Journal: The Journal of biological chemistry

Article Title: ATXN3 functions as a tumor suppresser through potentiating Galectin-9-mediated apoptosis in human colon adenocarcinoma.

doi: 10.1016/j.jbc.2024.107415

Figure Lengend Snippet: Figure 5. Targeted ATXN3 deletion inhibits colon cancer growth in mice. A–C, WT or ATXN3-KO HCT116 cells were injected subcutaneously into RAG1 mutant mice (n = 10). Tumor growth curve (A), photograph (B), and weight (C) are shown. D and E, the protein expression of Galectin-9, Ki67, Cleaved Caspase-3 in (B) tumor was detected by IHC and measured relative expression by image J software(n = 5). Scale bar: 200 mm. F–H, HCT116 cells were injected subcutaneously into RAG1 mutant mice and treated with Galectin-9 recombinant protein (n = 10). Tumor growth curve (F), photograph (G), and weight (H) are shown. I and J, the protein expression of Galectin-9, Ki67, Cleaved Caspase-3 in (G) tumor was detected by IHC and measured relative expression by image J software(n = 4). Scale bar: 200 mm. K–M, tumor growth curve (K) of RAG1 mutant mice injected subcutaneously with WT or ATXN3-KO HCT116 cells and stabilize overexpression of Galectin-9 by lentivirus (n = 20). Tumor photograph (L) and tumor weight (M) are shown. A, C, F, H, E, and J, 2- tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001. L and M: ordinary 1-way ANOVA. *p < 0.05, **p < 0.01,***p < 0.001.

Article Snippet: In order to achieve in vivo overexpression of the recombinant protein Galectin-9, mice were administered intraperitoneal injections of Galectin-9 recombinant protein (R&D Systems) every 2 days, with an average dosage of 1 mg per injection.

Techniques: Injection, Mutagenesis, Expressing, Software, Recombinant, Over Expression

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: Galectin-9 inhibits mycobacterial growth directly . A. Profile of Mtb H37Rv (Rv) grown at 37°C in Middlebrook 7H9 liquid medium with different concentration of Galectin-9 (Gal9, 0, 0.01, 0.1, 1, 10 μg/mL). Growth curve was measured using a Bioscreen Growth Curve Instrument. Optical density was measured at absorbance at 600 nm every 2 h. B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with10μg/mL galectin-9 (Gal9) or inactivated galectin-9 (Gal9 HK, heat killed at 95 [for 5 min). C. CFU of Mtb H37Rv (Rv) on Middlebrook 7H10 solid medium with or without 10μg/mL Galectin-9 (Gal9). Cultures were grown at 37°C for 4-8 weeks. D. Growth profile of Mycobacterium smegmatis (MS) in Middlebrook 7H9 liquid medium with different concentrations of Galectin-9 (Gal9, 0, 0.01, 0.1, 1 μg/mL). E. Concentrations of galectin-9 in sera of healthy donors (n = 40) and active TB patients (n = 40). F. Confocal microscopy of M. bovis BCG-DsRed (BCG-DsRed, red) and Galectin[9 (Anti-Gal9, green) in THP-1 cells. Nuclei was stained with DAPI (blue). G. Percent of cells with galectin-9 positive (gal9 + ) BCG in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. H. Confocal microscopy of Mtb H37Rv-GFP (Rv-GFP, green) and Galectin[9 (Anti-Gal9, red) in THP-1 cells. Nuclei were stained with DAPI (blue). I. Percent of cells with galectin9 positive (gal9 + ) Mtb H37Rv in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Data are representative of three independent experiments with similar results (F and H). Two-tailed unpaired Student’s t test (A-D, G, and I) or Mann-Whitney U test (E). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Concentration Assay, Confocal Microscopy, Staining, Infection, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and lactose (1 μg/mL). B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and D-glucose (10 μg/mL). C. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and AG (1 μg/mL). D. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with 1μg/mL CRD1 or CRD2 of galectin-9. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Two-tailed unpaired Student’s t test (A-D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Morphologic characteristics for Mtb H37Rv strain grown in liquid culture with or without anti-AG mAbs (1 μg/mL) observed by ×[2 magnifier. B. Bacterial shape of Mtb H37Rv strain treated as in (A) observed by acid fast staining under a Leica DM2500 microscope using the 100× oil microscopy. EMB, Ethambutol. Scale bar, 20μm. C. Ultrastructural morphology of Mtb H37Rv treated as in (A) analyzed by transmission electron microscopy (TEM). The cell wall was labeled with red arrows. D. Cell wall thickness of bacteria in (C). E. Schematic presentation of Mtb growth arrest by Galectin-9 or anti-AG antibodies. Data are representative of three independent experiments with similar results (A, B and C). Data are means ± SD of 11 bacteria, representatives of three independent experiments (D). Two-tailed unpaired Student’s t test (D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Staining, Microscopy, Transmission Assay, Electron Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

doi: 10.1038/s41467-022-28171-5

Figure Lengend Snippet: a GAL9 decreases the level of HBc protein. Immunoblotting (upper) and RT-PCR assays (lower) of HepG2 cells expressing HA-HBc and GFP-GAL9. b Bafilomycin A1 inhibits GAL9-induced HBc degradation. HepG2 cells were transfected with vectors encoding HA-HBc and GFP-GAL9. Cells were treated with bafilomycin A1 (BafA1, 100 µM) or MG132 (10 µM) 16 h prior to harvesting. c ATG5 depletion attenuates GAL9-induced HBc degradation. HepG2 transduced with control- (Ctrl) or ATG5-targeting siRNA were transfected with vectors encoding HA-HBc and GFP-GAL9. d GAL9 and HBc accumulation in cytoplasmic bodies. Confocal microscopic imaging of HepG2 cells expressing HA-HBc and HaloTag empty vector (upper) or HaloTag-GAL9 (lower). Nuclei were stained with DAPI. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . e GAL9 and HBc accumulation in intracellular membranes. Confocal micrographs show HepG2 cells expressing HA-HBc and HaloTag-GAL9. Intracellular membranes were stained with CellMask reagent. Expanded views are also shown. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . f GAL9 and HBc co-localize with LC3. Confocal micrographs show HepG2 cells expressing HT-GAL9, HA-HBc, and GFP-LC3. Expanded views are also shown. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . g IFN-β increases colocalization of HBc and LC3 in a GAL9-dependent manner. Confocal micrographs show HepG2 cells expressing HA-HBc in the presence or absence of IFN-β. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . The graph on the right shows the percentage of cells in which HBc localized to LC3-positive puncta ( n = 50 cells examined over three experiments, mean ± SD). ** P = 0.0027, **** P < 0.0001, two-tailed unpaired t -test. Scale bar, 10 µm. Bar charts in a – c indicate the ratio of HBc over Vinculin, as determined by densitometry, and are presented as a mean ± SD ( n = 3 experiments). **** P < 0.0001 ( a , b ), ** P = 0.0011 ( c ), two-tailed unpaired t -test. Immunoblots and micrographs are representative of experiments with similar results ( n ≥ 2). Source data are provided as a Source Data file.

Article Snippet: Alternatively, PHHs were transduced with lentiviral particles carrying gene-specific shRNA (Santa Cruz, #sc-35444-V for GAL9, #sc-78079-V for RNF13, #sc-29679-V for p62, #sc-41445-V for ATG5).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Transduction, Control, Imaging, Plasmid Preparation, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

doi: 10.1038/s41467-022-28171-5

Figure Lengend Snippet: a Viperin interacts with HBc and GAL9. NanoBRET analysis was performed on HepG2 cells expressing indicated HT proteins and NL-conjugated HBc ( n = 3 experiments, mean ± SD). **** P < 0.0001, two-tailed unpaired t -test. b GAL9 can bind HBc in the presence of viperin. Recombinant HBc and GAL9 were incubated with or without viperin and subjected to in vitro pull-down assay using anti-HBc antibody. c Viperin colocalization with GAL9 and HBc. Confocal microscopic imaging of HepG2 cells expressing GFP-GAL9, HT-viperin and HA-HBc. Nuclei were stained with DAPI. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . d , e Viperin depletion decreases the interaction between HBc and GAL9. HepG2 transduced with control- (Ctrl) or viperin-targeting siRNA were transfected with vectors encoding HA-HBc and GFP-GAL9. Cell lysates were precipitated with anti-HA antibody, followed by immunoblotting ( d ). Viperin knockdown was confirmed by RT-PCR ( e ). The mean ± SD of two independent determinations is plotted. ** P = 0.0059, two-tailed unpaired t -test. f GAL9 and HBc bind to different domains of viperin. Immunoprecipitation assays of HepG2 cells expressing truncated viperin mutants and HA-HBc or GFP-GAL9. Cell lysates were precipitated with anti-HA or anti-GFP antibodies, followed by immunoblotting. g Viperin depletion attenuates HBc localization on GAL9-positive puncta. HepG2 transduced with control- (Ctrl) or viperin-targeting siRNA were transfected with vectors encoding HA-HBc and GFP-GAL9. Nuclei were stained with DAPI. The graph on the right is the percentage of HBc-puncta-forming cells ( n = 50 cells examined over two independent experiments, mean ± SD). * P = 0.0313, two-tailed unpaired t -test. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . h HepG2.15.7 cells transduced with control- (Ctrl) or viperin-targeting siRNA were treated with IFN-β (1000 U/mL) for 24 h. Cells were stained with anti-GAL9 and anti-HBc antibodies ( n = 50 cells examined over two independent experiments, mean ± SD). ** P = 0.0063, two-tailed unpaired t -test. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . Immunoblots and micrographs are representative of experiments with similar results ( n ≥ 2). Source data are provided as a Source Data file.

Article Snippet: Alternatively, PHHs were transduced with lentiviral particles carrying gene-specific shRNA (Santa Cruz, #sc-35444-V for GAL9, #sc-78079-V for RNF13, #sc-29679-V for p62, #sc-41445-V for ATG5).

Techniques: Expressing, Two Tailed Test, Recombinant, Incubation, In Vitro, Pull Down Assay, Imaging, Staining, Transduction, Control, Transfection, Western Blot, Knockdown, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal: Nature Communications

Article Title: Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

doi: 10.1038/s41467-022-28171-5

Figure Lengend Snippet: a GAL9 interacts with p62. Immunoprecipitation assay was performed on HepG2 cells expressing FLAG-GAL9 and indicated HT-fused proteins. Cell lysates were precipitated with anti-FLAG antibody, followed by immunoblotting. b p62 is required for GAL9 activity. HepG2 cells were transduced with control- (Ctrl) or p62-targeting siRNA, and then transfected with vectors expressing HA-HBc and GFP-GAL9. Cells were subjected to immunoblotting analysis to detect the indicated proteins. c GAL9 promotes colocalization of p62 and HBc. Micrographs show HepG2 cells expressing FLAG-HBc (blue) with or without GFP-GAL9 (green). Endogenous p62 was stained in red. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . d , e The ubiquitin-binding domain (UBA) and LC3-interacting region (LIR) of p62 are required for GAL9-mediated HBc degradation. HepG2 cells were transduced with p62-targeting siRNA, and then transfected with vectors expressing HA-HBc, GFP-GAL9, and siRNA-resistant Myc-p62 (WT, ∆UBA, or ∆LIR). Cells were subjected to immunoblotting analysis to detect the indicated proteins. Bar charts in b , d , e indicate the ratio of HBc over Vinculin, as determined by densitometry, and are presented as a mean ± SD ( n = 3 experiments). **** P < 0.0001, two-tailed unpaired t -test. Immunoblots and micrographs are representative of experiments with similar results ( n ≥ 2). Source data are provided as a Source Data file.

Article Snippet: Alternatively, PHHs were transduced with lentiviral particles carrying gene-specific shRNA (Santa Cruz, #sc-35444-V for GAL9, #sc-78079-V for RNF13, #sc-29679-V for p62, #sc-41445-V for ATG5).

Techniques: Immunoprecipitation, Expressing, Western Blot, Activity Assay, Transduction, Control, Transfection, Staining, Ubiquitin Proteomics, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

doi: 10.1038/s41467-022-28171-5

Figure Lengend Snippet: a GAL9 induces HBc-complex ubiquitination. HepG2 cells expressing HA-HBc, GFP-GAL9, and Myc-Ubiquitin (Ub) were pulled down with anti-HA antibody, and the pulldowns were analyzed by immunoblotting. b GAL9 interacts with RNF13. Immunoprecipitation assay was performed on HepG2 cells expressing FLAG-GAL9 and HT-RNF13. Cell lysates were precipitated with anti-FLAG antibody, followed by immunoblotting. c Confocal microscopic imaging of HepG2 cells expressing GFP-GAL9 and HT-RNF13. Nuclei were stained with DAPI. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. . d Glycosylation of RNF13 is required for GAL9 interaction. HepG2 cells expressing HA-RNF13 were immunoprecipitated with anti-HA. The immunoprecipitates were incubated with recombinant GAL9 in the presence or absence of Protein Deglycosylation Mix II (5 units/µL) and then pulled down with GAL9 antibody for immunoblotting. e GAL9 CRD binds to RNF13. Immunoprecipitation assays of HepG2 cells expressing GAL9 CRD-deleted mutant and HT-RNF13. Cell lysates were precipitated with anti-GFP antibody, followed by immunoblotting. f RNF13 is a cofactor for ubiquitination of HBc complex. HepG2 cells expressing HA-HBc, GFP-GAL9, and Myc-Ub. Cells were transduced with siRNA targeting RNF13 at 24 h prior to DNA transfection. Cells were then immunoprecipitated with anti-HA antibody and subjected to immunoblotting. g Ubiquitination of RNF13 is increased by GAL9. HepG2 cells expressing HA-RNF13 were immunoprecipitated with anti-HA. The immunoprecipitates were incubated with indicated recombinant proteins in the presence or absence of GAL9 for 1 h. Samples were then electrophoresed and the gel was subsequently processed for CBB staining or immunoblotting with anti-Ub and UBE1 antibodies. h CRD-deleted GAL9 does not enhance the auto-ubiquitination of RNF13 and p62 recruitment. HepG2 cells expressing indicated proteins were precipitated with anti-HT antibody, followed by immunoblotting. Immunoblots and micrographs are representative of experiments with similar results ( n ≥ 2). Source data are provided as a Source Data file.

Article Snippet: Alternatively, PHHs were transduced with lentiviral particles carrying gene-specific shRNA (Santa Cruz, #sc-35444-V for GAL9, #sc-78079-V for RNF13, #sc-29679-V for p62, #sc-41445-V for ATG5).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation, Imaging, Staining, Glycoproteomics, Incubation, Recombinant, Mutagenesis, Transduction, Transfection

Journal: Nature Communications

Article Title: Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

doi: 10.1038/s41467-022-28171-5

Figure Lengend Snippet: a Induced GAL9 degrades intracellular HBc. HepG2-tet-GAL9 cells were transfected with pHBV genotype A or C, and then were treated with doxycycline (0.5 or 1 µg/mL) to induce GAL9. Cells were subjected to immunoblotting analysis to detect the indicated proteins. Bar charts indicate the ratio of HBc or HBs over Vinculin, as determined by densitometry, are presented as a mean ± SD ( n = 3 experiments). **** P < 0.0001, two-tailed unpaired t -test. b Induced GAL9 inhibits HBV particle release. HepG2-tet-GAL9 cells were transfected with pHBV (genotype C). Four hours after transfection, the cells were washed three times and treated with doxycycline. Three days after transfection, supernatants were harvested, followed by the detection of HBcAg and viral DNA. Bar charts are presented as a mean ± SD ( n = 3 experiments). *** P = 0.0008, **** P < 0.0001, two-tailed unpaired t -test. c – f GAL9 inhibits HBV replication. The experimental design is presented in c . Primary human hepatocytes (PHHs) were transduced with lentiviral vectors carrying a GFP empty vector or GFP-GAL9 2 days before HBV infection. Eleven days after infection, supernatants were subjected to HBcAg ELISA ( d ) and qPCR ( e ) to detect released infectious HBV. The cells were subjected to qPCR to detect the cccDNA ( f ). Bar charts are presented as a mean ± SD ( n = 3 experiments). *** P = 0.0001 ( d , e ), *** P = 0.0002 ( f ), two-tailed unpaired t -test. g – i GAL9 depletion attenuates IFN antiviral activity. The experimental design is shown in ( g ). PHHs transduced with control (Ctrl) or GAL9-targeting shRNA were infected with HBV in the presence or absence of IFN-β (100 U/mL). Eleven days after infection, the cells were subjected to qPCR to detect GAL9 expression ( h ). The supernatants were subjected to HBcAg ELISA to detect the released HBV ( i ). Bar charts are presented as a mean ± SD ( n = 3 experiments). * P = 0.0192 ( h ), * P = 0.035 ( i ), two-tailed unpaired t -test. Immunoblots are representative of experiments with similar results ( n ≥ 2). Source data are provided as a Source Data file.

Article Snippet: Alternatively, PHHs were transduced with lentiviral particles carrying gene-specific shRNA (Santa Cruz, #sc-35444-V for GAL9, #sc-78079-V for RNF13, #sc-29679-V for p62, #sc-41445-V for ATG5).

Techniques: Transfection, Western Blot, Two Tailed Test, Transduction, Plasmid Preparation, Infection, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, shRNA, Expressing

Journal: Nature Communications

Article Title: Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

doi: 10.1038/s41467-022-28171-5

Figure Lengend Snippet: a Molecular mechanism of selective autophagy of HBc by GAL9. Type I IFN induces expression of GAL9 and viperin, which bind to RNF13 and HBc, respectively. Viperin binds to HBc and GAL9, leading to the HBc-puncta formation. Auto-ubiquitination of RNF13 is enhanced by GAL9, and autophagosome formation occurs when p62 is recruited. b Detailed binding mechanism. Viperin binds to GAL9 and HBc in the SAM and C-terminal domains, respectively. The CRD domain of GAL9 is important for binding to RNF13 carbohydrate chains. Ubiquitination of RNF13 is recognized by the UBA domain of p62.

Article Snippet: Alternatively, PHHs were transduced with lentiviral particles carrying gene-specific shRNA (Santa Cruz, #sc-35444-V for GAL9, #sc-78079-V for RNF13, #sc-29679-V for p62, #sc-41445-V for ATG5).

Techniques: Expressing, Ubiquitin Proteomics, Binding Assay