Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Journal of biomedical science

Article Title: SLCO4A1-AS1 promotes colorectal tumourigenesis by regulating Cdk2/c-Myc signalling.

doi: 10.1186/s12929-022-00789-z

Figure Lengend Snippet: Fig. 2 Promoter hypomethylation promotes SLCO4A1-AS1 expression in CRC. a Schematic map illustrating a predicted CpG island and its DNA methylation probes in the promoter of SLCO4A1-AS1. TSS, transcription start site. b The β-value of methylation of SLCO4A1-AS1 was lower in tumour samples than in normal tissues according to the CRC dataset of TCGA. The β-value of methylation of SLCO4A1-AS1 was linearly related to SLCO4A1-AS1 expression in CRC tissues from TCGA (c) and cell lines from CCLE (d). e Relative expression of SLCO4A1-AS1 in CRC cell lines was measured using qRT-PCR (the left panel). The methylation levels of SLCO4A1-AS1 in CRC cell lines and leukocyte cells were determined by bisulfite sequencing PCR. A total of 5 individual clones were randomly picked for sequencing (the right panel). f The mean methylation levels of these CpG sites were negatively associated with the expression levels of SLCO4A1-AS1 in CRC cells. g SLCO4A1-AS1 expression in CRC cells treated with DNA methyltransferase inhibitor (5-aza-dC). h DNA methylation analyses of SLCO4A1-AS1 in paired CRC tissues and noncancerous tissues using methylation-specific PCR assay. N, adjacent noncancerous tissue; T, tumour tissue; M, DNA marker

Article Snippet: Genomic DNA was extracted from cancer cells or human leukocytes using a Genomic DNA Mini Preparation Kit (Beyotime, China) and then bisulfite-modified using an EpiJET Bisulfite Conversion Kit (Thermo Fisher, USA).

Techniques: Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Methylation Sequencing, Clone Assay, Sequencing, Marker

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: Dynamics of DNA methylation (5mC) and mRNA m 6 A methylation in tomato fruit ripening. a Images of wild-type (WT) fruit at different ripening stages and Cnr fruit at 42 DPA. DPA, days post-anthesis; scale bar = 1 cm. b Relative 5mC levels of WT and Cnr fruit shown in a . For 5mC assay, 100 ng of genomic DNA was detected in each sample by MethylFlash™ methylated DNA quantification kit. 5mC level in each sample was normalized to that of the positive control according to the manufacturer’s instructions. The plus sign represents the average in each box. c LC-MS/MS assay showing the amount of mRNA m 6 A in WT and Cnr fruit shown in a . Data are presented as mean ± standard deviation ( n = 3). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01; Student’s t test). NS, no significance

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: DNA Methylation Assay, Methylation, Positive Control, Liquid Chromatography with Mass Spectroscopy, Standard Deviation

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: SlALKBH2 is transcriptionally regulated by DNA methylation. a The 5mC levels in the differentially methylated region (DMR) of SlALKBH2 promoter in wild-type (WT) and Cnr mutant fruit based on the Tomato Epigenome Database ( http://ted.bti.cornell.edu/epigenome/ ). b The 5mC levels in the DMR of SlALKBH2 promoter in WT and sldml2 mutant fruit based on the DNA methylome database . a , b The numbers indicate the cytosine positions relative to the start codon. Black represents the methylation frequency of cytosines at the indicated positions. DPA, days post-anthesis. c Schematic of the dual-luciferase system used for promoter activity assay. The SlALKBH2 promoter was cloned into the dual-luciferase reporter vector to activate the expression of firefly luciferase (Fluc). The renilla luciferase (Rluc) driven by the CaMV 35S promoter served as an internal control. LB, left border; RB, right border; Ter, terminator. d – f Co-expression of SlDML2 (SlDML2-HA) with the dual-luciferase reporter vector in the Nicotiana benthamiana leaves increased the relative Fluc activity ( d ), facilitated the Fluc gene expression ( e ), and reduced the 5mC level in SlALKBH2 promoter ( f ) compared with the empty plasmid control (HA). d The representative image from a total of six images (left panel). The Fluc activity was normalized against the Rluc activity, followed by normalization against the control (right panel). Data are presented as means ± standard deviation ( n = 6). Asterisks indicate significant differences (*** P < 0.0001; Student’s t test). e Gene expression was determined by quantitative RT-PCR analysis. Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (* P < 0.05; Student’s t test). f The box plot showing 5mC levels of all cytosines ( n = 41) in the DMR analyzed by Sanger bisulfite sequencing. The plus sign represents the average level in each box

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: DNA Methylation Assay, Methylation, Mutagenesis, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Expressing, Standard Deviation, Quantitative RT-PCR, Methylation Sequencing

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: SlALKBH2 is necessary for normal tomato fruit ripening. a Genotyping of mutations mediated by CRISPR/Cas9 gene-editing system in slalkbh2-23 , slalkbh2-25 , and slalkbh2-28 mutants. Diagram showing the single guide RNAs (sgRNAs) containing different target sequences (T1, T2, and T3), which were designed to specifically target the exons of SlALKBH2 . The red letters indicate the protospacer adjacent motif (PAM). The transgenic plants in the second generation were genotyped by sequencing genomic regions flanking the target sites. Red arrows indicate the editing sites. Two mutants ( slalkbh2-23 and slalkbh2-28 ) have a homozygous 1-bp insertion, and one ( slalkbh2-25 ) has a homozygous 5-bp deletion caused by target T2 in the fourth exon of SlALKBH2 . b Ripening phenotype of slalkbh2 mutants. Fruit from wild-type (WT) and slalkbh2 mutants ( slalkbh2-23 , slalkbh2-25 , and slalkbh2-28 ) at 39, 42, 47, and 52 days post-anthesis (DPA) are shown. c LC-MS/MS assay showing the amount of mRNA m 6 A in WT and slalkbh2 mutant fruit at 39 DPA. Data are presented as mean ± standard deviation ( n = 3). d m 6 A-IP-PCR assay showing the relative m 6 A enrichment in SlDML2 mRNA in WT and slalkbh2 mutant fruit at 39 DPA. e SlDML2 gene expression in WT and slalkbh2 mutant fruit at 39 and 42 DPA. The ACTIN gene was used as an internal control. d , e Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (* P < 0.05, ** P < 0.01; Student’s t test). f Model for the relationship between DNA methylation and m 6 A mRNA methylation in fruit ripening. DNA methylation negatively regulates SlALKBH2 to mediate overall m 6 A mRNA methylation. The m 6 A modification promotes SlDML2 mRNA decay, thereby affecting DNA methylation and fruit ripening

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: CRISPR, Transgenic Assay, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Standard Deviation, Expressing, DNA Methylation Assay, Methylation, Modification

Journal: BMC Cancer

Article Title: Overcoming acquired resistance to HSP90 inhibition by targeting JAK-STAT signalling in triple-negative breast cancer

doi: 10.1186/s12885-019-5295-z

Figure Lengend Snippet: Upregulation of JAK-STAT signalling pathway in HSP90i-resistant clones. a Upregulation of genes involved in JAK-STAT signalling pathway. Heatmap represents the significantly upregulated genes involved in the JAK-STAT signalling pathway in HSP90i-resistant clones CR2 and CR3 compared to parental Hs578T cells. The colour scale indicates the significant log2 FC (q-value ≤0.05) in gene expression for individual clones compared to Hs578T cells from blue (− 4, downregulation) to red (+ 4, upregulation). b Increased IL6 expression in HSP90i-resistant clones CR2 and CR3. Media from Hs578T, CR2 and CR3 cells after treatment with ganetespib (30 nM) for 24 h were subjected to the ELISA assay in order to measure the IL6 present in the media. The graph represents the mean IL6 concentration relative to DMSO-treated Hs578T cells from two independent experiments each performed in duplicate. Error bars indicate SEM. IL6 levels were significantly higher in CR2 and CR3 than Hs578T cells, where p values < 0.01 by two-way ANOVA with cell line and ganetespib treatment as factors. Ganetespib treatment did not significantly affect IL6 levels in Hs578T, CR2 or CR3 cells

Article Snippet: Cells were seeded in 96-well plate and treated the next day with 30 nM ganetespib or DMSO control for 24 h. After treatment, the media from the wells were collected and enzyme-linked immunosorbent assay (ELISA) was performed to measure the amount of IL6 in the media using the Quantikine ELISA Human IL6 kit (R&D Systems).

Techniques: Clone Assay, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Intra-tracheal infection with the USA 300 strain of MRSA induced a significant increase in pulmonary Il21 mRNA ( A ) and IL-21 protein ( B ) 24 hr after infection. ( C ) PBS or 2 μg of IL-21 was administered i.t. to WT mice one day prior to MRSA infection, and lung MRSA CFU were quantitated 7 and 24 hr later. ( D, E ) Lung immunopathology was assessed in H and E-stained sections of lung tissue from naïve uninfected mice and mice pre-treated with PBS or IL-21 and then infected for 7 or 24 hr with MRSA (in upper left panel, the bar = 500 μm and inset bar = 10 μm) ( D ), and pathology ( E ) scores were assessed. ( F–K ) animals were infected with MRSA as above. ( F ) Total lung neutrophil cellularity was quantitated by flow cytometry after staining with Ly6G and CD11b. ( G ) RNA-Seq analysis was performed on total lung tissue mRNA (pools of 5 animals) isolated 7 or 24 hr after treatment of WT mice with PBS or IL-21. Boxed regions include genes mentioned in the text. ( H–K ) RT-PCR was used to assess expression of Gzma ( H ) and Gzmb ( I ) mRNA in lungs treated with IL-21 for 7 hr, and ELISA was used to assess the induction of granzyme B ( J ) and IFNγ protein ( K ) in corresponding bronchoalveolar lavage fluid at 7 hr. Data are representative of either three ( A, B, C, F, H–K ) or two ( D, E, G ) independent experiments and validation of RNA-Seq was performed by RT-PCR of mRNA from additional mice.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, RNA Sequencing, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A–B ) Total cellularity of lung was assessed in BAL fluid ( A ) and lung ( B ) 24 hr after IL-21 intratracheal instillation. ( C ) Percentage of neutrophils was assessed by flow cytometry at 24 hr after IL-21 treatment. ( D–E ) Levels of chemokines were measured in BAL fluid at 24 hr after IL-21 treatment.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Bone-marrow neutrophils were purified by negative selection using a neutrophil isolation kit (Miltenyi) and either not stimulated or stimulated in vitro with IL-21 (100 ng/ml) for 24 hr in the absence (control) or presence of peptidoglycan (PG) (2 or 5 μg/ml); IL-21R expression on gated Ly6G + CD11b + neutrophils was detected by flow cytometry (blue = isotype control; red = anti-IL-21R). ( B, C ) IL-21 lowers the MRSA CFU in a neutrophil-dependent fashion. Mice were treated i.p. with an isotype control mAb or neutrophil-depleted with anti-Ly6G (1A8 mAb), and the efficacy of neutrophil depletion is shown in BAL and lung ( B ). Mice were then treated with PBS or IL-21 i.t., infected i.t. with MRSA, and CFU quantitated at 24 hr ( C ). CFU quantitation was performed on the left single lobe, whereas immune populations and RNA were determined using the right lobes. ( D, E ) Levels of Gzmb and Gzma mRNAs in lung tissue of untreated or neutrophil-depleted mice (treated as in panel C ) were quantitated by RT-PCR. ( F ) Levels of Gzma and Gzmb mRNAs in purified cell-sorted lung neutrophils (>98% pure Ly6G + CD11b + ) from either untreated or IL-21-treated WT or Il21r KO mice were measured by RT-PCR and normalized to Rpl7 expression. ( G, H ) Lung neutrophils were elicited by i.t treatment with heat-killed S. aureus 24 hr prior to isolation, purified, stimulated in vitro for 4 hr with either PBS or IL-21 (100 ng/ml), and Gzma ( G ) and Gzmb ( H ) mRNAs assayed by RT-PCR and normalized to Rpl7 expression. ( I ) Purified HKSA-elicited lung neutrophils were incubated in vitro with MRSA for 3 hr in the presence of PBS or IL-21 either without or with the granzyme B inhibitor, Z-AAD-CMK. MRSA CFU was quantitated by plating serial dilutions on blood agar plates. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Isolation, In Vitro, Control, Expressing, Flow Cytometry, Infection, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Quantitation of IL-21R MFI in neutrophils treated with or without IL-21 in the presence or absence of peptidoglycan (PG). ( B ) Lung neutrophils from HKSA-stimulated mice were purified via a magnetic neutrophil purification kit (Miltenyi). Shown is CD11b versus Ly6G staining.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Quantitation Assay, Purification, Staining

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Human peripheral blood neutrophils were purified by a neutrophil negative selection kit (StemCell Technologies). Shown is CD56 versus CD66b and CD8 versus CD66b staining. ( B ) Western blotting of granzyme B from neutrophils, PBMC, or A549 cells containing 0, 1, or 5% PBMC extracts, confirming that the low level of contamination of purified neutrophils cannot account for the granzyme B expression observed in this population. Also shown is western blotting for actin as a loading control.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Staining, Western Blot, Expressing, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Purified peripheral blood neutrophils were stimulated with PBS (control) or IL-21 (100 ng/ml) for 4 hr, and IL21R mRNA was measured by real-time PCR and normalized to RPL7 expression ( A ), and IL-21R protein levels were measured by flow cytometry ( B ); left panel shows isotype control shaded and anti-IL21R black line, with a summary in the right panel). ( C ) RNA-Seq was performed on neutrophils after 4 or 24 hr incubation with PBS or IL-21 in the absence or presence of heat-killed S. aureus (10 6 /ml). We used HKSA rather than live bacteria in order to allow analysis at 24 hr, as live bacteria would have overgrown the system by then. Genes differentially expressed (fold-change >2.0) are shown. Shown is a representative RNA-Seq analysis. ( D – G ) Human peripheral blood neutrophils were stimulated for 4 hr in vitro in the presence or absence of IL-21 and GZMA ( D ), GZMB ( E ), GNLY ( F ), and IFNG ( G ) mRNA levels were quantitated by RT-PCR and normalized to RPL7 expression. ( H ) Purified human neutrophils were stimulated with IL-21 for 4 hr, fixed, permealized, and stained for intracellular granzyme B protein (gated on CD66b + cells); MFIs are summarized in right panel. ( I ) Granzyme B protein was measured by ELISA in supernatants from human peripheral blood neutrophils cultured for 24 hr in either the absence or presence of IL-21 (100 ng/ml). ( J, K ) Human neutrophils were incubated in vitro with either MRSA ( J ) or S. pyogenes ( K ) for 3 hr with PBS or IL-21 (100 ng/ml). MRSA and S. pyogenes CFU were quantitated by plating serial dilutions on blood agar plates. Results shown are representative of 3 independent experiments, except panel C shows one of two similar independent RNA-Seq experiments, each from a different donor.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Control, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, RNA Sequencing, Incubation, Bacteria, In Vitro, Reverse Transcription Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: Reactive oxygen species were measured by flow cytometry in CellRox Red loaded peripheral blood neutrophils that were stimulated at 37°C for 30 min with PBS, IL-21, HKSA, or HKSA +IL-21.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT and Il21r KO mice were infected intratracheally with MRSA (2 × 10 7 ) and CFU in the lung were quantitated at 4 and 24 hr post-infection. ( B ) WT and Il21r KO mice were infected in parallel with WT mice that had been treated with PBS or 2 μg IL-21, and CFU in the lung were quantitated at 7 and 24 hr post infection. ( C–E ) Lung immunopathology was assessed in H and E-stained lung sections (bar in upper left panel = 500 μm; bar in inset = 10 μm) ( C ); lung neutrophil cellularity as assessed by pathology score ( D ) and flow cytometry ( E ) were assessed at 4 and 24 hr post-infection. ( F ) WT or Il21r KO mice were pre-treated with an isotype control antibody or anti-Ly6G to deplete neutrophils, infected with MRSA, and CFU quantitated at 24 hr. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT mice were treated intratracheally with 50 μg of control Fc (from IgG1) or IL-21R-Fc protein for 2 days prior to infection with MRSA and lung CFU were quantitated at 4 and 24 hr post-infection with MRSA. ( B ) Histology of lungs in control Fc or IL-21R-Fc pre-treated mice at 4 and 24 hr post-infection (bar in upper left panel = 500 μm; bar in inset = 10 μm). ( C ) IFNα levels in BAL fluid were measured by ELISA 4 hr after infection in mice pretreated with control Fc or IL-21R-Fc. ( D ) Gzmb mRNA was measured in lungs of WT or Il21r KO mice pre-treated with isotype control or anti-IFNAR1 antibodies prior to MRSA infection, and normalized to Rpl7 mRNA expression. ( E ) Gzmb mRNA was measured in lungs of mice treated with either control Fc or IL-21R-Fc prior to MRSA infection and normalized to Rpl7 expression. ( F ) Like IL-21, IFNβ also induced GZMB mRNA in human peripheral blood neutrophils and normalized to RPL7 expression. ( G ) IFNβ induces increased in vitro killing of MRSA by human peripheral blood neutrophils and this was prevented by a granzyme B inhibitor, Z-AAD-CMK. ( H–J ) Co-cultures of CD4 + T cells and dendritic cells were stimulated with SEB either in the presence of control Fc or IL-21R-Fc, and mRNA was quantitated by RT-PCR after 24 hr and normalized to Rpl7 expression. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: MRSA infection, as detailed in the legend to . ( A, B ) Pathology and neutrophil infiltration scores were assessed from H and E-stained lung sections shown in . ( C ) RNA-Seq was performed on total lung mRNA either before infection (Ctrl) or at 4 hr after MRSA infection of mice pretreated with either Fc protein or an IL-21R-Fc fusion protein. Representative of 2 independent RNA-Seq experiments.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, RNA Sequencing

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Peripheral blood neutrophils from normal donors (NDs) or AD-HIES patients (PTs) were assayed using an in vitro MRSA killing assay in the absence or presence of IL-21 (100 ng/ml). ( B ) RT-PCR was used to quantitate GZMB mRNA in neutrophils 4 hr after in vitro stimulation without or with IL-21. Expression was normalized to RPL7 expression ( C ) RNA-Seq was performed on normal donor and AD-HIES patient neutrophils stimulated for 4 hr with PBS, IL-21, IFNβ, or IL-21 +IFNβ. Genes differentially expressed (fold-change >1.5) in two independent RNA-Seq analyses are shown. ( D ) RT-PCR normalized to RPL7 expression was used to validate the expression pattern of GBP1 and GBP2 in neutrophils from additional normal donors and AD-HIES patients. ( E–G ) Mutant Stat3 transgenic mice were treated i.t. with PBS or 2 μg IL-21, infected i.t. 24 hr later with MRSA, and at 7 hr post-infection lung MRSA CFU quantitated ( E ), and IFNα levels were measured in the serum ( F ) and BAL fluid ( G ).

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing, Mutagenesis, Transgenic Assay, Infection

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) In wild-type (WT) mice, MRSA produces SEB, which bridges between T cells and dendritic cells by interacting with TCR and MHCII, leading to the production of IL-21 by CD4 + T cells. IL-21 stimulates neutrophils to release granzymes, with enhanced killing of MRSA. Type I IFN (e.g., produced by dendritic cells, as shown in the cartoon) can also induce granzyme production to promote killing of MRSA, but IL-21 inhibits dendritic cell production of type I IFN. ( B ) In the absence of either IL-21R or a functional STAT3 signaling response, the IL-21-induced neutrophil granzyme production does not occur. However, IL-21-mediated repression of type I IFN production by dendritic cells also no longer occurs, which leads to enhanced production of type I IFN and hence increased killing of MRSA through this pathway. The functional cross-talk of and relative potency of the IL-21- and type 1 IFN-mediated pathways and levels of each cytokine influence the outcome.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Produced, Functional Assay

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet:

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vivo, Flow Cytometry, Blocking Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Isolation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 is overexpressed in OSCCs patient samples and OSCC-derived cell lines. Total RNA from fresh samples and cell lines was converted to cDNA and subjected to qPCR. For gene expression analysis of tissue samples relative quantification was based on the comparison of a pool of five normal oral tissues, while the spontaneously immortalized but non-transformed epithelial cell line HGK was used as reference for comparison with OSCC-derived cell lines. The levels of HPSE1 mRNA were significantly higher in OSCC cell lines compared with HGK cells (A) . The high expression levels of protein HPSE1 were confirmed on OSCC-derived cell lines by Western Blot analysis (B) . The levels of HPSE1 mRNA were also significantly higher in OSCC tissue samples compared to normal oral mucosa (C) . Representative images in a high-power field (200×) of immunohistochemical analysis for HPSE1 in Normal oral tissue (D) and OSCC tissue preparations confirmed its higher expression at protein level; with a distinct cytoplasmic distribution and intensity in oral cancer samples expressing both higher (E) and lower (F) levels of HPSE1. Results were statistically determined by ANOVA followed by Tukey multiple comparison test, where ** p < 0.005, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Derivative Assay, Expressing, Transformation Assay, Western Blot, Immunohistochemical staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 overexpression discriminates OSCC and normal samples and predicts low patient survival. (A) Kaplan–Meier cumulative curves for disease-free survival of patients with OSCC as a function of HPSE1 expression, showing a 70% probability of death in patients with higher expression of HPSE1 compared with those with low expression. (B) Receiver operating characteristic (ROC) curves showing the ability of the HPSE1 overexpression distinguishes OSCC tumors from normal oral samples. (C) Kaplan–Meier of multivariate survival analysis combining HPSE1 expression levels with pT stages, where score 1: HPSE1 lower expressing samples associated with pT1/2, score 2: HPSE1 lower expressing samples associated with pT3/4 or higher HPSE1 with pT1/2, and score 3: HPSE1 higher expressing samples associated with pT3/4. These results confirmed that HPSE1 overexpression was significantly associated with higher grade tumors.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 knockdown and upregulation efficiency in OSCC cells. The efficiency of endogenous HPSE1 modulation was verified by Immunofluorescence (A) , Western Blot (B) and RT-qPCR (C) analyzes. For the loss-of-function strategy, SCC-9 cells were transduced with shRNA expressing a vector sequence against HPSE1 (shRNA HPSE1−) and empty vector (scrRNA control). For the gain-of-function strategy, SCC-9 cells were transduced with a ORF clone in a shuttle vector to enhance HPSE1 expression (orfRNA HPSE1+), along with mock-transduced cells as described in Methods . shRNA HPSE1− cells showed a significant reduction and orfRNA HPSE+ cells showed a significant upregulation of HPSE1 in both mRNA and protein levels compared to scrRNA control and parental mock-transduced SCC-9 control cells (pSCC9), without targeting sequences. Results were statistically determined by ANOVA, followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Immunofluorescence, Western Blot, Quantitative RT-PCR, Transduction, shRNA, Expressing, Plasmid Preparation, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Downregulation of HPSE1 inhibits proliferation and enhances apoptosis of OSCC cells. Cells were subjected to MTT cell proliferation (A) , DNA content cell cycle analysis (B) , and apoptosis (C) assays. (A) Cell Proliferation Assay of the OSCC SCC9 cell line (Control) compared to empty vector SCC9 control clone (ScrRNA control), HPSE1 inhibitory/silencing SCC9-ShRNA HPSE1− clone, and the HPSE1 overexpressing SCC9-OrfRNA HPSE1+ clone. (B) Quantification of cell cycle analysis was performed by flow cytometry after staining with propidium iodide for the SCC-9 control cells and the respectives HPSE1-modulated clones. Abrogation of HPSE1 induced arrest of the SCC-9 cells cycle in G1 phase, and its upregulation significantly induced cell proliferation. (C) Flow cytometric analysis of apoptosis showed a remarkable increase in the number of apoptotic cells in HPSE1-silenced cells (ShRNA HPSE1−), while SCC-9 overexpressing HPSE1 (Orf-RNA HPSE1+) exhibited a reduction in apoptosis. (D) No significant differences in cell adhesion properties were observed among any transduced clones modulating HPSE1 expression, compared to parental mock-transduced cells (control). Plots compose experimental triplicate analysis and were statistically calculated using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: MTT Cell Proliferation, Cell Cycle Assay, Proliferation Assay, Plasmid Preparation, shRNA, Flow Cytometry, Staining, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 is associated with migration, invasion, ECM remodeling, and acquisition of EMT properties. (A) Photomicrographs of cell lines were taken 0, 24, and 48 h after wounding (40X). The average width of the lacunae was measured. Migration of SCC-9 cells was significantly decreased in HPSE1-silenced cells (ShRNA HPSE1−) and increased in HPSE1-upregulated clones (OrfRNA HPSE1+). Migration analysis based on this assay showed that cells with lower expression of HPSE1 closed the scratch wound significantly slower than the SCC9 Control cells. (B) Invasion of SCC-9 cells was significantly inhibited by HPSE1 knockdown, and significantly enhanced after its upregulation. (C) Analysing EMT markers, the downregulation of HPSE1 significantly induced the expression of E-Cadherin (E-CAD), while OrfRNA HPSE1+ SCC9 clones overexpressing heparanase had a significant increasing of Vimentin (VIM) and SNAIL expressions. (D) The upregulation of HPSE1 significantly enhanced the expressions of MMP2 and MMP9. All the graphs compile experimental triplicate analyses, and the results were obtained by ANOVA followed by Tukey assay, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Migration, shRNA, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 enhances tumor neo-vascularization and induces VEGFA expression. HUVEC cells on a Miogel 2D layer co-culture. Photomicrograph of HUVEC endothelial cells added with preconditioned medium from OSCC cell lines and their respective clones after 12 h of experiment: (A) HUVEC cells incubated with preconditioned medium of SCC9-OrfRNA HPSE1+ clone overexpressing HPSE1 (100×); (B) HUVEC cells incubated with preconditioned medium of parental SCC9 (Control) (100×); (C) HUVEC cells incubated with preconditioned medium of SCC9-ShRNA HPSE1- clone with reduction of HPSE1 expression (100×). (D) Quantification of measures of the vessel circumference perimeter for each one of the conditions; the graph compiles two experimental triplicate analysis. (E) Gene expression analysis of endothelial growth factor VEGFA by qRT-PCR; the graph compiles folded expression values of relative quantification by ddCT obtained through comparison of clones OrfRNA HPSE1+ and ShRNA HPSE1− relative to the control SCC9 cells (normal reference = 1). Normalization of the analysis was performed using the endogenous control gene, PPIA. Statistical tests were performed using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing, Co-Culture Assay, Clone Assay, Incubation, shRNA, Quantitative RT-PCR