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: Biomedicines

Article Title: In Vivo Ca V 3 Channel Inhibition Promotes Maturation of Glucose-Dependent Ca 2+ Signaling in Human iPSC-Islets

doi: 10.3390/biomedicines11030807

Figure Lengend Snippet: Effects of intravitreal infusion of NNC55-0396 on Ca V 3 currents in insulin-expressing cells of retrieved intraocular hiPSC-islet grafts. ( A ) Slow deactivating tail current traces evoked by a depolarizing voltage pulse from −80 mV to +20 mV for 5 ms back to −70 mV ( upper panel ) in an insulin-positive cell of intraocular hiPSC-islet grafts retrieved at 2 months post-transplantation before (red) and when exposed to NNC55-0396 during patch clamp recordings (blue). ( B ) Sample slow deactivating tail current traces ( left ) and quantification of the average density of slow deactivating tail currents ( right ) registered in insulin-positive cells of intraocular hiPSC-islet grafts retrieved at 2 months post-transplantation following intravitreal infusion of vehicle or NNC55-0396; n = 13 for group before intravitreal infusion of vehicle (before vehicle), n = 15 for group after intravitreal infusion of vehicle (after vehicle), n = 13 for group before intravitreal infusion of NNC55-0396 (before NNC55-0396) and n = 15 for group after intravitreal infusion of NNC55-0396 (after NNC55-0396). ( C ) RT-PCR analysis of cDNA generated by reverse transcription of mRNA in insulin-positive cells of intraocular hiPSC-islet grafts retrieved after patch clamp recordings as well as a human islet β cell, negative control (sterile ultrapure water), COS7 cell and HEK293 cell with specific primers for insulin (146 bp amplicon).

Article Snippet: Insulin mRNA in collected cells was detected using the QIAGEN OneStep RT-PCR Kit (Valencia, CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Transplantation Assay, Patch Clamp, Reverse Transcription Polymerase Chain Reaction, Generated, Negative Control, Amplification

Journal: Science signaling

Article Title: Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling

doi: 10.1126/scisignal.abb0619

Figure Lengend Snippet: (A) Schematic of the experimental procedures. Splenocytes were magnetically isolated and then activated with a mixture of anti-CD3 and anti-CD28 antibodies (αCD3/CD28) or Con with A. (B) Flow cytometry histograms showing the cell surface abundance of the activation marker CD69 on CD4+CD25− primary T cells from WT and KO mice after in vitro activation for 48 hours by Con A (5 μg/ml) (left) or by a mixture of anti-CD3 and anti-CD28 antibodies (right). Blue- and red-filled histograms show CD69 abundance on naïve WT and KO cells, respectively. Unfilled histograms show CD69 after activation. Histograms are representative of three independent experiments. (C) Flow cytometry analysis of the abundance of the activation marker CD25 on WT and KO CD4+CD25− T cells after stimulation as described in (B). Histograms are representative of three independent experiments. (D) Representative histograms showing forward scatter (FSC) as a measure of activation for WT (blue) and KO (red) CD4+CD25− T cells (n=3). (E) Analysis of the division of CFSE-labeled WT and KO CD4+CD25− T cells as measuring the dilution of CFSE 72 hours after activation by anti-CD3 and anti-CD28 antibodies. The percentage of dividing cells (left gate) and undivided cells (right gate) are shown. Data are representative of three experiments. (F) Ca2+ responses in WT (blue) and KO (red) thymocytes after TCR stimulation. The intracellular Ca2+ increase was observed in response to the binding of streptavidin (SA) to biotinylated anti-CD3e, which was previously added to the cells. Ionomycin (IM) was used as a positive control and denotes maximal Ca2+ responses. The number of cells assessed is shown. The inset bar graph illustrates the rise-time (tau) of fluorescence upon streptavidin treatment in WT and KO thymocytes. (G) Quantification of secreted cytokines as detected in the cell culture medium of CD4+CD25− T cells activated for 72 hours with Con A (5 μg/ml). The P values were calculated by two-tailed t-test and are denoted on the box charts (n=3-4). (H) Quantification of secreted cytokines as detected in the cell culture medium of CD4+CD25− T cells activated for 72 hours with a mixture of anti-CD3 and anti- CD28 antibodies (each at 5 μg/ml). The P values were calculated by two-tailed t-test and are denoted on each box chart (n = three or four samples).

Article Snippet: The following sets of magnetic beads were used for T cell isolation: Dynabeads Untouched Mouse T Cells (Life Technologies, #11413D), CD4 + CD25 + Regulatory T Cell Isolation Kit mouse (Miltenyi Biotech, #130-091-041), and Dynabeads FlowComp Mouse CD4 + CD25 + T reg Cells (Life technologies, #11463D).

Techniques: Isolation, Flow Cytometry, Activation Assay, Marker, In Vitro, Labeling, Binding Assay, Positive Control, Fluorescence, Cell Culture, Two Tailed Test

Journal: Science signaling

Article Title: Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling

doi: 10.1126/scisignal.abb0619

Figure Lengend Snippet: (A) Experimental scheme. (B) Bivariant cytographs showing infiltration of CD45+ hematopoietic cells into the livers of WT (blue) and KO (red) mice 24 hours after the administration of saline or Con A (40 mg/kg, i.v.). CD45 FMO refers to the ”fluorescence minus one” control, wherein cells were analyzed without staining with anti-CD45 antibody to enable accurate gating. Plots are representative of four to six experiments. (C) Quantification of the number of CD45+ hematopoietic cells based on the flow cytometry analysis shown in (B). Mean values are indicated by a dash. The data were collected from four independent experiments. (D) Bivariant cytographs showing the infiltration of CD45+CD4+ T cells into the livers of WT and KO mice 24 hours after administration of saline or Con A (40 mg/kg, i.v.). Plots are representative of four to six experiments. (E) Quantification of the number of CD45+CD4+ T cells based on the flow cytometry analysis shown in (D). Mean percentage values are indicated by a dash. The data were collected from four independent experiments. (F) Flow cytometry histograms of CD4+Foxp3+ cells from the livers of WT (blue) and KO (red) mice 24 hours after administration of saline or Con A. The Foxp3 histograms were obtained from a cell population determined by sequential gating for a FSC/SSC profile, CD45+ and CD4+ (see fig. S2 for the gating scheme). (G) Quantification of Foxp3+ Treg cells in the livers of WT (blue) and KO (red) mice from the experiments shown in (F). The P value was calculated by t test. The data were collected from four independent experiments. (H) Immunofluorescence microscopy of infiltrating CD4+ T cells in liver sections. (I) Representative SEM images of liver sections from the indicated mice at 850X magnification. Scale bar, 100 μm.

Article Snippet: The following sets of magnetic beads were used for T cell isolation: Dynabeads Untouched Mouse T Cells (Life Technologies, #11413D), CD4 + CD25 + Regulatory T Cell Isolation Kit mouse (Miltenyi Biotech, #130-091-041), and Dynabeads FlowComp Mouse CD4 + CD25 + T reg Cells (Life technologies, #11463D).

Techniques: Saline, Fluorescence, Control, Staining, Flow Cytometry, Immunofluorescence, Microscopy

Journal: Science signaling

Article Title: Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling

doi: 10.1126/scisignal.abb0619

Figure Lengend Snippet: (A) Left: qRT-PCR analysis measuring the relative expression of Foxp3 in WT and KO thymocytes activated with anti-CD3 and IL-2 at the indicated times. Right: Analysis of Foxp3 induction in KO thymocytes in the presence of 2 nM CP690550 (JAK inhibitor) at the 48-hour time-point. C, untreated control. Both bar graphs denote mean values and the error bars indicate the SEM (n = 3 experiments). The P value was calculated by t-test. (B) Analysis of Foxp3 and CD25 expression on CD4+ thymocytes from WT (blue) and KO (red) mice after activation with anti-CD3, IL-2, or both, as indicated. (C) Quantification of the frequencies of CD4+CD25+Foxp3+ Treg cells from the experiments shown in (B). Error bars indicate the SEM (WT: n = 3; KO: n = 4). Thymocytes were pooled from at least 2 mice in each experiment. The P values were calculated by two-tailed unpaired Student’s t-test. (D) I-V relationship of ITRPM7 in WT thymocytes (blue trace) obtained by whole-cell, patch clamp recordings, orange trace shows inhibition of ITRPM7 after perfusion of 2 μM FTY720 in the bath solution. Inset bar graph shows ITRPM7 current densities for each condition, obtained 5 min after break-in at 100 mV (n = 3 experiments). (E) I-V relationship of ITRPM7 in thymocytes during bath perfusion of 2 μM FTY720-P (S1PR agonist; orange trace). The control WT thymocyte current (blue trace) is identical to the ITRPM7 with perfused FTY720-P. MgCl2- treated cells (10mM, red trace) display inhibition of TRPM7 current. (F) Quantification by qRT-PCR analysis of the relative expression of Foxp3 in WT thymocytes activated for 48 hours with anti-CD3 antibody in presence of the indicated concentrations of FTY720 (left) or FTY720-P (right). Error bars indicate the SEM (n = 3 experiments). The indicated P values were calculated by unpaired Student’s t-test. (G) Representative histograms of Foxp3 staining in CD4+CD25+ WT thymocytes activated for 48 hours with anti-CD3 and IL-2 in the absence (blue) or presence of FTY720 (orange). Data are representative of three experiments. (H) Histograms representing intracellular p-STAT5 staining. Thymocytes from xxx mice were pretreated with FTY720 (orange) or left untreated (blue) and then stimulated for 15 min with the indicated concentrations of IL-2. (I) Quantification of pSTAT5+ thymocytes from the experiments shown in (H). Data are representative of three independent experiments in which the cells were cultured in triplicate. P values were calculated by Student’s t-test (#P = 0.005).

Article Snippet: The following sets of magnetic beads were used for T cell isolation: Dynabeads Untouched Mouse T Cells (Life Technologies, #11413D), CD4 + CD25 + Regulatory T Cell Isolation Kit mouse (Miltenyi Biotech, #130-091-041), and Dynabeads FlowComp Mouse CD4 + CD25 + T reg Cells (Life technologies, #11463D).

Techniques: Quantitative RT-PCR, Expressing, Control, Activation Assay, Two Tailed Test, Patch Clamp, Inhibition, Staining, Cell Culture

Journal: Science signaling

Article Title: Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling

doi: 10.1126/scisignal.abb0619

Figure Lengend Snippet: (A) RT-qPCR analysis of the relative abundance of Il2ra mRNA in WT and KO thymocytes. The relative fold change was calculated after normalization to the abundance of mRNA encoding beta-2-Microglobulin (b2m). The P value was calculated by two–tailed unpaired t-test (n = 3 experiments). (B) Flow cytometry analysis of the cell surface expression of IL-2Rα (CD25) in WT and KO thymocytes. The histogram is representative of seven independent experiments. (C) Overlaid flow cytometry histograms showing intracellular IL-2 staining in Thy1.2+ WT (blue) and KO (red) thymocytes. The gray line denotes an FMO control. The data are representative of three independent experiments. (D) Left: Frequency of IL-2+Thy1.2+ thymocytes obtained from WT (blue) and KO (red) mice. Right: Bar graph of the MFI of IL-2 staining in Thy1.2+ thymocytes from the indicated mice. The P value was calculated by two-tailed unpaired t-test, error bars represent SEM (n = 3). (E) Overlaid histograms show the intracellular p-STAT5 staining in Thy1.2+CD4+ T cells obtained from WT and KO thymocytes, as indicated. The data are representative of four independent experiments (F) Percentage of p-STAT5+ cells (left) and p-STAT5 MFI values (right) in freshly isolated WT and KO thymocytes. The P value was calculated by two-tailed unpaired t-test (n = 4). (G) MFI values of p-STAT5 in freshly isolated WT and KO thymocytes. The P value was calculated by two–tailed unpaired t-test (n = 4). (H) Representative histogram (of three independent experiments) showing intracellular staining of p-STAT5 in freshly isolated WT and KO splenic T cells. FMO control is shown as a black trace. (I) Representative Bivariant contour plots (of four independent experiments) showing Foxp3 and p-STAT5 staining in CD4+ WT and KO thymocytes before and after stimulation with the indicated concentrations of IL-2 at the indicated times. Analysis was confined to CD4+ thymocytes; for the gating strategy used, see fig. S5C. (J) The frequency of p-STAT5+ Treg cells as determined by the analysis shown in (I). Error bars indicate the SEM (n = 4 experiments); P values were calculated by t-test. (K) Foxp3 and pSTAT5 staining in WT and KO CD4+ splenocytes before and 5 min after stimulation with the indicated concentrations of IL-2. (L) The frequency of p-STAT5+ splenic Treg cells quantified from the analysis shown in (K). Error bars indicate the SEM (n = 4 experiments); P values were calculated by t-test.

Article Snippet: The following sets of magnetic beads were used for T cell isolation: Dynabeads Untouched Mouse T Cells (Life Technologies, #11413D), CD4 + CD25 + Regulatory T Cell Isolation Kit mouse (Miltenyi Biotech, #130-091-041), and Dynabeads FlowComp Mouse CD4 + CD25 + T reg Cells (Life technologies, #11463D).

Techniques: Quantitative RT-PCR, Two Tailed Test, Flow Cytometry, Expressing, Staining, Control, Isolation

Journal: Science signaling

Article Title: Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling

doi: 10.1126/scisignal.abb0619

Figure Lengend Snippet: (A) Experimental scheme. (B) Representative flow cytometry contour plots sequentially gated on CD4+ (top) and Foxp3+ Treg cells (bottom) in thymi isolated from WT (blue) and KO (red) mice. (C) Frequency of CD4+Foxp3+ Treg cells in thymi from WT (blue) and KO (red) mice. The mean value is denoted by an empty square and the median by a horizontal line. The P value was calculated by two-tailed, unpaired Student’s t-test. (D) Absolute numbers of CD4+Foxp3+ thymocytes in thymi from WT (blue) and KO (red) mice. The empty square denotes the mean and the horizontal line the median. (E) MFI values of FOXP3 in CD4+ WT (blue) and KO (red) thymocytes. P value was calculated by two-tailed paired t-test. (F) Contour plots showing the frequencies of CD4+ and Foxp3+ Treg cells in the spleens of WT (blue) and KO (red) mice. (G) Box charts show the frequency of CD4+Foxp3+ Treg cells in the spleens of WT (blue) and KO (red) mice. (H) Absolute numbers of CD4+Foxp3+ Treg cells in splenocytes isolated from WT (blue) and KO (red) mice. (I) Quantification of the MFI for FOXP3 in CD4+ splenic T cells from the indicated mice. P value was calculated by two-tailed paired t-test. (J) I-V relationship of ITRPM7 in CD4+CD25+ WT Treg (blue trace) and KO Treg (red trace) cells obtained by whole cell patch clamp recordings. The patch clamp configuration and the recording conditions are shown in fig. S3F. The box chart shows the ITRPM7 current densities quantified at 5 min after break-in at 100 mV in WT and KO Treg cells. See Materials and Methods for a description of the statistical parameters shown in the box chart. (K) The box chart shows the ITRPM7 current densities quantified at 5 min after break-in at 100 mV in Teff (CD4+CD25−) and Treg (CD4+CD25+) cells isolated from WT mice. The P value was calculated by two-tailed unpaired t-test. Each filled circle represents data obtained from an individual cell.

Article Snippet: The following sets of magnetic beads were used for T cell isolation: Dynabeads Untouched Mouse T Cells (Life Technologies, #11413D), CD4 + CD25 + Regulatory T Cell Isolation Kit mouse (Miltenyi Biotech, #130-091-041), and Dynabeads FlowComp Mouse CD4 + CD25 + T reg Cells (Life technologies, #11463D).

Techniques: Flow Cytometry, Isolation, Two Tailed Test, Patch Clamp

Journal: Science signaling

Article Title: Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling

doi: 10.1126/scisignal.abb0619

Figure Lengend Snippet: (A) The experimental scheme used for the suppression assays. (B) Analysis of CFSE-labeled WT T cell proliferation after 3 days of culture in the presence of Treg cells isolated from WT (blue) and KO (red) mice. The ratios indicate the relative numbers of CD4+CD25− Teff cells to CD4+CD25+ Treg cells plated on day 1. Histograms show the dilution of CFSE in Teff cells as measured by flow cytometry. The percentage of Teff cells showing dilution of the CFSE dye reflects the proportion of Teff cells that underwent cell division. Data are representative of four experiments. (C) Treg cells from the thymi and spleens of WT (blue) and KO (red) mice were identified by flow cytometry (Left: bivariant gating for CD4 and Foxp3) and further analyzed for the expression of the Treg cell functional markers CD39, CD73, and CTLA4 (histograms). (D) Percentages of positive cells (left) and MFI values (right) for CD39, CD73, and CTLA4 in thymic and splenic CD4+Foxp3+ Tregs from WT and KO mice. The data were collected from two independent experiments with a total sample size of 4 mice. The P value was calculated by one-tailed unpaired t-test. (E) Cell surface expression of neuropilin-1 (Nrp-1) on thymic and splenic Treg cells (CD4+Foxp3+) from WT (blue) and KO (red) mice. (F) Bar graphs showing the percentage of positive cells (left) and MFI values (right) for Nrp-1 in thymic and splenic T cells from WT and KO mice. The data were collected from two independent experiments with a total sample size of four mice. The P value was calculated by one-tailed unpaired t-test.

Article Snippet: The following sets of magnetic beads were used for T cell isolation: Dynabeads Untouched Mouse T Cells (Life Technologies, #11413D), CD4 + CD25 + Regulatory T Cell Isolation Kit mouse (Miltenyi Biotech, #130-091-041), and Dynabeads FlowComp Mouse CD4 + CD25 + T reg Cells (Life technologies, #11463D).

Techniques: Labeling, Isolation, Flow Cytometry, Expressing, Functional Assay, One-tailed Test

Journal: Science signaling

Article Title: Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling

doi: 10.1126/scisignal.abb0619

Figure Lengend Snippet: (A) Experimental scheme (left) and bone marrow (BM) transplantation scenarios (right). (B) The absolute numbers of thymocytes obtained from WT recipient mice after transplantation of BM from WT (blue), KO (red), or mixed (black) mice. The P value was calculated by one-way ANOVA. (C) Absolute numbers of CD45+TCRb+CD4+CD25+Foxp3+ Tregs in the thymi obtained from the indicated recipient mice (data analyzed by one-way ANOVA). (D) Absolute numbers of CD45+TCRb+CD4+CD25−Foxp3−Teffs in the thymi obtained from the indicated recipient mice (data analyzed by one-way ANOVA). (E) The ratio of Treg:Teff absolute numbers in the thymi of the recipient mice from each transplant. P values were calculated by one-way ANOVA. (F) Total numbers of splenocytes resulting from the transplantation of WT (blue), KO (red), and mixed (black) BM into WT recipient mice. (G) The absolute numbers of CD45+CD4+CD25+Foxp3+ Treg cells in the spleens obtained from the indicated recipient mice. (H) The absolute numbers of CD45+CD4+CD25−Foxp3− Teff cells in the spleens obtained from the indicated recipient mice. (I) The ratio of the absolute numbers of splenic Treg:Teff cells in mice that received the indicated BM transplants. P values were calculated by one-way ANOVA.

Article Snippet: The following sets of magnetic beads were used for T cell isolation: Dynabeads Untouched Mouse T Cells (Life Technologies, #11413D), CD4 + CD25 + Regulatory T Cell Isolation Kit mouse (Miltenyi Biotech, #130-091-041), and Dynabeads FlowComp Mouse CD4 + CD25 + T reg Cells (Life technologies, #11463D).

Techniques: Transplantation Assay

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A ) Perilysosomal fluorescence after applying GPN to GCaMP3-ML1-transfected BMDMs treated with LP for a total of 4 h including LPS pretreatment for 3 h (left) (actual LP treatment time is 1 h). Peak fluorescence (right) (n=6). ( B ) [Ca 2+ ] Lys in OGBD-loaded Mϕs treated with LP for a total of 4 h including LPS pretreatment for 3 h (right). Representative fluorescence images (left) (n=8). ( C ) [Ca 2+ ] i in Mϕs treated with LP for a total of 4 h including LPS pretreatment for 3 h, determined using Fluo-3-AM staining (right upper) or Fura-2 (right lower). Representative Fluo-3 images (left) (n=7 for BSA; n=6 for LPS; n=13 for LP). ( D-E ) IL-1β ELISA of culture supernatant after treating Mϕs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of BAPTA-AM (D, n=3) or MitoTEMPOL (E, n=3). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student’s t-test (A, B), one-way ANOVA with Tukey’s test (C), or two-way ANOVA with Sidak test (D, E) (ns, not significant). Scale bars, 20 μm.

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Fluorescence, Transfection, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A ) Cellular ROS in peritoneal Mϕs treated with LPS alone for 21 h, PA alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h, determined by confocal microscopy after CM-H2DCFDA loading (right). Representative confocal images (left) (scale bar, 50 μm). ( B ) IL-1β ELISA of culture supernatant after treating Mϕs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of NAC ( n =3). ( C ) [Ca 2+ ] i in Mϕs treated with LPS alone for 4 h or with LP for a total of 4 h including LPS pretreatment for 3 h in the presence or absence of NAC, determined by confocal microscopy after Fluo-3-AM loading (middle) or ratiometric measurement after Fura-2 loading (right). Representative Fluo-3 fluorescence images (left) (scale bar, 20 μm) ( n =13 for BSA, Fluo-3-AM; n =12 for LPS, Fluo-3-AM; n =26 for LP, Fluo-3-AM; n =17 for LP+NAC, Fluo-3-AM; n =19 for BSA, Fura-2; n =23 for LP, Fura-2; n =23 for LP+BAPTA-AM, Fura-2; n =21 for LP+NAC, Fura-2). ( D ) Mitochondrial ROS in Mϕs treated with PA alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of MitoTEMPOL, determined by flow cytometry after MitoSOX staining (right). Representative histograms (left) ( n =3). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A, C, D), or two-way ANOVA with Sidak test (B).

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Fluorescence, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A ) [Ca 2+ ] ER in GEM-CEPIA1e r- (left) or D1ER- transfected BMDMs (right) treated with LP for 1 h without extracellular Ca2+ after LPS pretreatment for 3 h. ( n =26 for BSA; n =25 for LP). ( B ) BMDMs transfected with GEM-CEPIA1er and loaded with OGBD were treated with LP for 1 h after LPS pretreatment for 3 h (right) or BSA alone for 4 h (left). Tracing of [Ca 2+ ] Lys and [Ca 2+ ] ER after change to a fresh medium without extracellular Ca 2+ . ( n =4 for BSA; n =4 for LP). ( C ) OGBD-loaded Mϕs were treated with LP for 1 h after LPS pretreatment for 3 h. Recovery of [Ca 2+ ] Lys after change to a fresh medium with or without Xestospongin C (Xesto C), dantrolene (Dan) or TPEN (right). Representative confocal images (left) ( n =9 for BSA; n =8 for LP; n =9 for LP+Recovery; n =6 for LP+Recovery+Xesto C; n =5 for LP+Recovery+Dan; n =8 for LP+Recovery+TPEN). ( D ) IL-1β ELISA of culture supernatant after treating Mϕs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h, in the presence or absence of Xesto C ( n =4). ( E ) PLA in Mϕs treated with LPS alone for 21 h, PA alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h, using Abs to VAPA and ORP1L (right). Representative fluorescence images (left) ( n =4). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by two-tailed Student’s t-test (A), one way ANOVA with Tukey’s test (C, E) or two-way ANOVA with Sidak test (D). Scale bar, 20 μm.

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A-C ) IL-1β ELISA of culture supernatant after treating Mϕs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of EGTA (A) ( n =4), 2-APB (B) ( n =4) or BTP2 (C) ( n =3). ( D ) [Ca 2+ ] ER in D1ER -transfected BMDMs treated with LP for 1 h in the presence or absence of BTP2 without removal of extracellular Ca 2+ after LPS pretreatment for 3 h ( n =19 for BSA; n =16 for LP; n =24 for LP+BTP2). ( E ) STIM1 aggregation (white arrows) and colocalization with ORAI1 (yellow arrows) at the middle and bottom levels of BMDMs that were transfected with YFP- STIM1 together with mCherry-Orai1 and then treated with LP in Ca 2+ -free KRB buffer for 1 h after LPS pretreatment for 3 h, determined by confocal microscopy (scale bar, 5 mm). Data shown as means ± SEM from more than 3 independent experiments. **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (D) or two-way ANOVA with Sidak test (A, B, C). Scale bar, 5 μm.

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Confocal Microscopy

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A ) [K + ] i in Mϕs treated with LPS alone for 21 h, PA alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h (right), determined by confocal microscopy after Potassium Green-2-AM loading. Representative Potassium Green-2 fluorescence images (left) ( n =4 for BSA; n =2 for PA; n =4 for LPS; n =4 for LP). ( B ) IL-1β ELISA of culture supernatant after treating Mϕs with LPS alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h, at [K + ] e of 5.4 (K + concentration in RPMI medium) or 60 mM ( n =3 each). ( C , D ) IL-1β ELISA of culture supernatant after treating Mϕs with LPS alone for 21 h, PA alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of charybdotoxin (CTX) or TRAM-34 (C) ( n =3), or several K + efflux channel inhibitors (D) ( n =3). ( E ) [Ca 2+ ] ER in D1ER - transfected BMDMs after LP treatment for 1 h in the presence or absence of TRAM-34 without extracellular Ca 2+ removal after LPS pretreatment for 3 h ( n =16). Data shown as means ± SEM from more than 3 independent experiments. **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A, E) or two-way ANOVA with Sidak test or with Tukey’s test (B, C, D). Scale bar, 20 μm.

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A-D ) Nystatin-perforated patch clamp and slope conductance of TRAM-34-sensitive I/V curve in Kcnn4 +/+ (A, B) or Kcnn4 - /- Mϕs (C, D) treated with LPS alone for 4 h or with LP for a total of 4 h including LPS pretreatment for 3 h (B, D). Representative I/V curves (A, C). ( n =13 for each Kcnn4 +/+ group; n =4 for Kcnn4 - /- : BSA; n =5 for Kcnn4 - /- : LPS; n =4 for Kcnn4 - /- : LP). ( E ) IL-1β ELISA of culture supernatant (upper) and IB using indicated Abs (lower) after treating Kcnn4 +/+ or Kcnn4 - /- Mϕs with PA alone for 21 h, LPS alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h. ( n =3). ( F ) [K + ] i in Kcnn4 +/+ or Kcnn4 - /- Mϕs treated with LP for a total of 21 h including LPS pretreatment for 3 h (right). Representative Potassium Green-2 images (left). ( n =5). ( G ) OGBD-loaded BMDMs were treated with LP for a total of 4 h including LPS pretreatment for 3 h. [Ca 2+ ] Lys recovery after LP removal with or without TRAM-34 (right). Representative fluorescence images (left). ( n =11 for BSA; n =8 for LP; n =6 for LP:(-); n =9 for LP:TRAM-34). ( H ) IB of Mϕs treated with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h using indicated Ab, after BN gel electrophoresis. ( I ) IB of SVF of WAT from mice fed HFD for 8 weeks using indicated Abs. ( n =3). (J) The number of ASC specks in WAT of mice in (I) identified by ASC immunofluorescence (right). Representative ASC specks (red arrow heads) (left). (scale bar, 20 μm) ( n =28 for Kcnn4 +/+ : HFD; n =22 for Kcnn4 - /- :HFD). ( K ) The number of CLS in WAT of mice in (I) identified by F4/80 immunohistochemistry (lower). Representative F4/80 immunohistochemistry (upper). (scale bar, 50 μm) ( n =12 for Kcnn4 +/+ :HFD; n =11 for Kcnn4 - /- :HFD). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (B, D, F, G), or two-way ANOVA with Sidak test (E). Scale bar, 20 μm.

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Patch Clamp, Enzyme-linked Immunosorbent Assay, Fluorescence, Nucleic Acid Electrophoresis, Immunofluorescence, Immunohistochemistry

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A ) Real-time RT-PCR of Kcnn4 using mRNA from Mϕs treated with PA alone for 21 h, LPS alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h ( n =3). ( B ) Real-time RT-PCR of Kcnn4 using mRNA from Mϕs of Kcnn4 +/+ or Kcnn4 -/- mice ( n =3). ( C ) TNF-α and IL-6 ELISA of culture supernatant after treating Mϕs from Kcnn4 +/+ or Kcnn4 -/- mice with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h ( n =3). ( D ) PLA in Mϕs treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of BAPTA-AM, using Abs to KCNN4 and ORAI1 (right). Representative fluorescence images (left) (scale bar, 20 μm) ( n =10 for BSA; n =16 for LP; n =10 for BSA+BAPTA-AM; n =16 for LP+BAPTA-AM). ( E ) IL-1β ELISA of culture supernatant after treating Mϕs from Kcnn4 +/+ or Kcnn4 -/- mice with PA, nigericin, ATP, LLOMe and MSU for 18 h, 45 min, 1 h, 45 min and 3 h, respectively, after LPS pretreatment for 3 h ( n =6). ( F ) Immunoprecipitation of Mϕs treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of TRAM-34 using anti-NEK7 Ab and protein G beads. After bead heating in a sample buffer, collected supernatant was subjected to immunoblotting (IB) using indicated Abs. (red arrow, band of the correct size) ( G ) Immunoprecipitation of Mϕs from Kcnn4 +/+ or Kcnn4 -/- mice treated with LP for a total of 21 h including LPS pretreatment for 3 h using anti-NEK7 Ab and protein G beads. After bead heating in a sample buffer, collected supernatant was subjected to IB using indicated Abs. (red arrow, band of the correct size) ( H ) Intraperitoneal glucose tolerance test (IPGTT) in Kcnn4 +/+ and Kcnn4 -/- mice fed HFD for 8 weeks (left). AUC (right) ( n =9). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A, D), two-tailed Student’s t -test (B, K, H), or two-way ANOVA with Sidak test (C, E).

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Fluorescence, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A ) IB of BMDMs treated with LPS alone without PA (‘- PA’) for indicated time period (left half) or with ‘PA’ (together with LPS) for indicated time period after LPS pretreatment for 3 h (right half) (hence, the numbers indicating LPS treatment time in the right half is 3 + PA treatment time), using indicated Abs. ( B ) IB of BMDMs treated with LPS alone for 0.5 h or ‘PA’ (together with LPS) for 1 or 18 h after LPS pretreatment for 3 h (hence, the numbers indicating LPS treatment time of 4 or 21 h is 3 + PA treatment time), using indicated Abs. ( C ) BMDMs were treated with LPS alone for 0.5 h, ‘PA’ (together with LPS) for 1 or 18 h after LPS pretreatment for 3 h (hence, the numbers indicating LPS treatment time of 4 or 21 h is 3 + PA treatment time) or nigericin for 45 min after LPS pretreatment for 3 h. IB using indicated Abs after DSS crosslinking. ( D ) BMDMs were treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of ASK1 (selonsertib) or JNK inhibitor (SP600125). IB using indicated Abs after DSS crosslinking. ( E ) IL-1β ELISA of culture supernatant after treating BMDMs with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of selonsertib or SP600125. ( F-H ) IB using indicated Abs (F, G) and IL-1β ELISA of culture supernatant (H) after treating BMDMs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of KN-93 or −92. ( n =3). Data shown as means ± SEM from more than 3 independent experiments. ***p< 0.001 by two-way ANOVA with Tukey’s test (F, I).

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: ER-to-lysosome Ca 2+ refilling followed by K + efflux-coupled store-operated Ca 2+ entry in inflammasome activation and metabolic inflammation

doi: 10.1101/2023.04.19.537448

Figure Lengend Snippet: ( A ) [Ca 2+ ] i (left), [K + ] i (middle) and [Ca 2+ ] ER (right) determined by Flou-3-AM staining, Potassium Green-2-AM staining and D1ER transfection, respectively, after treatment with LP for a total of 4 or 21 h including LPS pretreatment for 3 h ( n =10 for Fluo-3-AM; n =10 for Potassium Green-2-AM; n =50 for D1ER ). ( B ) IB of lysate of Mϕs from Kcnn4 +/+ and Kcnn4 -/- mice (left) or Trpm2 +/+ and Trpm2 -/- mice (right) treated with PA alone for 21 h, LPS alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h, using indicated Abs. ( C ) IB of BMDMs treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of ASK1 (selonsertib) or JNK inhibitor (SP600125) using indicated Ab. ( D ) IL-1β ELISA of culture supernatant after treating BMDMs with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of 5Z-7-oxozeaenol ( n =3). ( E ) IB after treating BMDMs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of selonsertib using indicated Abs. Data shown as means ± SEM from more than 3 independent experiments*p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A), or two-way ANOVA with Tukey’s test (D).

Article Snippet: Cytokine content in culture supernatants of BMDMs or peritoneal Mφs was determined using mouse ELISA kits (R&D Systems), according to the manufacturer’s instruction.

Techniques: Staining, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: List of ELISA tests, cell proliferation, and protein extraction kits used. For ELISA tests, dilution of the supernatants or cell lysate samples used and detection limits are also reported.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Enzyme-linked Immunosorbent Assay, Protein Extraction

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: C-Kit (CD117) mRNA (a, b, c) and protein (d, e, f) expression after ESW treatment in primary bronchial fibroblasts of COPD patients (a, d), primary bronchial fibroblasts of control smokers (b, e), and bronchial epithelial cells (c, f). In bronchial epithelium (16HBE) c-Kit increased at mRNA (c) and protein (f) levels. In primary bronchial fibroblasts of COPD patients, c-Kit increased at protein level (d). T -test was used for comparative purposes, and p values are reported in the graphs.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Expressing

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: Photomicrographs showing thyroid transcription factor-1 (TTF-1) expression (panels a, b), c-Kit (CD117) (c, d), and proliferating cell nuclear antigen (PCNA) (e, f) in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary disease (COPD). Arrows indicate positively stained cells mainly located in the alveolar septa. Bars = 50 microns.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Expressing, Staining

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: Photomicrographs showing alveolar type II epithelial cells (TTF-1+ cells, red color) coexpressing c-Kit (CD117) (brown color) (a, b) and PCNA (brown color) (c, d) in the peripheral lung tissue of a representative patient with COPD. Positive double-stained cells can be recognized in the alveolar septa, even though their presence was only rarely observed. Arrows indicate positively stained cells located in the alveolar septa. Bars = 50 microns.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Staining

Journal: Disease Markers

Article Title: Evaluation of Immune Infiltration Based on Image Plus Helps Predict the Prognosis of Stage III Gastric Cancer Patients with Significantly Different Outcomes in Northeastern China

doi: 10.1155/2022/2893336

Figure Lengend Snippet: Expression of CD4, CD8, CD20, CD56, CD68, CD117, and CD177 by immune cells infiltrated tumor tissues of GC patients. Representative images of immune markers staining in immune cells from GC samples are shown at ×200 (100 mm) original magnification. Positive expression of CD4 (a), CD8 (b), CD20 (c), CD56 (d), CD68 (e), CD117 (f), and CD177 (g) infiltrated in tumor tissues with a regular distribution. In order to show the distribution from immune cells more clearly, we used computerized imaging system Image-Pro Plus version 6.2 to highlight positive areas in different colors.

Article Snippet: After cleaned in distilled water, the paraffin sections were pretreated with citrate buffer, pH 6.0 (CD177) and EDTA Antigen Retrieval Solution, pH 8.0 (CD4, CD8, CD20, CD56, CD68, and CD117) for 3 min at 120°C in a pressure cooker, and endogenous peroxidase was inhibited with 3% H 2 O 2 in PBS for 10 min. Nonspecific actions in the sections were also blocked with goat serum (BOSTER, USA) for 1 h at room temperature.

Techniques: Expressing, Staining, Imaging

Journal: Disease Markers

Article Title: Evaluation of Immune Infiltration Based on Image Plus Helps Predict the Prognosis of Stage III Gastric Cancer Patients with Significantly Different Outcomes in Northeastern China

doi: 10.1155/2022/2893336

Figure Lengend Snippet: The schematic diagram of the distribution of immune cells. The blue points represent CD20 + B cells, the yellow points represent CD4 + T cells, the orange points represent CD117 + mast cells, the green points represent CD68 + macrophages, the pink points represent CD8 + T cells, the black points represent CD56 + NK cells, and the red points represent CD177 + neutrophils. Although not all immune cells are distributed according to this fixed law, this pattern can basically reflect the general characteristics of their distribution.

Article Snippet: After cleaned in distilled water, the paraffin sections were pretreated with citrate buffer, pH 6.0 (CD177) and EDTA Antigen Retrieval Solution, pH 8.0 (CD4, CD8, CD20, CD56, CD68, and CD117) for 3 min at 120°C in a pressure cooker, and endogenous peroxidase was inhibited with 3% H 2 O 2 in PBS for 10 min. Nonspecific actions in the sections were also blocked with goat serum (BOSTER, USA) for 1 h at room temperature.

Techniques:

Journal: Disease Markers

Article Title: Evaluation of Immune Infiltration Based on Image Plus Helps Predict the Prognosis of Stage III Gastric Cancer Patients with Significantly Different Outcomes in Northeastern China

doi: 10.1155/2022/2893336

Figure Lengend Snippet: Differences in immune marker positive area/total area between the two groups (survival time of patients in group A was less than 1 year and group B survival time was more than 5 years) by the rank sum test. (a) CD4 + T cells ( P < 0.001). (b) CD8 + T cells ( P < 0.001). (c) CD20 + B cells ( P < 0.001). (d) CD68 + macrophages ( P < 0.001). (e) CD117 + mast cells ( P < 0.001). (f) CD177 + neutrophils ( P < 0.001).

Article Snippet: After cleaned in distilled water, the paraffin sections were pretreated with citrate buffer, pH 6.0 (CD177) and EDTA Antigen Retrieval Solution, pH 8.0 (CD4, CD8, CD20, CD56, CD68, and CD117) for 3 min at 120°C in a pressure cooker, and endogenous peroxidase was inhibited with 3% H 2 O 2 in PBS for 10 min. Nonspecific actions in the sections were also blocked with goat serum (BOSTER, USA) for 1 h at room temperature.

Techniques: Marker