with either vehicle (0.1% DMSO), formoterol (30nM), PLX4032 (30nM) or GSK11202 (10nM). Harvested proteins were subjected to immunoblot analysis of (A) and (C) GTP-bound and total Mfn1 and (B) pERK1/2 or total ERK1/2. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 8. Gallein blocks the effect of formoterol on Mfn1 activity in glucose treated RPTC. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were co-treated with either vehicle (0.1% DMSO), formoterol (30nM), gallein (100nM) or gallein+formoterol. Cells were harvested and proteins were subjected to immunoblot analysis of GTP-bound and total Mfn1 after treatment with gallein. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.
Figure 9. Formoterol and gallein have no effect on Akt phosphorylation in glucose treated RPTC. RPTC were grown in 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. At 72 hr, cells were co-treated with either vehicle (0.1% DMSO), formoterol (30nM), gallein (100nM) or gallein+formoterol. Cells were harvested and proteins were subjected to immunoblot analysis of GTP-bound and total Mfn1 after treatment with gallein. Data represented as mean±SEM, n=6. Statistical significance was determined by two-way ANOVA and Tukey-Kramer post-hoc test. Different Greek letters signify statistical differences.