Journal: PLoS ONE

Article Title: Inflamed In Vitro Retina: Cytotoxic Neuroinflammation and Galectin-3 Expression

doi: 10.1371/journal.pone.0161723

Figure Lengend Snippet: Immunohistochemical staining of Gal-3 (red) expressing microglia. All microglia were detected using the microglia marker Iba1 (green). At 0 DIV no Gal-3-expressing cells were found (A, B). In controls, at 3, 4 and 7 DIV Iba1/Gal-3 co-expressing cells were found and only in the GCL (C, D, G, H, K, and L). LPS-treated retinas displayed larger numbers of Iba1/Gal-3 co-expressing cells that were located in the GCL, INL and OPL at 3, 4 and 7 DIV. Scale bar: 200 μm.

Article Snippet: Sections were then incubated with primary antibodies, rabbit anti-glial fibrillary acidic protein (GFAP, 1:2000, DAKO Cytomation, Glostrup, Denmark), rabbit anti-Iba1 (1:200, WAKO, Tokyo, Japan), rat anti-mouse ED1 (CD68, 1:1000, Nordic Biosite, Täby, Sweden), rat anti-mouse Gal-3 (1:500 WAKO, Tokyo, Japan), mouse anti-NeuN (1:100, Chemicon International, Temecula, CA, USA) or anti-goat Ki67 (1:100, Millipore, Temecula, CA, USA) at 4°C overnight, before subsequent rinsing and incubation in secondary antibodies for 2 h. Secondary antibodies used were Texas Red-conjugated donkey anti-rabbit antibody (1:200; Abcam, Cambridge, UK), Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Inc. Eugene, OR, USA) and Alexa Fluor 564 goat anti-rat (Molecular Probes, Inc. Eugene, OR, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Marker

Journal: PLoS ONE

Article Title: Inflamed In Vitro Retina: Cytotoxic Neuroinflammation and Galectin-3 Expression

doi: 10.1371/journal.pone.0161723

Figure Lengend Snippet: Quantification of fractions of cells expressing ED1 and Gal-3, respectively, of the total number of Iba1-stained cells. ANOVA analysis was performed for comparison between the LPS-treated group and the control groups. Data are expressed as mean±StDev (n = 3/group) *p<0.05, ***p<0.001.

Article Snippet: Sections were then incubated with primary antibodies, rabbit anti-glial fibrillary acidic protein (GFAP, 1:2000, DAKO Cytomation, Glostrup, Denmark), rabbit anti-Iba1 (1:200, WAKO, Tokyo, Japan), rat anti-mouse ED1 (CD68, 1:1000, Nordic Biosite, Täby, Sweden), rat anti-mouse Gal-3 (1:500 WAKO, Tokyo, Japan), mouse anti-NeuN (1:100, Chemicon International, Temecula, CA, USA) or anti-goat Ki67 (1:100, Millipore, Temecula, CA, USA) at 4°C overnight, before subsequent rinsing and incubation in secondary antibodies for 2 h. Secondary antibodies used were Texas Red-conjugated donkey anti-rabbit antibody (1:200; Abcam, Cambridge, UK), Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Inc. Eugene, OR, USA) and Alexa Fluor 564 goat anti-rat (Molecular Probes, Inc. Eugene, OR, USA).

Techniques: Expressing, Staining

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: 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

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) The C-terminus of HscA is exposed on the surface of phagosomes. ( A ) Immunostaining of phagosomes containing conidia of hscA-myc or hscA L -myc strains isolated from A549 cells. Scale bars, 2 μm. ( B ) Relative signal intensities of the respective emission fluorescence along the lines drawn across the phagosomes shown in ( A ). ( C ) Quantification of exposure distance from a representative experiment. Data represent the mean ± SD; n = 16 individual phagosomes were analyzed. ( D and E ) Biotinylation of host cell proteins by HscA-miniTurboID (HscA-mT). ( D ) A549 cells incubated with conidia of strains hscA-mT or mT - hscA were stained with streptavidin and an antibody against RAB7. ( E ) Phagosomes with positive streptavidin (Strep + ) signal were quantified. The number of independent experiments is indicated at the bottom of the bars. ( F and G ) GAL3 is recruited to damaged phagosomes containing A. fumigatus conidia in A549 cells after 8 hours of infection. ( F ) A549 cells incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. ( G ) Quantification of GAL3 + phagosomes containing conidia in A549 cells. ( H and I ) Phagosomal SYTOX is released to the nucleus upon damage of the phagosome membrane. ( H ) Representative A549 cells whose lysosomes were loaded with SYTOX-Green, were incubated with A. fumigatus for 16 h. CellMask and CFW were used to stain the cell membrane and A. fumigatus cell wall, respectively. ( I ) Cells with SYTOX signal in the nuclei were quantified. ( J – L ) HscA-dependent phagosomal damage in hMDMs. ( J ) hMDMs incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. DIC, differential interference contrast. ( K ) GAL3 + phagosomes and ( L ) RAB7 + phagosomes in hMDMs were quantified. Statistics: Error bars represent the mean ± SD. For C and E, p -values were calculated using unpaired two-tailed t test. For G, I, K, and L, p -values are calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Gray dots represent the calculated values from individual microscopic images (G, n = 42–47; I, n = 25–28; K and L, n = 46–54), and colored dots represent the summarized result of individual experiments ( n = 3).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Immunostaining, Isolation, Fluorescence, Incubation, Staining, Infection, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) Deletion of fungal HscA or human host p11 gene, or chelation of Ca 2+ , increased phagosome maturation. ( A ) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated phagosomal markers: RAB7 and ANXA2 on the top row, and GAL3 and LAMP1 on the bottom row. Yellow arrows label phagosomes positive for both markers, while white arrows mark phagosomes positive for a single marker. Scale bars, 5 μm. ( B and C ) Phagosomes positive for ( B ) RAB7 and ( C ) LAMP1 were quantified. ( D and E ) Activation of TFEB by p11 deletion or Ca 2+ chelation. ( D ) Immunostaining of A549 cells infected with WT conidia. Dashed-line circles indicate the regions of nuclei. Scale bars, 10 μm. ( E ) Cells with TFEB localized in the nuclei were quantified. Statistics: Error bars represent the mean ± SD; p values were determined using one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments for figures B and C is indicated at the base of each bar. For E, grey dots represent the calculated values from individual microscopic images ( n = 41–68) and colored dots represent summarized results from individual experiments ( n = 3 for BAPTA-AM-treated cells and n = 4 for untreated cells).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Immunostaining, Incubation, Marker, Activation Assay, Infection

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: (A) Presence of Y596/Y αD in HscA/Ssb proteins of human pathogenic fungi. The phylogenic tree was constructed using the alignment of HscA orthologs and Hsp70/Ssa proteins. Branches and names of fungal species are color-coded based on the amino acid residue present in the αD domain: green for Y and yellow for F. Branches corresponding to Hsp70/Ssa are highlighted in pink. ( B and C ) C. albicans and C. glabrata cause damages to phagosomes in hMDMs. (B) hMDMs were incubated with yeast cells of C. albicans (top row) or C. glabrata (bottom row) for 2 hours and immunostained with antibody against GAL3. Scale bars, 5 μm. (C) Phagosomes positive for GAL3 were quantified. CgΔssb1 , the mutant strain of C. glabrata lacking SSB1 . ( D and E ) Increased phagosome damage caused by S. cerevisiae expressing Ssb with an L-to-Y mutation. (D) hMDMs were incubated with yeast cells of Ssb Y protein producing S. cerevisiae ( Sc-SSB Y ) or the Ssb L protein producing S. cerevisiae ( Sc-SSB L ) for 2 hours and immunostained with antibodies against GAL3 and RAB7. Yeast cell wall was stained with CFW. Arrows indicate phagosomes positive for both markers. Regions indicated by dashed-line frames are enlarged on the right. Channel intensity plots show the fluorescence signal across the yellow lines. Scale bars, 5 μm. (E) The yeast cells containing phagosomes that are positive for GAL3 were quantified. Sc , S. cerevisiae WT strain; Sc-AD , S. cerevisiae strain transformed with control vector; Sc-AfHscA , S. cerevisiae strain producing A. fumigatus HscA; Sc-SSB F , S. cerevisiae strain producing Ssb F , Sc-AGA2 , S. cerevisiae strain producing Aga2p. Statistics: Error bars represent the mean ± SD of pooled calculated values, indicated by grey dots, from individual microscopic images ( n = 60 for C. albicans and C. glabrata ; n = 59 for CgΔssb1 ; n = 75–81 for strains of S. cerevisiae ). P- values were calculated using an unpaired two-tailed t test ( C ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( E ). Colored dots represent summarized results from individual experiments ( n = 3 for C and n = 4 for E).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Construct, Residue, Incubation, Mutagenesis, Expressing, Staining, Fluorescence, Transformation Assay, Control, Plasmid Preparation, Two Tailed Test