Fig 1: Exosome biogenesis related proteins under hypoxia stress.The expression of exosome biogenesis-related proteins Alix, Tsg101 and Rab11a in ischemic myocardium (A) primary cardiomyocytes (B) primary endothelial cells (C) and fibroblasts (D). Alix showed a higher expression in the ischemic myocardium and hypoxic cardiomyocytes. Rab11a was also higher in hypoxic cardiomyocytes. However, the hypoxic endothelial cells and fibroblasts did not show an increase in any of Alix, Tsg101 or Rab11a. * indicates P < 0.05 and NS indicates P > 0.05. The protein levels are shown as the experiment group divided by the control group.
Fig 2: (A) Cartoon illustrating the distribution of F-actin, polarized canalicular proteins ABCB11/MRP2 and RAB11A around the bile canalicular lumen in HepG2 cells. (B) Sequencing chromatogram of DNA fragment targeted by CRISPR-Cas9 in HepG2 UNC45A KO2 cells, aligned to WT sequences. Software Chromas and DNAMAN are used to analyze sequencing results. (C) Representative Western blot for UNC45A in HepG2 WT and HepG2 KO2 cells. (D) Representative Western blot for myosin Vb and UNC45A in HepG2 WT, HepG2 MYO5B KO, and 2 independent HepG2 UNC45A KO cell lines. (E) Quantification of relative myosin Vb expression by Western blot in HepG2 WT and 2 independent HepG2 KO cell lines. (F) Quantification of the relative mRNA expression of MYO5B gene in 2 independent HepG2 KO cell lines. (G) Representative Western blot showing the expression level of MYO5B (myosin Vb) and UNC45A in HepG2 WT cell lines, MYO5B-depleted HepG2 cells lines, UNC45A-depleted cell lines and UNC45A-depleted cells lines in which the a UNC45A WT-flag gene was re-introduced. (H) Double labeling of ABCC2 and RAB11A in HepG2 WT and HepG2 UNC45A KO cells. Scale bar: 10 µm. (I) F-actin staining in HepG2 WT and HepG2 UNC45A KO cells. Error bar indicates mean ± SD. Black dots indicate individual data points. t test, ****P < .0001.
Fig 3: The pathogenic mechanism of O2HE patient–associated UNC45A-p.V423D mutation. (A) Sequence alignment of UNC45 proteins fragment from Caenorhabditis elegans (CeUNC45: G5EG62), Drosophila melanogaster (DmUNC45; Q960B1), Danio rerio (DrUNC45A: F1QU23; DrUNC45B: Q6DGE9), Mus musculus (MmUNC45A: Q99KD5; MmUNC45B: Q8CGY6), Homo sapiens (HsUNC45A: Q9H3U1-2; HsUNC45B: Q8IWX7). Black box and star indicate V423 mutated site. Clustal omega27 and ENDscript server28 were used to make the alignment. (B) Ribbon presentation of the UNC45A protein modeling based on crystal structure of UNC45 (PDB ID: c4i2wA) showing its domains and mutated site. Different colors indicate different domains explained by word with the same color. The unc45a model was built by phyre2 intensive mode.29 The PhyMOL software (2.5) was used to color the domains and label the mutated site. (C) Sequencing chromatogram of DNA fragment with mutated site in Plenti-UNC45A-p.V423D-FLAG, aligned to UNC45A cds sequence (Q9H3U1-2). Software Chromas and DNAMAN are used to analyze sequencing results. (D) Representative Western blot for myosin Vb and UNC45A in Caco-2 UNC45A KO + unc45a-p. V423D-FLAG and that treated with MG132 (5 µmol/L, 16 hours). (E) Quantification of relative myosin Vb expression by Western blot in Caco-2 UNC45A KO + unc45a-p.V423D-FLAG and that treated with MG132. N = 3 independent experiments. Error bar indicates mean ± SD. Black dots indicate individual data points. t test, *P < .01. (F) Upper row shows a triple labelling of F-actin (white), myosin Vb (red), and FLAG (green). Lower row shows RAB11A (red) and FLAG (green) double labeling in Caco-2 UNC45A KO2 + UNC45a-p.V423D-FLAG cells. White arrows point to condensed RAB11A staining pattern in Caco-2 UNC45A KO cells, and yellow asterisks indicate F-actin-labeled microvilli (upper row)/peripheral RAB11A (lower row) in Caco-2 UNC45A KO + UNC45A-p. V423D FLAG cells. Scale bar: 10 µm.
Fig 4: Loss of UNC45A caused redistribution of RAB11A-positive RE in Caco-2 cells. (A) Immunofluorescence microscopy image of endogenous RAB11A in WT, Caco-2 MYO5B KO, and UNC45A KO2 cells. Scale bar: 10 µm. (B) Quantification of percentage of condensed RAB11A in Caco-2 WT, Caco-2 MYO5B KO, and Caco-2 UNC45A KO cells. N = 3 independent experiments. Around 500 cells of 3 cell types are analyzed in each experiment. (C) RAB11A labeling in Caco-2 UNC45A KO cells and that were treated with nocodazole (33 µmol/L 1.5 hours). Scale bar: 10 µm. (D) Quantification of percentage of condensed RAB11A in Caco-2 UNC45A KO cells and treated with nocodazole. N = 3 independent experiments. More than 100 cells of 2 cell types were analyzed in each experiment. (E–G) RAB1A and FLAG double labelling in Caco-2 UNC45A KO cells re-expressing UNC45A-FLAG (E) and re-expressing myc-myosin Vb-FLAG (F and G). White arrow shows the condensed RAB11A in Caco-2 UNC45A KO cells, and yellow asterisk indicates peripheral RAB11A in Caco-2 UNC45A KO + UNC45A-FLAG cells. (G) Representative Western blot for myosin Vb and FLAG in Caco-2 WT and Caco-2 WT + myc-myosin Vb-FLAG cells, those bands come from different position of the same membrane. Representative Western blot for myosin Vb and FLAG in Caco-2 UNC45A KO2 and Caco-2 UNC45A KO2 + myc-myosin Vb-FLAG cells, those come from different positions of the same membrane. (B) and (D) Error bar indicates mean ± SD. Black dots indicate individual data points. t test, ****P < .0001, ***P < .001.
Fig 5: RAB11A was a direct target of miR-944 in PA cells. (A) Bioinformatic analysis with miRDB database, starBase and TargetscanHuman database (TargetScanHuman 7.2) predicted that the 3'UTR of RAB11A possessed a sequence that was complementary to miR-944. (B) MiR-944 overexpression significantly suppressed the luciferase activity of WT-RAB11A 3'-UTR reporter, rather than that of MUT-RAB11A 3'-UTR reporter. (C, D) MiR-944 overexpression reduced the expression of RAB11A mRNA and protein in PA cells and miR-944 inhibitors increased the expression level of RAB11A mRNA and protein. **p < 0.01.
Supplier Page from Abcam for Anti-Rab11A antibody [EPR7587(B)]