Fig 1: Silencing of OPA3 expression decreases cell proliferation and epithelial–mesenchymal transition (EMT): (a,b) Cell proliferation in Panc-1 OPA3-shRNA cells was analyzed by clonogenic assay and quantified. (c) Cell proliferation in Panc-1 OPA3-shRNA cells was analyzed by cell counting. (d,e) Apoptosis in Panc-1 OPA-shRNA cells was analyzed by flow cytometry and quantified by Annexin-V positivity. (f) Expressions of EMT-related proteins in Panc-1 OPA3-shRNA cells were analyzed by immunoblotting. (g) Representative scheme depicting the role of OPA3 expression induced by oncogenic signaling in pancreatic cancer cells. Statistics: data are mean ± S.E.M. (n = 3); one-way ANOVA followed by Dunnett post hoc test (compared to “shCTL”) for (b) and (e) and two-way ANOVA for (c). * p < 0.05, ** p < 0.01, and *** p < 0.001. Abbreviations: 7-AAD: 7-aminoactinomycin D, K-ras: Kirsten RAS GTPase, NDUFAF1: NADH dehydrogenase 1 alpha subcomplex assembly factor 1, OPA3: optic atrophy protein 3, shCTL: control shRNA, and shOPA3: shRNA against human OPA3.
Fig 2: Silencing of OPA3 expression alters mitochondrial function in Panc-1 cells: (a,b) Real-time measurement of OCR and ECAR for Panc-1 cells using the Seahorse analyzer. (c,d) Lactate and glucose contents were quantified in culture medium of Panc-1 OPA3-shRNA cells. (e) Intracellular ATP content was quantified in Panc-1 OPA3-shRNA cells. (f,g) Real-time measurement of OCR and ECAR for HPNE/K-rasG12D cells using the Seahorse analyzer. HPNE/ K-rasG12D cell line was transfected with control siRNA (siCTL) or OPA3-siRNA for 24 h and then incubated with doxycycline for additional 24 h. Statistics: data are mean ± S.E.M. (n = 3); one-way ANOVA followed by Dunnett post hoc test (compared to “shCTL”) for (c–e). * p < 0.05, ** p < 0.01 and *** p < 0.001. Abbreviations: AA: antimycin A; ECAR: extracellular acidification rate; FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone; OCR: oxygen consumption rate; OPA3: optic atrophy protein 3; Rot: rotenone; shCTL: control shRNA; shOPA3: shRNA against human OPA3; siCTL: control siRNA; and siOPA3: siRNA against human OPA3.
Fig 3: Oncogenic K-ras activation induces OPA3 expression in HPNE cells. Inducible K-rasG12D cell line was incubated with doxycycline for various incubation times. The mRNA levels of (a) biogenesis, (b) mitophagy, (c) mitochondrial fusion, and (d) mitochondrial fission genes were measured by real-time PCR. (e,f) Inducible K-rasG12D cell line was incubated with doxycycline for various incubation times. OPA3 protein levels were analyzed by immunoblotting and quantified. (g) Immunohistochemistry analysis for OPA3 in human normal and pancreatic (PDAC) tumor tissues. (h) Score of OPA3 staining, measured by immunohistochemistry IHC, in human normal and PDAC tumor tissues. Statistics: data are mean ± S.E.M, one-way ANOVA followed by Dunnett post hoc test (compared to “OFF”) for (a–d) and (f), and unpaired T-test for (h). * p < 0.05, ** p < 0.01, and *** p < 0.001. Abbreviations: BNIP3L (Nix): BCL2 interacting protein 3 like; Drp1: dynamin related protein 1; Fis1: mitochondrial fission 1 protein; Mfn2: mitofusin-2; HPNE: human pancreatic nestin expressing cells; K-ras: Kirsten RAS GTPase; MID51: mitochondrial dynamics proteins of 51 kDa; Mito-PLD: mitochondrial phospholipase D; OPA1: optic atrophy protein 1; OPA3: optic atrophy protein 3; PDAC: pancreatic ductal adenocarcinoma; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; and Pink1: PTEN induced kinase 1.
Fig 4: Silencing of OPA3 expression does not modify mitochondrial morphology: (a) OPA3 mRNA levels in Panc-1 OPA3-shRNA cell lines were measured by real-time PCR. (b–d) OPA3 and phospho-Drp1 (ser616) protein levels in Panc-1 OPA3-shRNA cell lines were analyzed by immunoblotting and quantified. (e,f) Mitochondrial morphology was analyzed with MitoTracker red probe in Panc-1 OPA3-shRNA cells and HPNE/K-rasG12D cells (with or without doxycycline and siRNA against OPA3). Scale bars represent 20 μm (g) HPNE/ K-rasG12D cell line was transfected with control siRNA (siCTL) or OPA3 for 48 h and then incubated with doxycycline for additional 48 h. K-ras, OPA3, Drp1, and phospho-Drp1 (ser616) protein levels were analyzed by immunoblotting. Statistics: data are mean ± S.E.M. (n = 3); one-way ANOVA followed by Dunnett post hoc test (compared to “shCTL”) for (a) and (c,d). * p < 0.05 and ** p < 0.01. Abbreviations: Drp1: dynamin related protein 1, K-ras: Kirsten RAS GTPase, OPA3: optic atrophy protein 3, shCTL: control shRNA, shOPA3: shRNA against human OPA3, siCTL: control siRNA, and siOPA3: siRNA against human OPA3.
Supplier Page from Abcam for Anti-OPA3 antibody