Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: NRVMs were transduced with AdV5-CMV-CRYAB R120G -GFP and treated with 3 µM ruxolitinib or DMSO or 100 nM siRNAs targeting either JAK1 (siJak1), JAK2 (siJak2), STAT3 (siStat3) or scramble siRNA (scr). Thereafter, NRVMs were harvested or fixed after 4-6 days. A , Treatment with ruxolitinib or DMSO; Scale bar = 100 µm. B , Transfection with siJak1; Scale bar = 200 µm. C , Transfection with siJak2; Scale bar = 100 µm D , Transfection with siStat3, Scale bar = 20 µm. A , B , C , D , Western blots of protein extracts from treated NRVMs were stained with antibodies directed against indicated proteins. In the representative immunofluorescence images, aggregates are depicted in magenta (CRYAB R120G -GFP), cardiomyocytes in yellow (anti-cardiac troponin I), and nuclei in blue (DAPI). Quantification of aggregates in cardiomyocytes with NIS Elements or ImageJ software. Data were obtained from 1 (Western blot) or 2 (immunofluorescence) NRVM preparations with at least 3 wells per condition and at least 7 images per well for immunofluorescence. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transduction, Transfection, Western Blot, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: A , Heart weight-to-body weight ratio (HW/BW) in 1-, 4- and 7-month-old CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice. B , Ejection fraction (EF) in 7-month-old R120G and NTG mice. C , Representative Western blots of phosphorylated (P-)STAT3, STAT3 and indicated control in 1-, 4- and 7-month-old R120G and NTG mice. D , P-STAT3/STAT3 quantification of Western blots from 1-, 4- and 7-month-old R120G and NTG mice. E , Stat3 mRNA level determined by RT-qPCR from 7-month-old R120G and NTG mice. Western blot quantification was performed with Image Lab software. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Western Blot, Control, Quantitative RT-PCR, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice treated for 3 weeks with 75 mg/kg ruxolitinib (ruxo) or vehicle (veh) twice-daily oral gavage. Transthoracic echocardiography was performed at the start (before, 21-week-old) and the end of the treatment (veh or ruxo, 24-week-old). A , Scheme of experimental outline. B , WB and quantification of phosphorylated (P-) STAT3, STAT3 and indicated controls at the end of treatment. WB quantification was performed with Image Lab software. C, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW) before and after (veh/ruxo) treatment. D , Heart weight-to-body weight ratio (HW/BW) and body weight (BW) at the end of the treatment. E , Representative images and quantification of R120G and NTG mouse heart sections after vehicle or ruxolitinib treatment. CRYAB is depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 10 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one- way ( B ) or two-way ANOVA and Tukey’s multiple comparisons post-hoc analysis ( C, D ) or unpaired Student’s t-test ( E ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: Heterozygous (het) or homozygous (hom) Jak1 knockout (KO) was induced in MCM-transgenic (TG) mice crossed with CRYAB p.Arg120Gly TG (R120G) or non-transgenic (NTG) mice with tamoxifen chow. Transthoracic echocardiography was performed 26 weeks and hearts were extracted. A , Scheme of experimental outline. B , Jak1 mRNA levels determined by RT-qPCR. C , JAK1 protein levels determined by Western blot and normalized to ACTN2. Western blot quantification was performed with Image Lab software D, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW). E , Heart weight-to-body weight ratio (HW/BW) and body weight (BW). F , Representative images and quantification of mouse heart sections. Aggregates are depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 50 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one-way ( B ) or two- way ANOVA ( D , E ) with Tukey’s multiple comparisons post-hoc analysis or unpaired Student’s t-test ( C , F ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Knock-Out, Transgenic Assay, Quantitative RT-PCR, Western Blot, Software

Journal: Experimental Animals

Article Title: Systemic autoimmune abnormalities alter the morphology of mucosa-associated lymphoid tissues in the rectum of MRL/MpJ- Fas lpr/lpr mice

doi: 10.1538/expanim.23-0129

Figure Lengend Snippet: Immune cell composition of the rectal mucosa-associated lymphoid tissue (RMALT) in mice. Mice were analyzed at 34 weeks of age. (a) Gene expression of immune cell markers in the anorectal regions of MRL/MpJ- Fas +/+ (MpJ) and MRL/MpJ- Fas lpr/lpr (Lpr) mice via quantitative PCR. (b) Using immunofluorescence, the localization of CD3 + T cells, B220 + B cells, and IBA1 + macrophages in RMALTs were examined. White dotted lines indicate RMALTs. The nuclei were visualized using Hoechst 33342 staining. Bars=100 µ m. (c) Percentage of CD3 + T cells, B220 + B cells, and IBA1 + macrophages in RMALTs. Histoplanimetry. Significant differences between the strains are indicated by * P< 0.05, Mann-Whitney U test. Values=mean ± SE. n≥4.

Article Snippet: Purified total RNA was used as a template to synthesize cDNA using the ReverTra Ace quantitative polymerase chain reaction (qPCR) RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). qPCR analysis was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and gene-specific primers.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, MANN-WHITNEY

Journal: Experimental Animals

Article Title: Systemic autoimmune abnormalities alter the morphology of mucosa-associated lymphoid tissues in the rectum of MRL/MpJ- Fas lpr/lpr mice

doi: 10.1538/expanim.23-0129

Figure Lengend Snippet: The appearance of high endothelial venules (HEVs) and expression of chemokine molecules in the rectal mucosa-associated lymphoid tissues (RMALTs) of mice. Mice were analyzed at 30 weeks of age. (a) Localization and number of PNAd + HEVs in RMALTs of MRL/MpJ- Fas +/+ (MpJ) and MRL/MpJ- Fas lpr/lpr (Lpr) mice through immunohistochemistry and histoplanimetry. Insets magnify the area in the black square. Black arrowheads indicate HEVs. Bars=100 µ m. (b) Gene expression of chemokine ligands and their receptors in the anorectal parts of mice via quantitative PCR. (c) Localizations of CXCL13 and CXCL9 in RMALTs via immunofluorescence. (d) Ki67 + proliferative cells in RMALTs. White dotted lines indicate RMALTs. The nuclei were visualized using Hoechst 33342 staining. Scale Bars=100 µ m. Significant differences between the strains are indicated by * P< 0.05, Mann-Whitney U test. Significant differences between the strains are indicated by * P< 0.05, Mann-Whitney U test. Values=mean ± SE. n≥4.

Article Snippet: Purified total RNA was used as a template to synthesize cDNA using the ReverTra Ace quantitative polymerase chain reaction (qPCR) RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). qPCR analysis was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and gene-specific primers.

Techniques: Expressing, Immunohistochemistry, Gene Expression, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, MANN-WHITNEY

Journal: Experimental Animals

Article Title: Systemic autoimmune abnormalities alter the morphology of mucosa-associated lymphoid tissues in the rectum of MRL/MpJ- Fas lpr/lpr mice

doi: 10.1538/expanim.23-0129

Figure Lengend Snippet: Localization of M cells and goblet cells in the epithelium covering rectal mucosa-associated lymphoid tissues (RMALTs) in mice. Mice were analyzed at 30 weeks of age. (a) Localization of GP2 + M cells in the epithelium covering RMALTs of MRL/MpJ- Fas +/+ (MpJ) and MRL/MpJ- Fas lpr/lpr (Lpr) mice via immunofluorescence. Insets magnify the area in the white square including GP2 + M cells (yellow arrowheads). Yellow and white dotted lines indicate borders with the lumen and lamina propria, respectively. The nuclei were visualized using Hoechst 33342 staining. Bars=100 µ m. (b) Localization of periodic acid-Schiff (PAS) + goblet cells in the epithelium covering RMALTs. PAS staining. Black arrowheads indicate PAS + goblet cells. Insets magnify the area in the black square. Bars=100 µ m. (c) Gp2 gene expression levels and quantitative values for GP2 + M cells and goblet cells in the anorectal parts of mice were determined via quantitative PCR and histoplanimetry. Significant differences between the strains are indicated by * P< 0.05, Mann-Whitney U test. Values=mean ± SE. n≥4. Bars=100 µ m.

Article Snippet: Purified total RNA was used as a template to synthesize cDNA using the ReverTra Ace quantitative polymerase chain reaction (qPCR) RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). qPCR analysis was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and gene-specific primers.

Techniques: Immunofluorescence, Staining, Gene Expression, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Heliyon

Article Title: Inhibition of CTLA-4 accelerates atherosclerosis in hyperlipidemic mice by modulating the Th1/Th2 balance via the NF-κB signaling pathway

doi: 10.1016/j.heliyon.2024.e37278

Figure Lengend Snippet: The anti-CTLA-4 antibody promotes CD4 + T-cell infiltration and T-cell differentiation into Th1 cells in the aortic root. (A) GO enrichment analysis showing the significantly enriched biological processes in hyperlipidemic mice administered the anti-IgG antibody or anti-CTLA-4 antibody. n = 3 per group. (B) GSEA of RNA-seq data showing the enrichment of “regulation of Th1-type immune response” and “Th1 cell differentiation” in hyperlipidemic mice administered the anti-IgG antibody or anti-CTLA-4 antibody. NES: normalized enrichment score; FDR: adjusted p value. (C) Representative immunofluorescence staining of CD4 (red), α-SMA (green), and DAPI (blue) in mice in the indicated groups. The square represents the colocalization of CD4 with the smooth muscle marker α-SMA. The scale bars are 100 μm and 20 μm. (D) Quantitative analysis of CD4 fluorescence intensity in aortic roots in the indicated groups. The data are presented as the means ± SEM (n = 6). (E) Flow cytometric analysis of the distributions of CD4 + IFN-γ + T cells and CD4 + IL-4 + T cells in peripheral blood in the indicated groups. (F) Relative quantification of the percentages of CD4 + IFN-γ + T cells and CD4 + IL-4 + T cells among the sorted CD4 + T cells. The data are presented as the means ± SEM (n = 5). (G) The Th1/Th2 ratio was estimated by determining the ratio of CD4 + IFN-γ + IL-4 − cells to CD4 + IFN-γ − IL-4 + cells. The data are presented as the means ± SEM (n = 5). (H) Quantitative results were obtained by real-time PCR showing the relative mRNA expression of IFN-γ and IL-4 in the indicated groups. The data are presented as the means ± SEM (n = 6). *P < 0.05 vs . the anti-IgG group. (I – J) Quantitative analyses demonstrating the number of CD4 + IFN-γ + T cells and CD4 + IL-4 + T cells in aortic roots from each indicated experimental group. The data are presented as the means ± SEM (n = 4). *P < 0.05 vs . the anti-IgG group.

Article Snippet: One hundred microliters of mouse peripheral blood were collected in isotonic RPMI 1640 complete culture medium (R8758, Sigma-Aldrich) and stimulated with 2 μL of leukocyte activation cocktail (550583, BD Biosciences) in 5 % CO 2 at 37 °C for 4 h. Then, the cells were treated with anti-mouse CD3 (APC-Cy7-CD3, 1:200, 557596, BD Biosciences) and anti-mouse CD4 (BV510-CD4, 1:200, 563106, BD Biosciences) for 30 min and lysed in red blood cell lysis solution (130-094-183, Miltenyi Biotec) for 10 min. After being treated with fixation/permeabilization concentrate at room temperature for 1 h, total T lymphocytes were further stained with anti-mouse IFN-γ (FITC-IFN-γ, 1:100, 554411, BD Biosciences) and anti-mouse IL-4 (PE-IL-4, 1:100, 554435, BD Biosciences) for 30 min. Isotype controls were used for compensation and to confirm antibody specificity.

Techniques: Cell Differentiation, RNA Sequencing, Immunofluorescence, Staining, Marker, Fluorescence, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza modulated the neutrophil-associated TIME in the lungs. (A) Experimental setup. The mice were constructed as experimental metastasis models after a period of acclimatization. The mice were prepared as experimental metastasis models following an acclimatization period. Ginseng and Salvia miltiorrhiza extracts were administered daily to the relevant groups according to the schematic diagram, and lung tissues were collected on day 28. (B) Representative bioluminescence images of metastasis in the intravenously implanted LLC mice. (C) Representative H&E images of the tumor area in the lungs of the mice (scale bar: 50 μm). (D) Quantitative analysis of the lung tumor area ( n = 3). (E) Quantitative analysis of the number of lung metastatic nodules in tumor-bearing mice( n = 15). (F) Metastasis rates of LLC tumor-bearing mice ( n = 15). (G) GOBP analysis of intersecting genes in the control, model and G + S groups. (H) GSEA of genes related to neutrophil migration. (I) Representative images of immunohistochemical staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 50 μm). (J) Representative images of immunofluorescence staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 100 μm). All the data are presented as mean ± SEM. #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Construct, Control, Migration, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: CPT inhibited CD62E–mediated neutrophil adhesion and recruitment to metastases. (A) Representative images and quantification of HL–60 cell adhesion to HUVECs (scale bar: 200 μm). (B) Schematic diagram of the parallel plate flow chamber combined with the microscope. (C) Number of neutrophils firmly adhered to HUVECs. (D) Representative immunofluorescence images and quantification of CD62E in the lungs ( n = 3; scale bar: 50 μm). (E) Representative western blot images of CD62E levels in the lungs and quantitative data ( n = 3). GAPDH was used as a loading control. (F) The mRNA expression levels of TNF–α, ICAM1, and VCAM1 in lung tissues were determined by qRT–PCR ( n = 3). (G) The correlation between CD62E expression and neutrophil infiltration in LUAD and LUSC was analyzed via TIMER. (H) Representative images and quantification of Ly6G + neutrophils in lungs detected by flow cytometry ( n = 4). (I) Representative immunofluorescence images and quantification of Ly6G + neutrophils in the lungs (n = 3; scale bar: 50 μm). All the data are presented as mean ± SEM. ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Microscopy, Immunofluorescence, Western Blot, Control, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Neutrophil RNA sequencing analysis and correlation analysis of NETs and lung cancer. (A) Schematic diagram of neutrophil isolation and RNA sequencing from mouse lung tissues. (B-C) Heatmap analysis of neutrophil-related genes and KEGG analysis of altered genes. (D) Results of partial GOBP analysis of intersecting genes in the control, model and G + S groups. (E) The expression of HIST3H3 in different cancers and adjacent normal tissues was analyzed via TIMER. (F–H) The correlation between copy number alterations in HIST3H3, ELANE, and MPO and neutrophil infiltration was analyzed via TIMER. (I) The correlations between cumulative survival and HIST3H3, ELANE, MPO and neutrophil infiltration in lung cancer patients were analyzed via TIMER. A two-sided Wilcoxon rank-sum test was used. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: RNA Sequencing, Isolation, Control, Expressing

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza reduced the generation of NETs in lung tissues, and effective substances from ginseng were screened. (A) Representative immunohistochemical images of MPO expression in the lungs (scale bar: 100 μm). (B) Representative immunofluorescence images of H3cit and NE in the lungs (scale bar: 100 μm). (C–E) Quantitative analysis of MPO, H3cit and NE expression in the lungs ( n = 3). (F) Neutrophil MPO release was detected by ELISA. The chemical structure of Rg1 is shown on the right. (G) Representative images and quantification of extracellular DNA release after Sytox™ Green staining (scale bar: 50 μm). All the data are presented as mean ± SEM. ####p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reduced NET formation by inhibiting ROS production and ERK1/2 and MAPK phosphorylation. (A) Representative immunofluorescence images of the NET components H3cit and NE in neutrophils ( n = 3; scale bar: 50 μm). (B–C) Representative western blot images of H3cit expression in neutrophils and quantitative data. GAPDH was used as a loading control ( n = 3). (D-E) ROS production in dHL-60 cells (D) and neutrophils (E) was assessed by flow cytometry. The median fluorescence intensity of ROS was used for quantification. (F-G) Representative western blot images and quantification of p-ERK1/2, ERK1/2, p-MAPK and MAPK expression in dHL-60 cells (F) and neutrophils (G). GAPDH was used as a loading control. All the data are presented as mean ± SEM. # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Expressing, Control, Flow Cytometry, Fluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reversed NET-induced invasion, EMT, and adhesion of LLC cells and increased vascular permeability. (A) LLC cell proliferation was detected via a CCK8 assay. (B) Schematic diagram of NET isolation and coculture with LLC cells in the transwell system. (C) Images of the transwell invasion assay (scale bar, 200 μm). The number of infiltrated cells and the OD values were used for quantification. (D-E) Representative immunofluorescence images and quantification of MMP9 in the lungs (n = 3, scale bar 50 μm). (F) Representative immunofluorescence images of E-cadherin and N-cadherin in LLC cells treated with or without Rg1 or NETs. (G-I) Representative immunofluorescence images and quantification of E-cadherin and N-cadherin in the lungs ( n = 3, scale bar 50 μm). (J) Schematic diagram of the adhesion assay of LLC cells by NETs. (K) Representative fluorescence images of the adhesion assay for DiI-labeled LLC cells trapped within PMA- or Rg1-treated neutrophils. (L) RNA-seq analysis of neutrophils. GSEA of genes related to the regulation of vascular permeability. (M) Schematic diagram of the endothelial permeability assay. (N) The effects of Rg1 or NETs on vascular permeability were tested via an endothelial permeability assay. (O-P) Representative immunofluorescence images and quantification of CD31 and FITC-dextran in the lungs ( n = 3; scale bar, 50 μm). All the data are presented as mean ± SEM. # p < 0.05, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Permeability, CCK-8 Assay, Isolation, Transwell Invasion Assay, Immunofluorescence, Cell Adhesion Assay, Fluorescence, Labeling, RNA Sequencing, Control