Journal: bioRxiv

Article Title: Galectin-8 is a major ligand of LILRB4 prompting MDSC functions in the tumor microenvironment

doi: 10.1101/2022.07.27.501694

Figure Lengend Snippet: (A) Data analysis with TIDE shows that LGALS8 plays an important role in T cell dysfunction in tumor microenvironment. (B) Spearman correlations between Galectin-8 expression and TILs. (C) TIMER was used to calculated MDSC fraction and correlation with LGALS8expression in the indicated types of tumors from the TCGA dataset. (D) Schematic procedure of phagocytosis assay. (E) Phagocytosis assay (FC) shows that Galectin-8 inhibits the ability of phagocytosis. F. Transcriptome analysis of CD14+ monocytes exposed with Galectin-8. (F) Heatmap of transcriptome sequencing data. Samples of 2 groups including control and gal-8 were analyzed. (G) Volcano plot of transcriptome sequencing data. Significantly regulated genes were labeled.

Article Snippet: Reagents involving recombinant protein Galectin-8 (10301-HNAE; SinoBiological), APOE (APE-H5246; Acrobiosystems), CD3ε (10977-H02H; SinoBiological), CTLA-4 (CT4-H5255; Acrobiosystems), CD28 (CD8-H525a; Acrobiosystems), CD96 (TAE-H5252; Acrobiosystems), LAG-3 (LA3-H5255; Acrobiosystems), TIM-3 (TM3-H5258; Acrobiosystems), CD40 (CD0-5253; Acrobiosystems), ICOS (ICS-H5258; Acrobiosystems), OX40 (OX0-H5255; Acrobiosystems), TIGHT (TIT-H5254; Acrobiosystems), LY86 (10242-H02H; SinoBiological), LILRB4 (16742-H02H; SinoBiological), CD27 (CD7-H5254; Acrobiosystems), PD-1 (10377-H02H; SinoBiological), CD8b (11031-HCCH; SinoBiological) were also purchased from the indicated suppliers.

Techniques: Expressing, Phagocytosis Assay, Sequencing, Labeling

Journal: bioRxiv

Article Title: Galectin-8 is a major ligand of LILRB4 prompting MDSC functions in the tumor microenvironment

doi: 10.1101/2022.07.27.501694

Figure Lengend Snippet: (A) ELISA screening of potential Galectin-8 interactors revealed LILRB4. (B) 100X microscopic views of Immunofluorescence showing Galectin-8 binds to cell surface LILRB4. (C) CoIP of Galectin-8 and LILRB4. (D) Analysis of EC50 from ELISA test. Galectin-8 was coated by concentration scale, LILRB4-Fc was added to 1 μg/ml. (E) BLI assay showing the association-disassociation curve between Galectin-8 and LILRB4 with the kinetics constants. (F) Flow cytometry showing the fractions of MDSCs in PBMCs incubated with Gal-8, Gal-8 plus LILRB4 ectodomain, and APOE. (G) Flow cytometry showing the binding of Galectin8 to LILRB4 expressed on cell surface. (H) ELISA showing the binding between Galectin-8 and LILRB4 in presence of different concentrations of APOE.

Article Snippet: Reagents involving recombinant protein Galectin-8 (10301-HNAE; SinoBiological), APOE (APE-H5246; Acrobiosystems), CD3ε (10977-H02H; SinoBiological), CTLA-4 (CT4-H5255; Acrobiosystems), CD28 (CD8-H525a; Acrobiosystems), CD96 (TAE-H5252; Acrobiosystems), LAG-3 (LA3-H5255; Acrobiosystems), TIM-3 (TM3-H5258; Acrobiosystems), CD40 (CD0-5253; Acrobiosystems), ICOS (ICS-H5258; Acrobiosystems), OX40 (OX0-H5255; Acrobiosystems), TIGHT (TIT-H5254; Acrobiosystems), LY86 (10242-H02H; SinoBiological), LILRB4 (16742-H02H; SinoBiological), CD27 (CD7-H5254; Acrobiosystems), PD-1 (10377-H02H; SinoBiological), CD8b (11031-HCCH; SinoBiological) were also purchased from the indicated suppliers.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Concentration Assay, Flow Cytometry, Incubation, Binding Assay

Journal: bioRxiv

Article Title: Galectin-8 is a major ligand of LILRB4 prompting MDSC functions in the tumor microenvironment

doi: 10.1101/2022.07.27.501694

Figure Lengend Snippet: (A) Phosphorylation of SHP1 was upregulated by the interaction of Galectin8 and LILRB4 in THP-1 cells. (B) THP-1 NFκB reporter cells cocultured with Galectin-8-overexpressed 293 cells rather than control 293 cells showed lower activation of NFκB signal. (C) Immune blotting demonstrates Gal-8 inhibited NFκB while activated STAT3 in THP-1 cells. (D) Nuclear protein extraction of NC/shLILRB4 THP-1 cells and immune blotting revealing effect of LILRB4 on NFκB and STAT3. (E) Immune blotting of human CD14+ cells. (F) Immune blotting of SHP-1 inhibitor, TPI, inhibited STAT3 phosphorylation in human CD14+ cells.

Article Snippet: Reagents involving recombinant protein Galectin-8 (10301-HNAE; SinoBiological), APOE (APE-H5246; Acrobiosystems), CD3ε (10977-H02H; SinoBiological), CTLA-4 (CT4-H5255; Acrobiosystems), CD28 (CD8-H525a; Acrobiosystems), CD96 (TAE-H5252; Acrobiosystems), LAG-3 (LA3-H5255; Acrobiosystems), TIM-3 (TM3-H5258; Acrobiosystems), CD40 (CD0-5253; Acrobiosystems), ICOS (ICS-H5258; Acrobiosystems), OX40 (OX0-H5255; Acrobiosystems), TIGHT (TIT-H5254; Acrobiosystems), LY86 (10242-H02H; SinoBiological), LILRB4 (16742-H02H; SinoBiological), CD27 (CD7-H5254; Acrobiosystems), PD-1 (10377-H02H; SinoBiological), CD8b (11031-HCCH; SinoBiological) were also purchased from the indicated suppliers.

Techniques: Activation Assay, Protein Extraction

Journal: bioRxiv

Article Title: Quantification of differential toxin expressions and their relation to distinct lifespans of bacterial subpopulations associated with diverse host immune mechanisms

doi: 10.1101/2022.10.11.511682

Figure Lengend Snippet: (A), (C) and (E) depict representative time-lapse montages of lifetimes of SPN inside Gal3 (A) or LC3 (E) or dually (B) marked vacuoles. A549s stably expressing mStrawberry-Gal3 and GFP-LC3 were infected with DRAQ5 stained SPN and time-lapse imaging was performed at 30 min post infection. The stills in A, C and E correspond to the movies shown as S3 Movie, S4 Movie and S4 Movie, respectively. (B , D and F) Temporal quantification of Gal3, LC3 and SPN fluorescence intensities associated with PCVs either relative to fluorescence intensity of the cytosol ( I PCV /I Cyt ) (for LC3 and Gal3) or fluorescence signal of host cell nuclei ( I SPN /I Nuc ) (for SPN). Relative mean intensity values ~ 1.0 for any marker means disappearance of the signal of the corresponding marker. In (A) and (C) relative mean intensity values for bacteria ( I SPN /I Nuc ) remains > 1.0 for long period of time before finally disappearing. However, in (E) I SPN /I Nuc remained > 1.0 throughout the course of imaging, highlighting prolonged persistence of low Ply producing SPN in only LC3 marked compartments. (G) Mean degradation time of SPN:Ply-Low population subsets as determined by the time lapse fluorescence imaging. Statistical analysis was performed using one-way ANOVA followed by Tukey’s Multiple Comparison Test. *** p <0.001. (H) Schematic representation of different SPN population subsets arising due to expression of low Ply. In one population subset, following minute damage, Gal3 recruitment triggers endomembrane repair pathway which eventually results in lysosomal fusion and SPN killing. In another subset, damage followed by Gal3 association triggers autophagic sequestration of damaged PCV and subsequent lysosomal killing. Finally, extremely minute damage in the endomembrane leads to LC3 lipidation without association of any damage sensing markers and this population exhibits prolonged persistence.

Article Snippet: The following antibodies were used in this study were Galectin3 (R&D systems, 194805), Galectin 8 (R&D systems, AF1305), anti-goat IgG 633 (Invitrogen, A21082), anti-mouse IgG 555 (Invitrogen, A21422).

Techniques: Stable Transfection, Expressing, Infection, Staining, Imaging, Fluorescence, Marker, Bacteria, Comparison

Journal: bioRxiv

Article Title: Quantification of differential toxin expressions and their relation to distinct lifespans of bacterial subpopulations associated with diverse host immune mechanisms

doi: 10.1101/2022.10.11.511682

Figure Lengend Snippet: (A) Percentage of association of Δ ply mutant with Gal3, Gal8 and LC3 in comparison to WT SPN. Statistical analysis was performed using Students t-test. *** p <0.001. (B) Quantitative analysis showing increased association of Δ ply mutant with LC3 in absence of damage sensing marker Gal3, upon treatment with different conc. of monensin. n ≥ 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s Multiple Comparison Test. *** p <0.001. (C) Cell viability at different conc. of monensin. Triton X-100 (0.1%) served as a positive control. Statistical analysis was performed using one-way ANOVA followed by Tukey’s Multiple Comparison Test. ns, non-significant, *** p <0.001. (D) Representative time-lapse montage of Δ ply mutant showing prolonged persistence post monensin treatment (50 μM). A549s stably expressing mStrawberry-Gal3 and GFP-LC3 were infected with DRAQ5 stained Δ ply mutant. Following monensin treatment cells were monitored using confocal microscope at an interval of 60 min for extended hours. The stills correspond to the movie shown as S6 Movie. (E) Temporal quantification of Gal3, LC3 and SPN fluorescence intensities either relative to fluorescence in the cytosol ( I PCV /I Cyt ) (for LC3 and Gal3) or fluorescence signal of A549 nuclei ( I SPN /I Nuc ) (for SPN). I PCV /I Cyt values were always found to be close to 1.0 for Gal3, exhibiting no association of Gal3 with PCVs. Contrarily I PCV /I Cyt for LC3 and I SPN /I Nuc for SPN were > 1.0, revealing prolonged persistence of SPN inside LC3 marked vacuole for extended hours.

Article Snippet: The following antibodies were used in this study were Galectin3 (R&D systems, 194805), Galectin 8 (R&D systems, AF1305), anti-goat IgG 633 (Invitrogen, A21082), anti-mouse IgG 555 (Invitrogen, A21422).

Techniques: Mutagenesis, Comparison, Marker, Bacteria, Positive Control, Stable Transfection, Expressing, Infection, Staining, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Quantification of differential toxin expressions and their relation to distinct lifespans of bacterial subpopulations associated with diverse host immune mechanisms

doi: 10.1101/2022.10.11.511682

Figure Lengend Snippet: (A) Normalized histogram of total time of pore formation, t C’ (in min), on PCV for escape to the cytosol, by high-Ply producing subpopulation among WT SPN. (B) Normalized histogram of cytosolic escape time t p (in min), after Gal8 recruitment. The continuous red line represents the theoretical best-fitted curve ( b = 2, ( N-N 0 ) =1 3, k = 0.107 min −1 ) to the experimental histogram. (C) Normalized histogram represents the total degradation time t Vac (in min) of WT SPN subpopulations with moderate or low Ply production within PCV. (D) Normalized histogram of degradation time t v (in min) of WT SPN within vacuoles is plotted for times < 400 min. The theoretically best-fitted line in red ( b = 2, ( N-N 0 ) = 10, k = 0.0412 min −1 ) is shown against the cyan histogram. (E) Normalized histogram of degradation times t m (in min) measured after the LC3 recruitment, for LC3 + Gal3 - marked subpopulation among the SPN:Ply-Low strains.

Article Snippet: The following antibodies were used in this study were Galectin3 (R&D systems, 194805), Galectin 8 (R&D systems, AF1305), anti-goat IgG 633 (Invitrogen, A21082), anti-mouse IgG 555 (Invitrogen, A21422).

Techniques:

Journal: Current Biology

Article Title: Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

doi: 10.1016/j.cub.2020.05.083

Figure Lengend Snippet:

Article Snippet: Goat polyclonal anti-galectin 8 , R and D Systems , Cat# AF1305; RRID: AB_2137229.

Techniques: Virus, Recombinant, Protease Inhibitor, Plasmid Preparation, Silver Staining, Synthesized, Expressing, Software, Nickel Column, Electron Microscopy

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: paraffin sections of anterior chamber angle from a normal human eye were immunostained with anti-Gal8 antibody. (i) anti-Gal8 IgG reacted intensely with cells on the trabecular beams (arrows) and with cells in the juxtacanalicular portion of TM (arrowheads). Staining was also observed in the ECM of both portions of TM (JCT and CS) and in the wall of Schlemm’s canal. (ii) No staining was observed when the sections were not exposed to the primary antibody. SC : Schlemm’s canal, JCT : juxtacanalicular TM; CS beams : corneoscleral beams. Bar: 25 µm. B: (i) RT-PCR . Total RNA (1.0 µg) from confluent cultures of normal human TM cells was subjected to RT-PCR. The expected 191 bp fragment was amplified using Gal8- specific-primers. In each case, no components were amplified when reaction mixtures lacked reverse transcriptase (RT). (ii) qRT-PCR . Total RNA was subjected to Taq-Man RT-PCR using Gal8 specific primers. Original amplification plots of Gal8 and GAPDH mRNAs genes are shown (Ct 37.37 and 33.38 for Gal8 and GAPDH, respectively). N = 3 for each experiment; all experiments were performed twice using TM cells from two different donors with reproducible results. (iii and iv) Western Blot Analysis . Protein extracts from confluent cultures of normal human TM cells were incubated with lactogel beads and eluted first with sucrose, and then with lactose. Eluted proteins were electrophoresed, the protein blot of the gel was stained with Ponceau S (iii) and was then processed for immunostaining with goat anti-Gal8 (iv). Both the total cell extract ( T ) and the lactose eluate ( L ) contained a major 36-kDa anti-Gal8 reactive component. This component was not detected in the unbound fraction ( UB ) and in the sucrose eluate ( S ).

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Reverse Transcription, Quantitative RT-PCR, Western Blot, Incubation, Immunostaining

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: Normal human TM cells were incubated on microtiter wells coated with BSA, fibronectin, or Gal8 in DPBS, in the presence and absence of a function-blocking anti-β 1 integrin antibody (JB1A), or control mouse IgG. Following incubation at 37°C for 30 min, cells were fixed and stained with crystal violet. Attached cells in fibronectin-coated wells are set as 100% (positive control); attached cells in other wells are presented as percent of positive control. Data are expressed as mean±SEM and analyzed with one-way ANOVA. * P <0.05 vs IgG; ** P <0.01 vs media or IgG; *** P <0.001 vs media. B and C: Cell spreading assay. TM cells were fixed with 4% paraformaldehyde after adhesion for 30 min. F-actin was stained with rhodamine-labeled phalloidin and cell nuclei were labeled with DAPI. Random fields of each experimental condition were photographed, and spread areas of individual cells were quantified with ImageJ. Representative micrographs of TM cells incubated in the presence and the absence of anti-β 1 integrin antibody are shown in C. Data are presented as Box–whisker plot (after Tukey) and analyzed with one-way ANOVA. *** P <0.001 vs media or IgG. This experiment was performed three times with reproducible results. Bar: 100 µm.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Incubation, Blocking Assay, Control, Staining, Positive Control, Labeling, Whisker Assay

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: Normal human TM cells were plated on eight-chamber glass slides coated with 20 µg/ml of recombinant human Gal8 (i–iii), 20 µg/ml of fibronectin (iv–vi), or 100 µg/ml of poly-L-lysine (vii–ix) in serum-free DMEM at 37°C for 30 min (i, iv, vii), 1 hr (ii, v, viii), and 2 hr (iii, vi, ix). Following the incubation period, cells were fixed with 4% paraformaldehyde and stained with rhodamine-labeled phalloidin. Bar: 50 µm. B: Quantification of stress fiber formation. Random fields were photographed, and cells with robust stress fibers were counted. N = 225 to 362. Data are expressed as mean±SEM and analyzed with one-way ANOVA. *** P <0.001 vs poly-L-lysine at different time points. This experiment was performed three times with reproducible results.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Recombinant, Incubation, Staining, Labeling

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A and C: Serum-starved human TM cells were incubated on chamber glass slides coated with recombinant human Gal8 in the presence of the Rho inhibitor, C3 transferase or ROCK inhibitor, Y27632, at different concentrations. After 2 hr, cells were stained with rhodamine-labeled phalloidin, and cells with robust stress fibers were enumerated. Data are expressed as mean±SEM. B and D: Cells were treated C3 transferase at 2 µg/ml (B) or Y27632 at 20 µM (D), stained with rhodamine-labeled phalloidin, and random fields were photographed. Note that cells treated with Y27632 or C3 transferase are not spread and exhibit dendrite-like structures. Bar: 50 µm.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Incubation, Recombinant, Staining, Labeling

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: Gal8 induces phosphorylation of MLC2 in a time-dependent manner. Normal human TM cells were incubated on 100-mm dishes coated with Gal8 for 0.5, 1, and 2 hr. Following incubation, cells were lysed, and protein extracts were subjected to electrophoresis in 12% SDS-PAGE gels. Blots were probed with anti-phosphorylated myosin light chain 2 (ppMLC2) (Thr18/Ser19) antibody. The blots were subsequently stripped and reprobed with anti-MLC antibody. A representative Western blot is shown in the top panel. Images were acquired by Odyssey Infrared Imaging System, and band intensity was quantified by ImageJ (bottom panel). N = 3. B: Phosphorylation of MLC2 is inhibited by Rho and ROCK inhibitors. Normal human TM cells were serum-starved overnight and treated with C3 transferase (2 µg/ml) and Y27632 (20 µM) for 4 hr. Treated cells were detached and plated on Gal8-coated dishes for 2 hr in the presence or absence of inhibitors and were then examined for the expression levels of ppMLC2 as described in the legend to panel A. Top: A representative Western blot; bottom: quantification of ppMLC2. Data are expressed as mean±SEM and analyzed with one-way ANOVA. *** P <0.001 vs control. N = 3.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Phospho-proteomics, Incubation, Electrophoresis, SDS Page, Western Blot, Imaging, Expressing, Control

Journal: Molecular and Cellular Biology

Article Title: Invading Bacterial Pathogens Activate Transcription Factor EB in Epithelial Cells through the Amino Acid Starvation Pathway of mTORC1 Inhibition

doi: 10.1128/mcb.00241-22

Figure Lengend Snippet: TFEB activation in infected epithelial cells is dependent on the amino acid-dependent inhibition of mTORC1. (a) Scramble and DEPDC5-KD HeLa cells infected with Shigella flexneri, grown in complete DMEM or starved of amino acids in KRB for 4 h. TFEB subcellular location was visualized by staining with an anti-TFEB antibody. White boxes outline zoomed images below. (b) Quantification of TFEB cellular localization from a. Data are represented as mean ± SEM from four independent experiments (*, P < 0.05; **, P < 0.01; ****, P < 0.0001). (c) Scramble and DEPDC5-KD HeLa cells infected with Salmonella enterica Typhimurium, grown in complete DMEM or starved of amino acids in KRB for 2 h. TFEB subcellular location was visualized by staining with an anti-TFEB antibody. White boxes outline zoomed images below. (d) Quantification of TFEB cellular localization from c. Data are represented as mean ± SEM from four independent experiments (**, P < 0.01; ****, P < 0.0001). (e) Protein expression and cellular localization of TFEB in scramble and DEPDC5-KD HeLa cells infected with Shigella flexneri for up to 4 h, as indicated. (f) Quantification of average Galectin-8 puncta per cell from g. Data are represented as mean ± SEM from 4 independent experiments (***, P < 0.001; ns, not significant). (g) Scramble and DEPDC5-KD HeLa cells infected with Shigella flexneri or grown in complete DMEM for 1 h. Membrane damage was visualized by staining with an anti-Galectin-8 antibody.

Article Snippet: TABLE 1 Antibody target Use a (dilution) Manufacturer; catalog no. TFEB WB (1:1,000); IF (1:100) Cell Signaling; 4240S TFE3 WB (1:1,000) Millipore Sigma; HPA023881 GAPDH WB (1:10,000) Cell Signaling; 5174S H3 WB (1:1,000) Invitrogen; AHO1432 Galectin-8 IF (1:100) R&D Systems; MAB1305 IκBα WB (1:1,000) Abcam; ab32518 NF-κB p65 IF (1:100) Cell Signaling; 6956S Open in a separate window a IF, immunofluorescence; WB, Western blotting.

Techniques: Activation Assay, Infection, Inhibition, Staining, Expressing, Membrane

Journal: Molecular and Cellular Biology

Article Title: Invading Bacterial Pathogens Activate Transcription Factor EB in Epithelial Cells through the Amino Acid Starvation Pathway of mTORC1 Inhibition

doi: 10.1128/mcb.00241-22

Figure Lengend Snippet: Membrane damage induced by invading bacteria is required for TFEB nuclear import during infection. (a) WT HeLa cells infected with WT Shigella flexneri for 1 or 4 h. Membrane damage and/or membrane remnants were visualized by staining with an anti-Gal-8 antibody. White squares indicate zoomed areas, and white arrowheads indicate Galectin-8-targeted bacteria. (b) WT HeLa cells infected with either WT or ΔIcsA or ΔIcsB Shigella flexneri for 1 h. MOI of 10 for WT Shigella, 1,200 for ΔIcsA Shigella, and 600 for ΔIcsB Shigella. (c) Quantification of Galectin-8 puncta from b. Data are represented as mean ± SEM from four independent experiments (*, P < 0.05). (d) Quantification for TFEB nuclear import from e. Data are represented as mean ± SEM from four independent experiments (*, P < 0.05; **, P < 0.01). (e) WT HeLa cells infected with either WT or ΔIcsA or ΔIcsB Shigella flexneri for 4 h. (f and g) Protein expression and cellular localization of TFEB in WT HeLa cells infected with either WT or ΔIcsA or ΔIcsB Shigella flexneri for 1 h (f) or 4 h (g). (h) Protein expression and cellular localization of TFEB in WT HeLa cells infected with either WT or Δhly Listeria monocytogenes for 1 h.

Article Snippet: TABLE 1 Antibody target Use a (dilution) Manufacturer; catalog no. TFEB WB (1:1,000); IF (1:100) Cell Signaling; 4240S TFE3 WB (1:1,000) Millipore Sigma; HPA023881 GAPDH WB (1:10,000) Cell Signaling; 5174S H3 WB (1:1,000) Invitrogen; AHO1432 Galectin-8 IF (1:100) R&D Systems; MAB1305 IκBα WB (1:1,000) Abcam; ab32518 NF-κB p65 IF (1:100) Cell Signaling; 6956S Open in a separate window a IF, immunofluorescence; WB, Western blotting.

Techniques: Membrane, Bacteria, Infection, Staining, Expressing

Journal: Molecular and Cellular Biology

Article Title: Invading Bacterial Pathogens Activate Transcription Factor EB in Epithelial Cells through the Amino Acid Starvation Pathway of mTORC1 Inhibition

doi: 10.1128/mcb.00241-22

Figure Lengend Snippet: Bacterial invasion and replication, but not bacterium-induced membrane damage, are unaffected by the absence of TFEB in nonimmune cells. (a) Protein expression of TFEB in WT, TFEB-KO, and TFEB-KD HeLa cells. (b) WT and TFEB-KO HeLa cells infected with Shigella flexneri for 1 h. Membrane damage was visualized by staining with an anti-Gal-8 antibody. White squares indicate zoomed area and white arrowheads indicate Galectin-8-targeted bacteria. (c) Quantification of Galectin-8 puncta from b. (d) WT and TFEB-KO HeLa cells infected with Shigella flexneri for 1 h. Membrane damage was visualized by staining with an anti-Gal-8 antibody. (e) Quantification of Galectin-8 puncta from d. Data are represented as mean ± SEM from 3 independent experiments (*, P < 0.05; ***, P < 0.001). (f and g) Quantification of CFU values from gentamicin protection assay of WT HeLa, TFEB-KO, Scramble HeLa and TFEB-KD cells infected with Shigella flexneri (f) or Salmonella enterica Typhimurium (g) for 1 or 4 h, as indicated. (h and i) Quantification of bacterial replication (CFU 4 h/1 h) of f and g, respectively. Data are shown as mean ± SEM from 3 independent experiments.

Article Snippet: TABLE 1 Antibody target Use a (dilution) Manufacturer; catalog no. TFEB WB (1:1,000); IF (1:100) Cell Signaling; 4240S TFE3 WB (1:1,000) Millipore Sigma; HPA023881 GAPDH WB (1:10,000) Cell Signaling; 5174S H3 WB (1:1,000) Invitrogen; AHO1432 Galectin-8 IF (1:100) R&D Systems; MAB1305 IκBα WB (1:1,000) Abcam; ab32518 NF-κB p65 IF (1:100) Cell Signaling; 6956S Open in a separate window a IF, immunofluorescence; WB, Western blotting.

Techniques: Membrane, Expressing, Infection, Staining, Bacteria

Journal: Molecular and Cellular Biology

Article Title: Invading Bacterial Pathogens Activate Transcription Factor EB in Epithelial Cells through the Amino Acid Starvation Pathway of mTORC1 Inhibition

doi: 10.1128/mcb.00241-22

Figure Lengend Snippet: Comprehensive list of antibodies utilized for immunofluorescence and Western blotting

Article Snippet: TABLE 1 Antibody target Use a (dilution) Manufacturer; catalog no. TFEB WB (1:1,000); IF (1:100) Cell Signaling; 4240S TFE3 WB (1:1,000) Millipore Sigma; HPA023881 GAPDH WB (1:10,000) Cell Signaling; 5174S H3 WB (1:1,000) Invitrogen; AHO1432 Galectin-8 IF (1:100) R&D Systems; MAB1305 IκBα WB (1:1,000) Abcam; ab32518 NF-κB p65 IF (1:100) Cell Signaling; 6956S Open in a separate window a IF, immunofluorescence; WB, Western blotting.

Techniques: Immunofluorescence, Western Blot

Journal: EBioMedicine

Article Title: Three kinds of corneal host cells contribute differently to corneal neovascularization

doi: 10.1016/j.ebiom.2019.05.026

Figure Lengend Snippet: Neovascularization-related factors in corneal tissues identified by immunoblotting and ELISA. (a) Detection of MMP-2, fibronectin, COL8A2, CRYAA, COL12A1, laminin α5, galectin-8, neuropilin-2, Bcl-2, MMP-9, Bax, TIMP-2, TIMP-1, VEGF-A, CD31, VEGF-C, and beta-actin in corneal extracts by immunoblotting. C1-C5, vascularized corneal tissues of patients; N1-N3, normal corneal tissues. (b) Detection of MMP-2, TIMP-2, galectin-8, VEGF-C, VEGF-A, COL8A2, Bcl-2, MMP-9, total laminin, COL12A1, fibronectin, CD31, Bax, and neuropilin-2 in corneal extracts by ELISA. Values represent the mean ± SD. Vessel (+), vascularized corneal tissues of patients; Vessel (−), non-vascularized corneal tissues of patients; Normal, normal control corneal tissues. N = 33 for vessel (+) group; n = 21 for vessel (−) group; n = 9 for normal group.

Article Snippet: The ELISA kits used in this study include MMP-2 (ml058669; Mlbio, Shanghai, China), TIMP-2 (E-EL-H1453c; Elabscience, Houston, USA), galectin-8 (CSB-EL012894HU; CUSABIO, Wuhan, China), VEGF-C (CSB-E04759h; CUSABIO, Wuhan, China), VEGF-A (CSB-E11718h; CUSABIO, Wuhan, China), COL8A2 (ml204018; Mlbio, Shanghai, China), Bcl-2 (E-EL-H0114c; Elabscience, Houston, USA), MMP-9 (CSB-E08006h; CUSABIO, Wuhan, China), COL12A1 (ml241608; Mlbio, Shanghai, China), fibronectin (ml023813; Mlbio, Shanghai, China), CD31 (E-EL-H1640c; Elabscience, Houston, USA), Bax (E-EL-H0562c; Elabscience, Houston, USA), neuropilin-2 (CSB-EL016092HU; CUSABIO, Wuhan, China), and total laminin (E-EL-H0128c; Elabscience, Houston, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay