Fig 1: Exosome purification and characterization.A, schematic describing exosome purification procedure. B, size distribution plot of purified exosomes, as determined by NTA. C, immunofluorescence image of purified exosomes, labeled with an Alexa Fluor 647-conjugated anti-CD63 monoclonal antibody, captured using the Particle Metrix PMX-220 ZetaView camera. D, electron micrograph of negative-stained, purified exosomes. Bar, 100 nm. E, immunoblot analysis of equal proportions of 293F cell and exosome lysates using antibodies specific for the exosomal markers CD81, CD9, CD63, E-cadherin (E-cad), N-cadherin (N-cad), GRP78/BiP, calreticulin (CALR), ERGIC-3, GM130, HSP60, and HSP90. The amount and ratio of cell and exosome lysates were selected empirically to show the different enrichment of the CD81, CD9, and CD63 proteins, were kept constant in all immunoblots, by proportion equaled a 10-fold overloading of exosomes relative to cells, and by amount of protein equaled 15 µg cell lysate protein/lane and 0.15 µg exosome lysate protein/lane.
Fig 2: ATF5 knockdown reversed mitochondrial function in Mut-H USCs.A ATF5 knockdown decreased UPRmt related gene expression. USCs were transfected with siRNA specific to ATF5 for 48 h and the mRNA levels of UPRmt related genes (ATF5, HSP70, HSP60 and LONP1) expression were quantified by RT-qPCR. B Mitochondrial membrane potential was increased by ATF5 knockdown. USCs were transfected with siRNA specific to ATF5 for 48 h and mitochondrial membrane potential was measured using fluorescence probe JC-1 assay system. Red color indicates cells with normal mitochondrial membrane potential while green indicates cells with loss of mitochondrial membrane potential. C Quantification plot for B. D Intracellular ROS was decreased by ATF5 knockdown. USCs were transfected with siRNA specific to ATF5 for 48 h and stained with DCFH-DA. Green color indicate ROS-positive cell population. E Quantification plot for D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar = 50 um.
Fig 3: OPTN alleviates Aßo-induced dysfunction of mitochondrial autophagy. A, B BV2 cells were treated with Aßo in the absence or presence of transfection with plvx-IRES-OPTN. A mKeima fluorescence was evoked using two excitation filters (438 ± 12 nm and 550 ± 15 nm) and a 610LP emission filter. B Expression levels of HSP60 and OPTN were detected by western blotting. ß-actin served as an internal control. The data represent the means ± S.E. of independent experiments. BV2 cells treated with vehicle group or Aßo and overexpressing OPTN were compared with group Aßo alone *P < 0.05, **P < 0.01, ***P < 0.001
Fig 4: Pharmacologic activation of GPER inhibits leukemic cell survival via mitochondrial-related apoptosis.A CCK-8 assay of cell viability in the leukemic cells treated with corresponding concentrations of GPER agonist G-1 for 48 h, IC50 values were calculated on basis of drug concentrations that causes 50% cell viability. B Leukemic cells were treated with 1 µM G-1 for the corresponding times. C CCK-8 assay of cell viability in the primary blasts and PBMNCs from HD treated with G-1 for 48 h. D, E FCM of apoptosis in the cell lines treated with G-1 for 24 h, and western blotting of the indicated proteins. F Western blot analysis of Cyto C in the mitochondria fractions and cytosolic fraction of the cells treated with indicated drugs for 24 h. HSP60 or GAPDH were used as an internal control. G Western blot analysis of GPER in OCI-AML2 cells infected with two independent sh RNAs targeting GPER. H FCM of apoptosis in the cells treated with 1 µM G-1 for 24 h. The data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01. ns, not significant.
Fig 5: FOXG1 improves mitochondrial function in NPC cells. (A) The copy number of mitochondrial DNA in C666-1 and SUNE-1 cells was detected via reverse transcription-quantitative PCR. (B) Relative ATP/ADP ratio in C666-1 and SUNE-1 cells. (C) Mitochondrial membrane potential was detected via flow cytometry; red fluorescent mitochondria was found in the upper right quadrant and green fluorescent mitochondria in the lower right quadrant; decreased Red/Green ratio represented a decrease in mitochondrial membrane potential. (D) Expression levels of mitochondrial markers (SDHA, HSP60 and PDH) in C666-1 and SUNE-1 cells were detected via western blot analysis. **P<0.01 vs. Control, si-NC or pcDNA3.1-NC group. NC, negative control; si, small interfering RNA; FOXG1, forkhead-box gene 1; SDHA, succinate dehydrogenase complex flavoprotein subunit A; PDH, pyruvate dehydrogenase; HSP60, heat shock protein 60.
Supplier Page from Abcam for Anti-Hsp60 antibody [EPR18245-93]