Corrigendum to “An ATP synthase beta subunit is required for internalization of dsRNA into shrimp cells” [Fish Shellfish Immunol. 106 (2020) 948–958] (Fish and Shellfish Immunology (2020) 106 (948–958), (S1050464820306264), (10.1016/j.fsi.2020.09.010))

• Main Authors: Poohrawind Sanitt, Sakol Panyim, Apinunt Udomkit
• Other Authors: Mahidol University
• Format: Erratum
• Published: 2020
• Subjects: Agricultural and Biological Sciences; Environmental Science
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/60359

© 2020 Elsevier Ltd The authors regret for not including the legends of figures in the original article. The missing figure legends are now provided below. The authors would like to apologise for any inconvenience caused. Fig. 1: Internalization of labeled dsHSD into shrimp hepatopancreatic cells.Primary hepatopancreatic cells from L. vannamei were challenged with different concentrations of fluorescein-UTP labeled dsHSD. (A) The signals of dsHSD (green) were shown in the left panels, and the merging between the dsHSD signals and Rhodamine Phalloidin staining of actin (orange) to indicate cell boundary was shown in the right panels. (B) Competitive internalization was performed by the pretreatment with 100 μg/ml and 500 μg/ml of unlabeled dsHSD, and the intensity of dsHSD signals were calculated as a percentage to that of the control cells using image wizard analysis. (C) The competitive internalization was analyzed by using different dsRNA (dsRab7) and plasmid DNA. Fig. 2: Colocalization between cell surface fluorescein-UTP-labeled dsHSD and ATP synthase beta subunit.Primary cell culture from hepatopancreas were incubated on ice with 10 μg of fluorescein-UTP-labeled dsHSD for 2.5 min followed by detection of the labeled dsHSD (green) and ATP synthase beta subunit (red) on ice. After cell fixation with 1% formaldehyde, the images were captured using LSM800 confocal microscope. The colocalization spots were indicated by white arrows. The cell boundary was indicated using Rhodamine Phalloidin staining (orange). Fig. 3: Time course colocalization between labeled dsHSD and cell surface ATP synthase beta subunit.Shrimp primary hepatopancreatic cells were incubated with 10 μg of labeled dsHSD at 2.5 min time course prior to detection of the labeled dsHSD and ATP synthase beta subunit as described in Fig. 2. (A) Colocalization signals (yellow) between ATP synthase beta subunit (red) and dsHSD (green) at 2.5 min in non-permeabilized cells (left panels) and permeabilized cells (right panels). (B) Line graphs comparing total colocalization spots counted from permeabilized cells and cell surface colocalization spots counted from non-permeabilized cells at different time courses. (C) Quantitative colocalization analysis was performed by using Pearson colocalization coefficient (PCC) and Mander colocalization coefficient (MCC) at each time course. Fig. 4: Interference of dsHSD internalization by anti-ATP synthase beta subunit monoclonal antibody and angiostatin.(A) Shrimp primary hepatopancreatic cells were challenged with mouse anti-human ATP synthase beta subunit monoclonal antibody on ice prior to challenge with the labeled dsHSD. The signal of dsHSD (green) was detected by rabbit anti-fluorescein-UTP polyclonal antibody, and actin was visualized by Rhodamine Phalloidin staining (orange). The enlargement of the images in square boxes was illustrated in the right panels (B) Quantitative analysis of the signal intensities as a percentage to that of the control cells was performed using image wizard analysis. (C) The cells were challenged with different amounts of an ATP inhibitor, angiostatin prior to dsHSD internalization, and the quantitative analysis was performed. Fig. 5: Pathway of dsRNA internalization. Shrimp primary hepatopancreatic cells were treated with endocytic inhibitors before incubating with the labeled dsHSD. (A) Colocalization signals in the dsHSD treated cells between the labeled dsHSD (green) and Rab5 (orange; top row) or Rab7 (orange; bottom row) were seen as yellow spots (right panel of both rows). Bar graphs show quantitated intensity of fluorescein-UTP-labeled dsHSD in the cells that were pre-treated with either 15 or 30 μM chlorpromazine (B) or 800 nM bafilomycinA (C) comparing with dsHSD-treated cells that were not pre-treated with endocytic inhibitors as a control. Fig. 6: Functional assay of ATP synthase beta subunit-mediated dsRNA internalization on RNAi activity.(A) Shrimp hepatopancreatic cells were initially challenged with 10 μg/ml of either dsHSD, dsRab7 or dsYHV to investigate whether the cells could show specific knockdown. Gene transcript levels were determined by RT-PCR (left panels), and the band intensities were calculated by ImageJ program and presented as bar graphs (right panels). (B) The cells were blocked with anti-ATP synthase beta subunit monoclonal antibody at 4 °C prior to challenge with dsHSD. Gene transcript levels were determined by RT-PCR comparing between the control and the dsHSD-challenged group either without or with ATP synthase beta subunit blocking by antibody. The results were shown as mean ± SEM from three independent experiments.