Journal: Hepatology Communications

Article Title: Secretion of the HBV small surface antigen is driven by an ER autophagic pathway

doi: 10.1097/HC9.0000000000000939

Figure Lengend Snippet: The SHBs resides in the ER and late endosomal compartments. (A) Western blot (WB) analysis of culture medium (CM or EXT) and whole cell lysates (WCLs or INT) from either HepG2 parental cells or the HepG2-SHBs stable cell line. Distinct 27/24 kD bands are detectable using 2 distinct SHBs antibodies (Virostat and Biosynth) in both the EXT (CM) and INT (WCLs) in HepG2-SHBs stable cells, but not from the parental cell line. (B) Quantitation of extracellular SHBs by CLIA. (C–F) Immunofluorescence (IF) staining of HepG2-SHBs stable cells showing the intracellular distribution of the SHBs (red) with different compartment markers (green) including (C) the endoplasmic reticulum (ER, PDI), (D) the Golgi (GM130), (E) early endosomes (E. Endo, EEA1), and (F) multivesicular bodies (MVB)s/late endosomes (CD63). Intensity line scan quantitation of SHBs within the different compartments of the provided images. At least 10 cells were analyzed per condition, and the experiment was performed 3 times. p -value ****, <0.0001. Scale bar, 10 μm. Abbreviations: CD63, cluster of differentiation 63; CLIA, chemiluminescent immunoassay; CM, culture medium; EEA1, early endosome antigen 1; ER, endoplasmic reticulum; EXT, external/secreted; GM130, Golgi matrix protein 130; HepG2-SHBs, HepG2 cell line stably expressing SHBs; IF, immunofluorescence; INT, internal; MVB, multivesicular body; PDI, protein disulfide isomerase; SHBs, small hepatitis B surface antigen; WB, western blot; WCLs, whole cell lysates.

Article Snippet: Extracellular SHBs were quantified by a HBsAg chemiluminescence immunoassay (CLIA) (Autobio Diagnostics, Zhengzhou, China, CL0310-2).

Techniques: Western Blot, Stable Transfection, Quantitation Assay, Immunofluorescence, Staining, Expressing

Journal: Hepatology Communications

Article Title: Secretion of the HBV small surface antigen is driven by an ER autophagic pathway

doi: 10.1097/HC9.0000000000000939

Figure Lengend Snippet: VSV-G, but not SHBs protein, is visible in the Golgi under a low-temperature block. IF images of HepG2-SHBs cells profiling colocalization of vesicular stomatitis virus G protein (VSVG)-GFP (A–C) or SHBs (D, E) with Golgi. Cells were incubated at 20 °C for either (B, E) 2 hours or (C, F) 4 hours before fixation and staining. Corresponding intensity line scan quantitation of VSV-G or SHBs with the Golgi for each image. At least 10 cells were analyzed per condition, and the experiment was performed 3 times. Scale bar, 10 μm. Abbreviations: GFP, green fluorescent protein; HepG2-SHBs, HepG2 cell line stably expressing SHBs; IF, immunofluorescence; SHBs, small hepatitis B surface antigen; VSVG, vesicular stomatitis virus G protein.

Article Snippet: Extracellular SHBs were quantified by a HBsAg chemiluminescence immunoassay (CLIA) (Autobio Diagnostics, Zhengzhou, China, CL0310-2).

Techniques: Blocking Assay, Virus, Incubation, Staining, Quantitation Assay, Stable Transfection, Expressing, Immunofluorescence

Journal: Hepatology Communications

Article Title: Secretion of the HBV small surface antigen is driven by an ER autophagic pathway

doi: 10.1097/HC9.0000000000000939

Figure Lengend Snippet: Development of a novel SHBs-tagged probe to observe trafficking in live cells. (A, B) Schematic diagrams and domain maps of (A) GFP-tagged SHBs WT fusion protein (GFP-WT) and (B) truncated form missing the C-terminus (GFP-ΔHCR) of SHBs. Corresponding fluorescence images of these fusion proteins expressed in HepG2-SHBs stable cells co-stained with an MVB marker (red, white arrowheads). (C) WB analysis of the HepG2 parental and the HepG2-SHBs stable expressing cells (lanes 1, 2), co-expressing either the GFP-WT protein (lanes 3, 4) or the truncated GFP-ΔHCR protein (lanes 5, 6). Lane 3: Parental cells transiently expressing only full-length wild-type GFP-SHBs, which shows minimal secretion. Lane 4: HepG2-SHBs stable cells expressing full-length untagged SHBs, along with transiently expressed wild-type GFP-SHBs, supports ~50% secretion, though the protein localizes to an undefined compartment (see Supplemental Figure S3, http://links.lww.com/HC9/C332 ). Lane 5: Parental cells transiently expressing GFP-ΔHCR alone without co-expression of untagged SHBs for dimerization, secretion from these cells remains minimal. Lane 6: HepG2-SHBs stable cells expressing full-length untagged SHBs and GFP-ΔHCR show optimal secretion. (D) Densitometric quantitation of the ratios from 3 independent experiments of GFP-tagged WT and truncated ΔHCR proteins secreted into the culture medium (EXT) versus intracellular (INT) from WB. (E) Quantitation of secreted GFP constructs from HepG2 parental and HepG2-SHBs stable cells. p -value *, <0.05. p -value **, <0.01. p -value ***, <0.001. Scale bar, 10 μm. Abbreviations: AU, arbitrary units; EXT, external/secreted; GFP, green fluorescent protein; GFP-ΔHCR, GFP-tagged SHBs lacking the C-terminal hydrophobic region; GFP-WT, GFP-tagged wild-type SHBs; HepG2-SHBs, HepG2 cell line stably expressing SHBs; INT, internal; MVB, multivesicular body; SHBs, small hepatitis B surface antigen; WB, western blot.

Article Snippet: Extracellular SHBs were quantified by a HBsAg chemiluminescence immunoassay (CLIA) (Autobio Diagnostics, Zhengzhou, China, CL0310-2).

Techniques: Fluorescence, Staining, Marker, Expressing, Quantitation Assay, Construct, Stable Transfection, Western Blot

Journal: Hepatology Communications

Article Title: Secretion of the HBV small surface antigen is driven by an ER autophagic pathway

doi: 10.1097/HC9.0000000000000939

Figure Lengend Snippet: Intracellular trafficking of the SHBs probe bypasses the Golgi and matches the localization of wild-type, untagged SHBs. (A–C) Stills from time-lapse videos of the HepG2-SHBs stable cells expressing the mCh-ΔHCR SHBs probe and GFP-tagged marker proteins for (A) MVBs (CD63), (B) lysosomes (LAMP1), and (C) Golgi (GM130) for 24 hours before the initiation of image acquisition. Images were acquired every 10 minutes for over 60 hours. Images shown are at T=0 hour that indicates the start of image acquisition, and every 10 hours afterward. White arrows point to red mCh-ΔHCR protein that can be seen accumulating into large spherical organelles reminiscent of MVBs (A), or lysosomes (B), while never appearing to transit through the Golgi. (C) Large pools of mCh-ΔHCR in red represent nascent ER-residing probe that eventually transits out into autophagosomes/lysosomes. The original videos are available in the supplemental materials (Supplemental Movies 4A–C, http://links.lww.com/HC9/C332 ). Scale bar, 10 μm. Abbreviations: CD63, cluster of differentiation 63; ER, endoplasmic reticulum; GFP, green fluorescent protein; GM130, Golgi matrix protein 130; HepG2-SHBs, HepG2 cell line stably expressing SHBs; LAMP1, lysosomal-associated membrane protein 1; mCh-ΔHCR, monomeric cherry–tagged SHBs lacking the C-terminal hydrophobic region; MVB, multivesicular body; SHBs, small hepatitis B surface antigen.

Article Snippet: Extracellular SHBs were quantified by a HBsAg chemiluminescence immunoassay (CLIA) (Autobio Diagnostics, Zhengzhou, China, CL0310-2).

Techniques: Expressing, Marker, Stable Transfection, Membrane

Journal: Hepatology Communications

Article Title: Secretion of the HBV small surface antigen is driven by an ER autophagic pathway

doi: 10.1097/HC9.0000000000000939

Figure Lengend Snippet: Transit of the SHBs from the ER to late endosome/lysosome compartments is mediated by a conventional autophagic pathway as well as directly to the lysosome. (A) IF images of HepG2-SHBs cells transiently expressing the autophagic adaptor protein LC3-GFP (green), fixed and stained for SHBs (red). Arrows within enlargements point to structures showing colocalization. (B) Stills from live cell imaging of mCh-ΔHCR and LC3-GFP in HepG2-SHBs cells (Supplemental Movie 5B, http://links.lww.com/HC9/C332 ). Enlargements show LC3-GFP associating with mCh-ΔHCR structures (arrows). (C) WB analysis of culture medium (EXT) and cell lysates (INT) from HepG2-SHBs cells treated with either control (siNT) or targeted siRNA to the key autophagic protein ATG5 (siATG5) in the presence (Baf-A1) or absence (DMSO) of the lysosome inhibitor. (D–F) Densitometric quantitation of WB following siRNA treatment: (D) intracellular ATG5 levels, (E) intracellular (INT) SHBs, and (F) extracellular (EXT) SHBs. (G) CLIA quantification of extracellular SHBs. (H, I) IF staining of HepG2-SHBs cells expressing LC3-GFP and treated with (H) siRNA (siNT) or (I) siATG5; enlargements show SHBs associated (arrows) or not associated (arrowheads) with LC3-GFP. (J) Quantitation of the colocalization expressed as percentage of cells with LC3-GFP co-localizing with SHBs vesicles in cells treated with siNT or siATG5 (K, L). IF staining of HepG2-SHBs cells treated with (K) siNT or (L) siATG5; enlargements show SHBs associated with lysosomes (arrows) or not associated with lysosomes (arrowheads). (M) Quantitation of cells with colocalization between SHBs and lysosomes in cells treated with siNT control or siATG5. p -value **, <0.01. p -value ***, <0.001. p -value ****, <0.0001. Scale bar, 10 μm. Abbreviations: ATG5, autophagy-related 5; AU, arbitrary units; Baf-A1, bafilomycin-A1; CLIA, chemiluminescent immunoassay; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; EXT, external/secreted; GFP, green fluorescent protein; HepG2-SHBs, HepG2 cell line stably expressing SHBs; IF, immunofluorescence; INT, internal; LC3, microtubule-associated proteins 1A/1B light chain 3B; mCh-ΔHCR, monomeric cherry–tagged SHBs lacking the C-terminal hydrophobic region; N.S., not significant; SHBs, small hepatitis B surface antigen; siATG5, small interfering RNA targeting ATG5; siNT, non-targeting small interfering RNA; WB, western blot.

Article Snippet: Extracellular SHBs were quantified by a HBsAg chemiluminescence immunoassay (CLIA) (Autobio Diagnostics, Zhengzhou, China, CL0310-2).

Techniques: Expressing, Staining, Live Cell Imaging, Control, Quantitation Assay, Stable Transfection, Immunofluorescence, Small Interfering RNA, Western Blot

Journal: Hepatology Communications

Article Title: Secretion of the HBV small surface antigen is driven by an ER autophagic pathway

doi: 10.1097/HC9.0000000000000939

Figure Lengend Snippet: Expression of ER-phagy receptors is required for efficient secretion of the SHBs via the endosomal/autophagic pathway. (A) Representative WB of extracellular (EXT) or intracellular (INT) protein levels in HepG2-SHBs cells following treatment with control siRNA (siNT) or siRNAs targeted to reticuon-3 (RTN3), atlastin GTPase3 (ATL3), Sec62, cell-cycle progression protein 1 (CCPG1), or testis-expressed 264 (Tex264). (B) Densitometric quantitation from 3 independent WB for each of the proteins normalized to NT siRNA control. Densitometric quantitation of INT SHBs (C) and (D) EXT SHBs by WB. (E) Quantitation of SHBs by CLIA in culture medium. (F) IF staining of SHBs (green) and MVB (red) in HepG2-SHBs cells treated with control (NT) siRNA or siRNAs targeting either ATL3, Sec62, or Tex264. (G) A representative WB of culture medium (EXT) and intracellular protein (INT) from HepG2-SHBs stable cells treated with siATL3 or siNT in the presence or absence of Baf-A1. (H) Quantitation of western blots (n=3 independent replicates) for knockdown efficiency of siATL3 normalized to siNT control. Quantitation of western blots (n=3 independent replicates) for intracellular (INT) SHBs (I) and extracellular (EXT) SHBs (J). Quantitative measurement by CLIA of SHBs in culture medium (K). p -value *, <0.05; p -value **, <0.01; p -value ***, <0.001; p -value ****, <0.0001. Scale bar, 10 μm. Abbreviations: ATL3, atlastin 3; AU, arbitrary units; Baf-A1, bafilomycin-A1; CCPG1, cell-cycle progression gene 1; CLIA, chemiluminescent immunoassay; ER, endoplasmic reticulum; EXT, external/secreted; HepG2-SHBs, HepG2 cell line stably expressing SHBs; IF, immunofluorescence; INT, internal; MVB, multivesicular body; RTN3, reticulon 3; Sec62, ER protein Sec62; SHBs, small hepatitis B surface antigen; siATL3, small interfering RNA targeting ATL3; siNT, non-targeting small interfering RNA; Tex264, testis-expressed 264; WB, western blot.

Article Snippet: Extracellular SHBs were quantified by a HBsAg chemiluminescence immunoassay (CLIA) (Autobio Diagnostics, Zhengzhou, China, CL0310-2).

Techniques: Expressing, Control, Quantitation Assay, Staining, Western Blot, Knockdown, Stable Transfection, Immunofluorescence, Small Interfering RNA

Journal: Hepatology Communications

Article Title: Secretion of the HBV small surface antigen is driven by an ER autophagic pathway

doi: 10.1097/HC9.0000000000000939

Figure Lengend Snippet: Hepatocellular secretory pathways utilized by SHBs. Illustration of the 2 SHBs pathways observed in this current study. Created with BioRender.com. Abbreviations: AP, autophagic pathway; ATG5 KD, autophagy-related 5 knockdown; ATL3, atlastin 3; Baf-A1, bafilomycin-A1; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; Lys, lysosome; MVB, multivesicular body; SHBs, small hepatitis B surface antigen.

Article Snippet: Extracellular SHBs were quantified by a HBsAg chemiluminescence immunoassay (CLIA) (Autobio Diagnostics, Zhengzhou, China, CL0310-2).

Techniques: Knockdown