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

Journal: Upsala Journal of Medical Sciences

Article Title: Surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men

doi: 10.1080/03009734.2019.1592266

Figure Lengend Snippet: Surface glycosylation of seminal prostasomes: lectin- and immune-transmission electron microscopy. A: Lectin-TEM using SNA. Inserts show enlarged characteristic pattern of SNA-reactivity to each sample group. B: Lectin-TEM using ConA. Inserts show enlarged characteristic pattern of ConA-reactivity to vesicles in each sample group. In O, staining of some proteinaceous material was also observed (arrowheads). C: Immune-TEM using anti-galectin-3 antibodies. Inserts show enlarged characteristic pattern of anti-gal-3-reactivity to each sample group. Micrographs show most characteristic patterns obtained. Although differences in the reactivity of particular vesicles could be noticed, it does not affect the general reactivity of the sample (as in IEC when taking all vesicles into account). (N = seminal prostasomes from normozoospermic men; O = seminal prostasomes from oligozoospermic men).

Article Snippet: Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK) and biotinylated goat anti-galectin-3 (gal-3) antibodies from R&D Systems (Minneapolis, MN, USA); 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), D-lactose, and methyl- alpha D-mannopyranoside were from Sigma (St. Louis, MO, USA).

Techniques: Transmission Assay, Electron Microscopy, Staining

Journal: The EMBO Journal

Article Title: Microglial colonization of the developing mouse brain is controlled by both microglial and neural CSF-1

doi: 10.1038/s44318-025-00625-8

Figure Lengend Snippet: ( A ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP, MAC2 and OPN at the CSA ( n = 3). Close-ups are 75 µm wide. ( B ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP and GPNMB at the CSA ( n = 4). ( C ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ pups showing co-expression of GFP, MAC2 and OPN at the EDWM ( n = 3). ( D ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ pups showing co-expression of GFP and GPNMB at the EDWM ( n = 3). ( E ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP and EdU (open arrowheads) at the CSA. ( F ) Percentage of OPN-positive ameboid microglia at the CSA from E14.5 embryos ( n = 13; from at least two distinct litters). ( G ) Percentage of EdU-positive microglia at the CSA at E14.5 amongst OPN-positive or OPN-negative ameboid microglia ( n = 9; from one litter) ( P = 0.0197). ( H ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ embryos showing co-expression of GFP and EdU (open arrowheads) at the EDWM. ( I ) Percentage of OPN-positive ameboid microglia at the EDWM from P7 pups ( n = 13; from at least two distinct litters). ( J ) Percentage of EdU-positive ameboid microglia at the EDWM at P7 amongst OPN-positive or OPN-negative ameboid microglia ( n = 8; from two distinct litters) ( P = 0.0272). Data were presented as mean ± SEM. Two-sided unpaired Mann–Whitney test were performed to assess differences ( G , J ). * P < 0.05. Scale bars: 100 µm ( A – D , H ); 50 µm ( E ). Close-ups are 100 µm ( A – D ) and 75 µm ( E , H ) wide. CSA cortico-striato-amygdalar boundary, EDWM early-dorsal white matter, H Hoechst. See Figs. and . .

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated in the same blocking solution with the following primary antibodies overnight at 4 °C: rat anti-CD206 (1/200; Bio-Rad Cat# MCA2235, RRID:AB_324622), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID:AB_10000240), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504), chicken anti-IBA1 (1/400; Synaptic Systems Cat# 234 009, RRID:AB_2891282), rabbit anti-KI67 (1/200; Abcam Cat# ab15580, RRID:AB_443209), rat anti-Lyve1 (ALY7) (1/200; Thermo Fisher Scientific Cat# 14-0443-82, RRID:AB_1633414), rat anti-GAL3 (MAC2) (1/1000; Cedarlane Cat# CL8942AP; RRID:AB_10060357), goat anti-OPN (1/400; R and D Systems Cat# AF808, RRID:AB_2194992), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330; RRID:AB_2112934), rabbit anti-P2RY12 (1/500; AnaSpec; EGT Group Cat# 55043 A, RRID:AB_2298886), and anti-VGlut2 (1/2000; Millipore Cat# AB2251-I, RRID:AB_2665454).

Techniques: Immunolabeling, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Microglial colonization of the developing mouse brain is controlled by both microglial and neural CSF-1

doi: 10.1038/s44318-025-00625-8

Figure Lengend Snippet: ( A ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP, MAC2, and OPN at the CSA in E14.5 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at E12.5) ( n control = 5; n mutant = 4). ( B ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP and GPNMB at the CSA in E14.5 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at E12.5). ( C ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP, MAC2 and OPN at the CSA in P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at P3) ( n control = 3; n mutant = 3). ( D ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP and GPNMB at the CSA in P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at P3). ( E ) Immunolabeling of coronal brain sections from control and Cx3cr1 creER/+ ;Csf1 fl/fl mice showing absence of expression of OPN in cortical microglia (IBA1-positive) at P7 (tamoxifen administered at P3) while some white matter microglia do express OPN (solid arrowheads). Close-ups are 200 µm wide. ( F ) Proportion of proliferative microglia in control and Cx3cr1 creER/+ ;Csf1 fl/fl neocortex of P7 mice (tamoxifen administered at P3)( n control = 3 (2 F;1 M) ; n mutant = 4 (2 F;2 M); from one litter). ( G ) Microglial density (IBA1-positive cells/mm²) in control and Cx3cr1 creER/+ ;Csf1 fl/fl neocortex of P7 mice (tamoxifen administered at P3)( n control = 3 (2 F;1 M) ; n mutant = 4 (2 F;2 M); from one litter). ( H ) RNAscope experiments on coronal brain sections from P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl pups showing Csf1 expression in IBA1-positive ameboid microglia at the CSA (solid arrowheads) after tamoxifen administration at P1 and P3. ( I ) Normalized quantification of RNAscope experiments on coronal brain sections from P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos showing decreased Csf1 expression in mutant mice. Left column shows all quantified cells and right column the mean expression per animal ( n control = 3 (1 F,2 M); n mutant = 3 (2 M,1 F); from two distinct litters). ( J ) Immunolabeling of coronal brain sections from control and Cx3cr1 creER/+ ;Csf1 fl/fl mice showing co-expression of IBA1 and OPN in ameboid microglia within the EDWM accumulation at P7 (tamoxifen administered at P1 and P3) ( n control = 3 (1 F,1 M) ; n mutant = 3 (2 F;1 M); from one litter)(all cells, left: P < 0.0001; mean per animal, right: P = 0.10). ( K ) Microglial density (IBA1-positive cells/mm²) in control and Cx3cr1 creER/+ ;Csf1 fl/fl EDWM of P7 mice (tamoxifen administered at P1 and P3) ( n control = 9 (3 F;6 M); n mutant = 6 (3 F;3 M); from three distinct litters) ( P = 0.0028). ( L ) Number of OPN-positive microglia at the EDWM at P7 ( n control = 9 (3 F;6 M); n mutant = 6 (3 F;3 M); from three distinct litters) ( P = 0.0016). Data were presented as mean ± SEM. Two-sided unpaired Mann–Whitney test were performed to assess differences ( F , G , J – L ). ns not significant, ** P < 0.01, *** P < 0.001. Scale bars: 100 µm ( A – D , H , I ); 200 µm ( E ). CSA cortico-striatal-amygdalar boundary, EDWM early-dorsal white matter, H Hoechst, Ncx neocortex.

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated in the same blocking solution with the following primary antibodies overnight at 4 °C: rat anti-CD206 (1/200; Bio-Rad Cat# MCA2235, RRID:AB_324622), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID:AB_10000240), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504), chicken anti-IBA1 (1/400; Synaptic Systems Cat# 234 009, RRID:AB_2891282), rabbit anti-KI67 (1/200; Abcam Cat# ab15580, RRID:AB_443209), rat anti-Lyve1 (ALY7) (1/200; Thermo Fisher Scientific Cat# 14-0443-82, RRID:AB_1633414), rat anti-GAL3 (MAC2) (1/1000; Cedarlane Cat# CL8942AP; RRID:AB_10060357), goat anti-OPN (1/400; R and D Systems Cat# AF808, RRID:AB_2194992), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330; RRID:AB_2112934), rabbit anti-P2RY12 (1/500; AnaSpec; EGT Group Cat# 55043 A, RRID:AB_2298886), and anti-VGlut2 (1/2000; Millipore Cat# AB2251-I, RRID:AB_2665454).

Techniques: Immunolabeling, Expressing, Control, Mutagenesis, RNAscope, MANN-WHITNEY