Journal: International Journal of Molecular Sciences

Article Title: Chemerin-156 is the Active Isoform in Human Hepatic Stellate Cells

doi: 10.3390/ijms21207555

Figure Lengend Snippet: Alpha-smooth muscle actin (α-SMA) and galectin-3 in LX-2 cells expressing chemerin isoforms. ( a – c ) LX-2 cells were transfected with an insertless plasmid (C) and plasmids to express huChem-155, 156 and 157. Cellular α-SMA was analyzed by immunoblot at 24, 48 and 72 h post-transfection. Coomassie stained membrane served as control; ( d – f ) Galectin-3 in the cells described in a–c; ( g – i ) Galectin-3 in the cell media of LX-2 cells described in a–c. N = 3; * p < 0.05, ** p < 0.01, *** p < 0.001, the number in ( h ) in brackets is the p -value.

Article Snippet: The antibodies used were obtained from the following sources: human chemerin, R&D Systems (Wiesbaden-Nordenstadt, Germany); GAPDH, New England Biolabs GmbH (Frankfurt am Main, Germany); galectin-3, BD Biosciences (Heidelberg, Germany); α-SMA and CMKLR1, Abcam (Cambridge, UK); GPR1, Bioss (Woburn, MA, USA) and Abnova (Taipei City, Taiwan).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Chemerin-156 is the Active Isoform in Human Hepatic Stellate Cells

doi: 10.3390/ijms21207555

Figure Lengend Snippet: Secretome of HSCs and LX-2 cells. ( a ) IL-6 in the supernatant of HSCs of three different donors and LX-2 cells; ( b ) Pentraxin 3 (PTX3); ( c ) IL-8; ( d ) galectin-3; and ( e ) chemerin in the cell culture media of these cells.

Article Snippet: The antibodies used were obtained from the following sources: human chemerin, R&D Systems (Wiesbaden-Nordenstadt, Germany); GAPDH, New England Biolabs GmbH (Frankfurt am Main, Germany); galectin-3, BD Biosciences (Heidelberg, Germany); α-SMA and CMKLR1, Abcam (Cambridge, UK); GPR1, Bioss (Woburn, MA, USA) and Abnova (Taipei City, Taiwan).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Chemerin-156 is the Active Isoform in Human Hepatic Stellate Cells

doi: 10.3390/ijms21207555

Figure Lengend Snippet: Effect of huChem-157 on IL-6, IL-8, pentraxin 3 (PTX3) and galectin-3 in primary human HSCs of three donors. ( a ) HSCs were incubated with increasing concentrations of recombinant huChem-157 for 24 h and IL-6; ( b ) IL-8; ( c ) PTX3 and ( d ) galectin-3 were measured by ELISA.

Article Snippet: The antibodies used were obtained from the following sources: human chemerin, R&D Systems (Wiesbaden-Nordenstadt, Germany); GAPDH, New England Biolabs GmbH (Frankfurt am Main, Germany); galectin-3, BD Biosciences (Heidelberg, Germany); α-SMA and CMKLR1, Abcam (Cambridge, UK); GPR1, Bioss (Woburn, MA, USA) and Abnova (Taipei City, Taiwan).

Techniques: Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Molecular cell

Article Title: Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

doi: 10.1016/j.molcel.2022.06.012

Figure Lengend Snippet: Figure 2. CNN2 translocates to damaged lysosomes and is ubiquitylated for timely dissociation (A) Dynamic association of endogenous CNN2 with damaged lysosomes in HeLa cells. Immunofluorescence of CNN2 and Gal3 as lysosomal damage marker after mock or LLOMe treatment for indicated time periods. Note CNN2 translocation and dissociation before Gal3 clearance. (B) Graph represents the percentage of CNN2 and Gal3-positive vesicles among all Gal3-positive vesicles per cell. More than 30 cells were quantified per condition in each experiment (n = 4 biologically independent experiments). One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test, ** p = 0.0036, *** p = 0.0002, and **** p < 0.0001. Error bars represent the mean ± SEM. (C) Schematic domain structure of CNN2 with positions of identified ubiquitylation sites indicated. CH domain, calponin homology domain; ABS1/2, actin- binding sites. (D) HeLa cells expressing CNN2-GFP wild type or harboring lysine-to-arginine substitutions in the CH domain (CH-KR) following mock or LLOMe treatment as indicated. Note that CNN2 wild type dissociates from LAMP1 vesicles within 3 h, but the ubiquitylation mutants persist. See Figure S3A for CNN2-GFP 5xKR covering the 5 ubiquitylation sites detected by MS. (E) Quantification of (D). Percentage of LAMP1 vesicles positive for CNN2. More than 20 cells were quantified. One-way ANOVA with Tukey’s multiple comparison test, *** p = 0.0003 and **** p < 0.0001; ns, not significant. Error bars represent the mean ± SD. (F) Live-cell imaging of HeLa cells expressing CNN2-GFP CH-KR and mCherry-Gal3. Lysosomes were loaded with photosensitizer AIPcS2a, irradiated in the indicated area, and chased over the course of 1 h. See Figure S3E for wild type and CNN2 5xKR imaging data. (A, D, and F) Scale bars, 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat monoclonal anti-Gal3 (IF, 1:500) Santa Cruz Biotechnology Cat#sc-23938; RRID:AB_627658 Rabbit monoclonal anti-LAMP1 (IF, 1:500) Cell Signaling Technology Cat#9091; RRID:AB_2687579 Rabbit polyclonal anti-CNN2 (IF, 1:500; WB, 1:1000) Thermo Fischer Scientific Cat#PA5-61878; RRID:AB_2639249 Mouse monoclonal anti-CNN2 (WB, 1:1000) Origene Cat#TA503688; RRID:AB_11124845 Rabbit polyclonal anti-LC3 (IF, 1:500; WB, 1:1000) MBL Cat#PM036; RRID:AB_2274121 Rabbit polyclonal anti-p62 (IF, 1:500; WB, 1:1000) Sigma-Aldrich Cat#P0067; RRID:AB_1841064 Mouse monoclonal anti-p62 (WB, 1:1000) Abnova Cat#A00008878 Mouse monoclonal anti-HSP27 (IF, 1:500) Thermo Fischer Scientific Cat#MA3-015; RRID:AB_325463 Rabbit polyclonal anti-HSP27 (WB, 1:1000) Thermo Fischer Scientific Cat#PA5-78010; RRID:AB_2735924 Rabbit polyclonal anti-TAX1BP1 (WB, 1:1000) Sigma Cat#HPA024432; RRID:AB_1857783 Mouse monoclonal anti-p97 (WB, 1:2000) Santa Cruz Biotechnology Cat#sc-57492; RRID:AB_793927 Mouse monoclonal anti-P4D1 (WB, 1:1000) Cell Signaling Technology Cat#3936; RRID:AB_331292 Mouse monoclonal anti-Tubulin (WB, 1:5000) Sigma-Aldrich Cat#T-5168; RRID:AB_477579 Mouse monoclonal anti-GFP (WB, 1:10000) Roche Cat#11814460001; RRID:AB_390913 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (WB, 1:10000) Biorad Cat#170-6515; RRID:AB_11125142 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (WB, 1:10000) Biorad Cat#1706516; RRID:AB_11125547 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 568 (IF, 1:500) Invitrogen Cat#A11011; RRID:AB_143157 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 488 (IF, 1:500) Life technologies Cat#A11034; RRID:AB_2576217 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#A21071; RRID:AB_2535732 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#10696113 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 594 (IF, 1:500) Thermo Fisher Scientific Cat#A-11032; RRID:AB_2534091 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#10246252 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 568 (IF, 1:500) Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121 (Continued on next page) e1 Molecular Cell 82, 2633–2649.e1–e7, July 21, 2022

Techniques: Marker, Translocation Assay, Comparison, Binding Assay, Expressing, Live Cell Imaging, Irradiation, Imaging

Journal: Molecular cell

Article Title: Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

doi: 10.1016/j.molcel.2022.06.012

Figure Lengend Snippet: Figure 7. p97 and HSPB1 cooperate in removing ubiquitylated CNN2 from lysosomes (A) HSPB1 is trapped on CNN2 in the absence of p97 activity. Proximity biotinylation analyzed by western blot in indicated conditions of lysosome damage (LLOMe) and p97 inhibition (NMS-873). Cells expressing CNN2-APEX2 were treated with LLOMe for 1 h and chased for 2 h after washout prior to the addition of H2O2 to trigger biotinylation. (B) Loss of p97 or HSPB1 function impairs CNN2 dissociation in a non-additive manner. The time course of CNN2 localization to damaged lysosomes upon p97 inhibition (NMS-873), HSPB1 depletion, or a combination of both as indicated. Scale bars, 10 mm. (C) Quantification of (B). The graph represents the percentage of CNN2 and Gal3-positive vesicles among all Gal3-positive vesicles per cell. More than 30 cells were quantified per condition in each experiment (n = 3 biologically independent experiments). Two-way ANOVA with Tukey’s multiple comparison test, **** p < 0.0001, *** p = 0.0004, * p = 0.0118. Error bars represent the mean ± SEM. (D) HSPB1 acts downstream of ubiquitylation together with p97. HeLa cells stably expressing CNN2-GFP were LLOMe treated for 1 h and chased for 2 h prior to denaturing lysis upon indicated treatments after p97 inhibition (NMS-873), HSPB1 siRNA, or a combination of both as indicated. Ubiquitylation of CNN2 was assessed by western blot after immunoprecipitation using GFP nanobodies. Note that loss of HSPB1, or p97 inhibition, leads to the increased accumulation of ubiquitylated CNN2 after LLOMe-induced damage, but that effects are not additive. (E) Model. After lysosome damage, various resident proteins become ubiquitylated to serve as an anchor point for autophagy-receptor-mediated recruitment of the LC3-decorated phagophore. CNN2 is recruited by associating with the p62 autophagy receptor and then stabilizes actin filaments that assist phagophore formation. CNN2 needs to be subsequently ubiquitylated and removed by p97 with the help of HSPB1 to allow efficient phagophore formation.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat monoclonal anti-Gal3 (IF, 1:500) Santa Cruz Biotechnology Cat#sc-23938; RRID:AB_627658 Rabbit monoclonal anti-LAMP1 (IF, 1:500) Cell Signaling Technology Cat#9091; RRID:AB_2687579 Rabbit polyclonal anti-CNN2 (IF, 1:500; WB, 1:1000) Thermo Fischer Scientific Cat#PA5-61878; RRID:AB_2639249 Mouse monoclonal anti-CNN2 (WB, 1:1000) Origene Cat#TA503688; RRID:AB_11124845 Rabbit polyclonal anti-LC3 (IF, 1:500; WB, 1:1000) MBL Cat#PM036; RRID:AB_2274121 Rabbit polyclonal anti-p62 (IF, 1:500; WB, 1:1000) Sigma-Aldrich Cat#P0067; RRID:AB_1841064 Mouse monoclonal anti-p62 (WB, 1:1000) Abnova Cat#A00008878 Mouse monoclonal anti-HSP27 (IF, 1:500) Thermo Fischer Scientific Cat#MA3-015; RRID:AB_325463 Rabbit polyclonal anti-HSP27 (WB, 1:1000) Thermo Fischer Scientific Cat#PA5-78010; RRID:AB_2735924 Rabbit polyclonal anti-TAX1BP1 (WB, 1:1000) Sigma Cat#HPA024432; RRID:AB_1857783 Mouse monoclonal anti-p97 (WB, 1:2000) Santa Cruz Biotechnology Cat#sc-57492; RRID:AB_793927 Mouse monoclonal anti-P4D1 (WB, 1:1000) Cell Signaling Technology Cat#3936; RRID:AB_331292 Mouse monoclonal anti-Tubulin (WB, 1:5000) Sigma-Aldrich Cat#T-5168; RRID:AB_477579 Mouse monoclonal anti-GFP (WB, 1:10000) Roche Cat#11814460001; RRID:AB_390913 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (WB, 1:10000) Biorad Cat#170-6515; RRID:AB_11125142 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (WB, 1:10000) Biorad Cat#1706516; RRID:AB_11125547 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 568 (IF, 1:500) Invitrogen Cat#A11011; RRID:AB_143157 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 488 (IF, 1:500) Life technologies Cat#A11034; RRID:AB_2576217 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#A21071; RRID:AB_2535732 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#10696113 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 594 (IF, 1:500) Thermo Fisher Scientific Cat#A-11032; RRID:AB_2534091 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#10246252 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 568 (IF, 1:500) Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121 (Continued on next page) e1 Molecular Cell 82, 2633–2649.e1–e7, July 21, 2022

Techniques: Activity Assay, Western Blot, Inhibition, Expressing, Comparison, Stable Transfection, Lysis, Immunoprecipitation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: (A) The expression of MUC16 protein in HCECs separately transduced with two lentiviral-based shRNA vectors (shMUC16-1 or shMUC16-2) were examined through a Western blot analysis. The a-tubulin was used as a loading control. (B) Representative flow cytometric analyses of fluorescence intensity and MUC16 expressions in transduced HCECs followed by fluorescein staining were shown in contour plots. (C) Representative flow cytometric analyses of fluorescence intensity and MUC16 expression in HCECs cultured in KSFM ( left ) or MPM ( right ) followed by fluorescein staining were presented in contour plots. (D) The frozen tissue sections prepared from fluorescein-stained corneal epithelia were stained with GAL3 ( red , immunofluorescence), and the cell nuclei were counterstained with DAPI ( blue , immunofluorescence).

Article Snippet: To detect the expression of GAL3, cell suspensions were fixed and incubated with anti-galectin-3 (GAL3) mouse antibody (no. A3A12; Novus Biologicals).

Techniques: Expressing, Transduction, shRNA, Western Blot, Control, Fluorescence, Staining, Cell Culture, Immunofluorescence