mouse anti gal3  (Santa Cruz Biotechnology)

Journal: Neuroprotection

Article Title: Environmental enrichment modulates chronic poststroke inflammation and links white matter TREM2‐positive microglia in recovery in mice

doi: 10.1002/nep3.70028

Figure Lengend Snippet: Infarct size correlations with peri‐infarct immunofluorescence analysis of ionized calcium binding adaptor molecule 1 (Iba1), galectin‐3 (Gal3), and purinergic receptor P2Y12 (P2RY12). (A) Representative immunostaining of 4′,6‐diamidino‐2‐phenylindole (DAPI), Iba1, P2RY12, and Gal3 in a standard environment (SE) mouse at peri‐infarct area (at ×20 magnification). (B) Representative immunostaining of DAPI, Iba1, P2RY12, and Gal3 in an enriched environment (EE) mouse at peri‐infarct area (at ×20 magnification). (C) Quantification of indirect infarct area measurements. (D) Correlation of infarct area with Neuroscore per group. (E) Iba1 coverage quantification measured as the percentage of image covered by Iba1 area (%area). (F) Correlation of infarct area with Iba1 coverage. (G) Gal3 coverage quantification measured as the percentage of image covered by Iba1 + Gal3 + area (%area). (H) Correlation of infarct area with Gal3 coverage. (I) P2RY12 coverage quantification measured as the percentage of image covered by Iba1 + P2RY12 + area (%area). (J) Correlation of infarct area with P2RY12 coverage. Peri‐infarct area is shown as dashed red lines in A and B. In (C, E, G, and I), values are expressed as individual experimental replicates with mean ± SEM. In (D, F, H, and J), values are expressed as individual experimental replicates with simple linear regression lines; Pearson correlation's r value with p ‐value are also shown. In (C, E, G, and I), unpaired t ‐test was performed. n = 4/6 mice in SE and n = 7 mice in EE (2 mice in SE were not behaviorally characterized). P ‐values and r values are expressed with 3 decimals. P ‐values were not corrected for multiple comparisons.

Article Snippet: They were then incubated at 4°C overnight with one of the following antibodies: Iba1 (1:500, rabbit, Cat# 016‐26721; Wako), Gal3 (1:750, goat, Cat# AF1197; R&D Systems) P2RY12 (1:200, rat, Cat# S16007D; Biolegend).

Techniques: Immunofluorescence, Binding Assay, Immunostaining

Journal: Neuroprotection

Article Title: Environmental enrichment modulates chronic poststroke inflammation and links white matter TREM2‐positive microglia in recovery in mice

doi: 10.1002/nep3.70028

Figure Lengend Snippet: Infarct size correlations with white matter immunofluorescence analysis of ionized calcium binding adaptor molecule 1 (Iba1), galectin‐3 (Gal3), and purinergic receptor P2Y12 (P2RY12). (A) Representative immunostaining of 4′,6‐diamidino‐2‐phenylindole (DAPI), Iba1, P2RY12, and Gal3 (at ×20 magnification) in a standard environment (SE) mouse at white matter area (corpus callosum + external capsule). (B) Representative immunostaining of DAPI, Iba1, P2RY12, and Gal3 in an enriched environment (EE) mouse at white matter area (at ×20 magnification). (C) Iba1 coverage quantification measured as the percentage of image covered by Iba1 area (%area). (D) Gal3 coverage quantification measured as the percentage of image covered by Iba1 + Gal3 + area (%area). (E) P2RY12 coverage quantification measured as the percentage of image covered by Iba1 + P2RY12 + area (%area). (F) Correlation of infarct area with Iba1 coverage. (G) Correlation of infarct area with Gal3 coverage. (H) Correlation of infarct area with P2RY12 coverage. White matter area is shown as dashed red lines in (A, B). In (C–E) values are expressed as individual experimental replicates with mean ± SEM. In (F–H) values are expressed as individual experimental replicates with simple linear regression lines; Pearson correlation's r value with p ‐value are also shown. In (C–E) unpaired t ‐test was performed n = 6 mice in SE and n = 7 mice in EE. p ‐Values and r values are expressed with 3 decimals. p ‐Values were not corrected for multiple comparisons.

Article Snippet: They were then incubated at 4°C overnight with one of the following antibodies: Iba1 (1:500, rabbit, Cat# 016‐26721; Wako), Gal3 (1:750, goat, Cat# AF1197; R&D Systems) P2RY12 (1:200, rat, Cat# S16007D; Biolegend).

Techniques: Immunofluorescence, Binding Assay, Immunostaining

Journal: Neuroprotection

Article Title: Environmental enrichment modulates chronic poststroke inflammation and links white matter TREM2‐positive microglia in recovery in mice

doi: 10.1002/nep3.70028

Figure Lengend Snippet: Quantification of peri‐infarct myelin debris, white matter myelin loss, and their correlations with microglial markers. (A) Representative myelin staining in a standard environment mouse (at ×20 magnification). (B) Enlarged views of infarct contralateral cortical (green square) and peri‐infarct (red square) myelin in a standard environment (SE) mouse. (C) Representative myelin staining in an enriched environment mouse (at ×20 magnification). (D) Enlarged views of infarct contralateral cortical (green square) and peri‐infarct (red square) myelin in an enriched environment (EE) mouse. (E) Myelin debris coverage quantification measured as the percentage of peri‐infarct image covered by Black Gold Myelin dark debris area (%area). (F) Correlation of infarct area with myelin debris coverage. (G) Myelin loss quantification measured as the percentage of myelin lost at corpus callosum in ipsilateral versus contralateral infarct. (H) Correlation of infarct area with myelin loss. (I) Correlations of myelin debris with ionized calcium binding adaptor molecule 1 (Iba1), galectin‐3 (Gal3), purinergic receptor P2Y12 (P2RY12), cluster of differentiation 68 (CD68), and triggering receptor expressed on myeloid cells 2 (TREM2) coverages at peri‐infarct. (J) Correlations of myelin loss with Iba1, Gal3, P2RY12, CD68, and TREM2 coverages at white matter. In (E, G) values are expressed as individual experimental replicates with mean ± SEM. In (F, H, I, J), values are expressed as individual experimental replicates with simple linear regression lines; Pearson correlation's r value with p ‐value are also shown. In (E, G) unpaired t‐test was performed. n = 6 mice in SE and n = 7 mice in EE. p ‐Values and r values are expressed with 3 decimals. p ‐Values were not corrected for multiple comparisons.

Article Snippet: They were then incubated at 4°C overnight with one of the following antibodies: Iba1 (1:500, rabbit, Cat# 016‐26721; Wako), Gal3 (1:750, goat, Cat# AF1197; R&D Systems) P2RY12 (1:200, rat, Cat# S16007D; Biolegend).

Techniques: Staining, Binding Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Spinal cord injury (SCI) increases galectin-3 (GAL3) expression in spinal neurons. (A) Relative GAL3 mRNA expression in the spinal cord after SCI. One-way ANOVA, n = 3/group. (B) Western blot analysis of GAL3 protein after SCI. (C) Statistical data show relative GAL3 protein expression after SCI. One-way ANOVA, n = 3/group. (D) Enzyme-linked immunosorbent assay (ELISA) detection of GAL3 protein levels in rat serum after SCI. One-way ANOVA, n = 6/group. (E) Immunofluorescence microscopy reveals GAL3 co-localization with NeuN post-SCI. (F) Fluorescence intensity of GAL3 after SCI. One-way ANOVA, n = 3/group. (G,H) Immunofluorescence double staining of GAL3 and GFAP (G) or IBA1 (H) after SCI. (I) Determination of optimal glutamate concentration and duration using CCK8 assay. (J) Relative GAL3 mRNA expression in the glutamate-stimulated spinal cord neurons. Unpaired Student’s t -test, n = 3/group. (K) Western blot analysis of GAL3 protein in neuronal injury model. (L) Relative GAL3 protein expression in neuronal injury model. Unpaired Student’s t -test, n = 3/group. (M) ELISA detection of GAL3 in cell supernatant of neuronal injury model. Unpaired Student’s t -test, n = 3/group. (N) Immunofluorescence microscopy showing GAL3 expression in neuronal injury model. (O) Quantification of GAL3 fluorescence intensity in neuronal injury model. Unpaired Student’s t -test, n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Fluorescence, Double Staining, Concentration Assay, CCK-8 Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) contributes to spinal cord injury (SCI)-induced motor impairment. (A) The mRNA level of GAL3 after siR-GAL3 treatment. Unpaired Student’s t -test, n = 3/group. (B) Western blot shows the protein level of GAL3 after siR-GAL3 treatment. (C) Statistical data show the knockdown of GAL3 by siRNA. Unpaired Student’s t -test, n = 3/group. (D) Enzyme-linked immunosorbent assay (ELISA) shows the secretory GAL3 in the supernatant of neurons after siRNA treatment. Unpaired Student’s t -test, n = 3/group. (E) The Basso-Beattie-Bresnahan (BBB) locomotor scores were increased after siR-GAL3 or inhibitor treatment. Two-way Repeated Measures ANOVA, n = 8/group. (F) The inclined plane angles were increased after siR-GAL3 or inhibitor treatment. Two-way Repeated Measures ANOVA, n = 8/group. When SCI + siR-GAL3 group was compared with SCI + Vehicle group, ** P < 0.01, *** P < 0.001; when SCI + TD139 group was compared with SCI + Vehicle group, # P < 0.05, ### P < 0.001; when SCI + GAL3 group was compared with SCI + Vehicle group, + P < 0.05,++ P < 0.01.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) is closely related to programmed cell death after spinal cord injury (SCI). (A) The four datasets before the batch effect were removed. (B) The four datasets after the batch effect were removed. (C) Volcano map shows DEGs in the SCI dataset. (D) Biological Process (BP) analysis of Gene Set Enrichment Analysis (GSEA) in the SCI dataset. Each column represents the P -value score of the pathway between the Sham group and the SCI group, with red indicating upregulation of the pathway in the SCI group, and blue indicating downregulation. (E) Protein-Protein Interaction Networks (PPI) analysis of differentially expressed genes (DEGs) in SCI dataset. In the PPI nodes, red signifies an increase in expression level, while blue indicates a decrease. The intensity of the color corresponds to the magnitude of the differential expression, with darker shades representing a higher differential expression multiple.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Expressing, Quantitative Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) regulates neuronal autophagy. (A) Western blot analysis of GAL3 and neuronal autophagy markers ATG7, P62, and LC3 II/I in neurons. (B-E) Quantification of western blot detection of GAL3 (B) , ATG7 (C) , P62 (D) , and LC3 II/I (E) in neurons. One-way ANOVA, n = 3/group. (F) Western blot analysis of GAL3 and neuronal autophagy markers ATG7, P62, and LC3 II/I in the spinal cord of rats. (G–I) Quantification of western blot detection of ATG7 (G) , P62 (H) , and LC3 II/I (I) in the spinal cord of rats. One-way ANOVA, n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Sequencing analysis of spinal cord neurons with galectin-3 (GAL3) knocked down. (A) Volcano map shows differential expression genes (DEGs) in the neuron dataset. (B) Biological process (BP) analysis of Gene Set Enrichment Analysis (GSEA) in the neuron dataset. Each column represents the P -value score of the pathway between the Sham group and the spinal cord injury (SCI) group, with red indicating upregulation of the pathway in the SCI group, and blue indicating downregulation. (C) The Protein-Protein Interaction Networks (PPI) analysis of DEGs in the neuron dataset. In the PPI nodes, red indicates that the expression level increases and blue indicates that the expression level decreases. The darker the color, the greater the differential expression multiple.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Sequencing, Quantitative Proteomics, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) interacts with Cell-division-cycle-42 (CDC42) to regulate neuronal autophagy. (A) Intersected 29 core nodes from the neuron dataset with 22 core nodes from the spinal cord injury (SCI) dataset by the Venn diagram. (B) Correlation analysis between GAL3 and CDC42 expression level in SCI dataset. (C) Correlation analysis between GAL3 and CDC42 expression level in the neuron dataset. (D) Co-immunoprecipitation (Co-IP) shows a direct interaction between GAL3 and CDC42 in the glutamate-induced neuronal damage model. (E) Western blot shows the expression of CDC42, ATG7, P62, and LC3 II/I. (F–I) Quantification of western blot detection of CDC42 (F) , ATG7 (G) , P62 (H) , and LC3 II/I (I) . One-way ANOVA, n = 3/group. (J) Enzyme-linked immunosorbent assay (ELISA) detection of CDC42 in cell supernatant of GAL3-injury model. Unpaired Student’s t -test, n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Cell-division-cycle-42 (CDC42) contributes to spinal cord injury (SCI)-induced motor function impairment. (A) The mRNA level after siR-CDC42 treatment. Unpaired Student’s t -test, n = 3/group. (B) Enzyme-linked immunosorbent assay (ELISA) shows the secretory CDC42 in the supernatant of neurons after siRNA treatment. Unpaired Student’s t -test, n = 3/group. (C) The Basso-Beattie-Bresnahan (BBB) locomotor scores were increased after siR-CDC42 and ML141 treatment. Two-way Repeated Measures ANOVA, n = 8/group. (D) The inclined plane angles were increased after siR-CDC42 and ML141 treatment. Two-way Repeated Measures ANOVA, n = 8/group. When SCI + siR-CDC42 group was compared with SCI + Vehicle group, * P < 0.05, ** P < 0.01, *** P < 0.001; when SCI + ML141 group was compared with SCI + Vehicle group, ## P < 0.01, ### P < 0.001. (E,F) Detection of the protein expression level of galectin-3 (GAL3) (E) and CDC42 (F) in serum of healthy volunteers and SCI patients by ELISA. Unpaired Student’s t -test, n = 8/group. *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: In vivo (Athens, Greece)

Article Title: Expression of Galectin 3 and Activating Transcription Factor 3 in Nigral Dopaminergic Neurons of 6-Hydroxydopamine Induced Parkinsonian Rat Model.

doi: 10.21873/invivo.13938

Figure Lengend Snippet: Figure 2. Gal3 expression was upregulated in the ipsilateral nigral dopaminergic neurons. (A) Photographs of Gal3 immunohistochemistry in the contralateral (Cont) and ipsilateral (Ispi) substantia nigra (SN) after 6‐hydroxydopamine (6‐OHDA) lesion. Gal3 immunoreactivity was upregulated in the ipsilateral SN. (B) High magnification image of the area designated by the black square inside of (A). Morphologically typical multipolar neurons with large cell body and many prominent processes (arrows) and neuroglia with small cell bodies and many long and slender radiating processes (arrowheads) were positive for Gal3 in the ipsilateral SN. (C) Double immunofluorescence images of Gal3 and TH and their merged image, in the ipsilateral SN at 5 dpl. Gal3 was expressed in many nigral dopaminergic neurons (arrows). (D) Double immunofluorescence images of Gal3 and Iba1 and their merged image. Gal3 was also expressed in many microglial cells (arrows). (E) Double immunofluorescence images of Gal3 and GFAP and their merged image. None of the cells immunolabeled for Gal3 were colocalized with GFAP‐positive astrocytes (arrows). (F) Quantitative analysis showing the average number of neurons expressing Gal3 per unit area (mm2) at each time point. Scale bars represent 200 μm in (A), 50 μm in (B and E). Data are expressed as means±SEM (n=6, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). dpl, Days post‐lesion.

Article Snippet: Antibody Catalog Manufacturer Host Description Concentration number IHC IF Gal3 AF‐1197 R&D system Goat Wide range of cell type 1:500 1:50 ATF3 Sc‐188 Santa Cruz Rabbit Wide range of cell type 1:1000 1:100 TH MAB5280 Millipore Mouse Tyrosine hydroxylase 1:1000 1:500 Iba1 AB283319 Abcam Mouse Microglia marker 1:200 GFAP AB279289 Abcam Mouse Astrocyte marker 1:200 FG AB153 Millipore Rabbit Fluorogold 1:500 1:100 Gal3, Galectin 3; ATF3, activating transcription factor 3; TH, tyrosine hydroxylase; Iba1, ionized calcium‐binding adapter molecule 1; GFAP, glial fibrillary acidic protein; FG, fluorogold; IHC, immunohistochemistry; IF, immunofluorescence. antibodies against TH (1:500), or rabbit polyclonal antibodies against FG (1:100).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Immunolabeling

Journal: In vivo (Athens, Greece)

Article Title: Expression of Galectin 3 and Activating Transcription Factor 3 in Nigral Dopaminergic Neurons of 6-Hydroxydopamine Induced Parkinsonian Rat Model.

doi: 10.21873/invivo.13938

Figure Lengend Snippet: Figure 4. Co‐localization of Gal3 and ATF3 in the 6‐OHDA insulted dopaminergic neurons. (A) Representative photomicrographs showing triple immunofluorescence labeling for FG, Gal3, and ATF3 and their merged image in the ipsilateral SN after 6‐OHDA lesion at 5 dpl. (B) High magnification image of the area designated by the white square inside of (A). ATF3 and Gal3 were colocalized in the dopaminergic neurons that are labeled with FG, i.e., neurons that have been retrogradely insulted with 6‐OHDA. Scale bar represents 200 μm and 50 μm in (A) and (B), respectively. dpl, Days post‐lesion; Ipsi, ipsilateral.

Article Snippet: Antibody Catalog Manufacturer Host Description Concentration number IHC IF Gal3 AF‐1197 R&D system Goat Wide range of cell type 1:500 1:50 ATF3 Sc‐188 Santa Cruz Rabbit Wide range of cell type 1:1000 1:100 TH MAB5280 Millipore Mouse Tyrosine hydroxylase 1:1000 1:500 Iba1 AB283319 Abcam Mouse Microglia marker 1:200 GFAP AB279289 Abcam Mouse Astrocyte marker 1:200 FG AB153 Millipore Rabbit Fluorogold 1:500 1:100 Gal3, Galectin 3; ATF3, activating transcription factor 3; TH, tyrosine hydroxylase; Iba1, ionized calcium‐binding adapter molecule 1; GFAP, glial fibrillary acidic protein; FG, fluorogold; IHC, immunohistochemistry; IF, immunofluorescence. antibodies against TH (1:500), or rabbit polyclonal antibodies against FG (1:100).

Techniques: Immunofluorescence, Labeling

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