Structured Review

Cayman Chemical ionomycin
Activation of BK by NS1619 facilitates ML1-mediated Ca 2+ release. ( A,B ) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. ( C ) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca 2+ content was not affected. ( D,E ) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). ( F ) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca 2+ content. ( G ) GECO-TRPML1 responses to <t>Ionomycin</t> (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.
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1) Product Images from "BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases"

Article Title: BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

Journal: Scientific Reports

doi: 10.1038/srep33684

Activation of BK by NS1619 facilitates ML1-mediated Ca 2+ release. ( A,B ) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. ( C ) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca 2+ content was not affected. ( D,E ) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). ( F ) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca 2+ content. ( G ) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.
Figure Legend Snippet: Activation of BK by NS1619 facilitates ML1-mediated Ca 2+ release. ( A,B ) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. ( C ) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca 2+ content was not affected. ( D,E ) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). ( F ) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca 2+ content. ( G ) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.

Techniques Used: Activation Assay, Expressing

BK upregulation promotes lysosomal Ca 2+ release in ML4 fibroblasts carrying F408Δ mutation. ( A ) BK overexpression increased ML-SA1 (10 μM)-induced GECO-TRPML1-F408Δ response in HEK293T cells. ( B ) BK overexpression did not alter ML-SA1 (10 μM)-induced GECO-TRPML1-R403C response in HEK293T cells. ( C ) Statistical analysis of GECO response to ML-SA1 (10 μM) showing that BK overexpression significantly increased GECO-TRPML1-F408Δ signal but not others. ( D ) BK overexpression did not alter GPN (200 μM)-induced GECO responses. ( E ) BK overexpression did not alter Ionomycin (1 μM)-induced GECO responses. ( F,G ) BK overexpression enhanced GECO-TRPML1-F408Δ response to ML-SA1 (10 μM) in TRPML1-F408Δ human fibroblasts, suggesting that BK upregulation facilitates TRPML1-F408Δ activity. ( H,I ) GECO-TRPML1-F408Δ responses to GPN (200 μM) ( H ) and Ionomycin (1 μM) ( I ) were not altered in TRPML1-F408Δ human fibroblasts by BK overexpession, suggesting BK overexpression did not alter lysosomal Ca 2+ content and GECO- TRPML1-F408Δ expression level in TRPML1-F408Δ human fibroblasts, respectively.
Figure Legend Snippet: BK upregulation promotes lysosomal Ca 2+ release in ML4 fibroblasts carrying F408Δ mutation. ( A ) BK overexpression increased ML-SA1 (10 μM)-induced GECO-TRPML1-F408Δ response in HEK293T cells. ( B ) BK overexpression did not alter ML-SA1 (10 μM)-induced GECO-TRPML1-R403C response in HEK293T cells. ( C ) Statistical analysis of GECO response to ML-SA1 (10 μM) showing that BK overexpression significantly increased GECO-TRPML1-F408Δ signal but not others. ( D ) BK overexpression did not alter GPN (200 μM)-induced GECO responses. ( E ) BK overexpression did not alter Ionomycin (1 μM)-induced GECO responses. ( F,G ) BK overexpression enhanced GECO-TRPML1-F408Δ response to ML-SA1 (10 μM) in TRPML1-F408Δ human fibroblasts, suggesting that BK upregulation facilitates TRPML1-F408Δ activity. ( H,I ) GECO-TRPML1-F408Δ responses to GPN (200 μM) ( H ) and Ionomycin (1 μM) ( I ) were not altered in TRPML1-F408Δ human fibroblasts by BK overexpession, suggesting BK overexpression did not alter lysosomal Ca 2+ content and GECO- TRPML1-F408Δ expression level in TRPML1-F408Δ human fibroblasts, respectively.

Techniques Used: Mutagenesis, Over Expression, Activity Assay, Expressing

BK upregulation reduces lipofuscin accumulation in NPA cells by promoting TRPML1-mediated Ca 2+ release. ( A,B ) Abnormal lipofuscin accumulation in NPA human fibroblasts and its rescue by BK overexpression or NS1619 (15 μM, 16 h) treatment. Expression of TRPML1-DD/KK or Syt VII DN reversed the rescue effect of BK upregulation. More than 35 cells were analyzed for each condition. ( C–E ) Impaired ML-SA1 (10 μM)-mediated GECO-TRPML1 response in NPA human fibroblasts and its rescue by NS1619 (15 μM, ~60 s) pretreatment ( C ) or BK overexpression ( D ). Paxilline (PAX, 3 μM) treatment reversed the rescue effect of NS1619 or BK overexpression. ( F,G ) GECO-TRPML1 responses to GPN (200 μM) (F) and Ionomycin (1 μM) ( G ) were not altered in all the treatments, suggesting lysosomal Ca 2+ content, or GECO-TRPML1 expression level was not affected.
Figure Legend Snippet: BK upregulation reduces lipofuscin accumulation in NPA cells by promoting TRPML1-mediated Ca 2+ release. ( A,B ) Abnormal lipofuscin accumulation in NPA human fibroblasts and its rescue by BK overexpression or NS1619 (15 μM, 16 h) treatment. Expression of TRPML1-DD/KK or Syt VII DN reversed the rescue effect of BK upregulation. More than 35 cells were analyzed for each condition. ( C–E ) Impaired ML-SA1 (10 μM)-mediated GECO-TRPML1 response in NPA human fibroblasts and its rescue by NS1619 (15 μM, ~60 s) pretreatment ( C ) or BK overexpression ( D ). Paxilline (PAX, 3 μM) treatment reversed the rescue effect of NS1619 or BK overexpression. ( F,G ) GECO-TRPML1 responses to GPN (200 μM) (F) and Ionomycin (1 μM) ( G ) were not altered in all the treatments, suggesting lysosomal Ca 2+ content, or GECO-TRPML1 expression level was not affected.

Techniques Used: Over Expression, Expressing

2) Product Images from "A Novel PHOX/CD38/MCOLN1/TFEB Axis Important For Macrophage Activation During Bacterial Phagocytosis"

Article Title: A Novel PHOX/CD38/MCOLN1/TFEB Axis Important For Macrophage Activation During Bacterial Phagocytosis

Journal: bioRxiv

doi: 10.1101/669325

TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).
Figure Legend Snippet: TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).

Techniques Used: Activation Assay, Infection

3) Product Images from "Chronic circadian misalignment accelerates immune senescence and abbreviates lifespan in mice"

Article Title: Chronic circadian misalignment accelerates immune senescence and abbreviates lifespan in mice

Journal: Scientific Reports

doi: 10.1038/s41598-020-59541-y

The long-term non-adjustive ADV condition accelerates generation of senescence-associated (SA) T cells and germinal center B cells. ( A , B ) Flow cytometry of senescence-associated T (SA-T) and follicular helper T (Tfh) cells in CD4 + TCRβ + -gated spleen cells ( A ), and percentages and cell numbers of SA-T (CD4 + TCRβ + CD44 + PD-1 + ), CD153 + SA-T, Tfh (CD4 + TCRβ + CXCR5 + PD-1 + ), and regulatory T (Treg, CD4 + TCRβ + CD25 + ) cells (B, n = 12) in spleens of LD- and ADV-conditioned mice. ( C , D ) Flow cytometry of SA-T and Tfh cells in CD4 + TCRβ + -gated mLN cells ( C ) and percentages and cell numbers of SA-T, CD153 + SA-T, Tfh, and Treg (D, n = 12–16) in mLNs of LD- and ADV-conditioned mice. ( E ) Whole mLN cells from LD- and ADV-conditioned mice were stimulated with PMA and ionomycin for 3 hours. Percentages of IFN-γ–, IL-4–, and IL-17A–producing helper T cells in CD4 T cells were shown (n = 12). ( F ) Flow cytometry of germinal center B cell (GC-B) and IgG1 + and IgA + class-switched B cells in mLNs of LD- and ADV-conditioned mice. ( G ) Cell numbers of GC-B (CD19 + B220 + CD95 + GL7 + ), IgG1 B cells (CD19 + B220 + IgG1 + ), and IgA B cells (CD19 + B220 + IgA + ) in mLN from LD- and ADV-conditioned mice (n = 12). Data are means ± SD. Two-tailed Student’s t -test, * P
Figure Legend Snippet: The long-term non-adjustive ADV condition accelerates generation of senescence-associated (SA) T cells and germinal center B cells. ( A , B ) Flow cytometry of senescence-associated T (SA-T) and follicular helper T (Tfh) cells in CD4 + TCRβ + -gated spleen cells ( A ), and percentages and cell numbers of SA-T (CD4 + TCRβ + CD44 + PD-1 + ), CD153 + SA-T, Tfh (CD4 + TCRβ + CXCR5 + PD-1 + ), and regulatory T (Treg, CD4 + TCRβ + CD25 + ) cells (B, n = 12) in spleens of LD- and ADV-conditioned mice. ( C , D ) Flow cytometry of SA-T and Tfh cells in CD4 + TCRβ + -gated mLN cells ( C ) and percentages and cell numbers of SA-T, CD153 + SA-T, Tfh, and Treg (D, n = 12–16) in mLNs of LD- and ADV-conditioned mice. ( E ) Whole mLN cells from LD- and ADV-conditioned mice were stimulated with PMA and ionomycin for 3 hours. Percentages of IFN-γ–, IL-4–, and IL-17A–producing helper T cells in CD4 T cells were shown (n = 12). ( F ) Flow cytometry of germinal center B cell (GC-B) and IgG1 + and IgA + class-switched B cells in mLNs of LD- and ADV-conditioned mice. ( G ) Cell numbers of GC-B (CD19 + B220 + CD95 + GL7 + ), IgG1 B cells (CD19 + B220 + IgG1 + ), and IgA B cells (CD19 + B220 + IgA + ) in mLN from LD- and ADV-conditioned mice (n = 12). Data are means ± SD. Two-tailed Student’s t -test, * P

Techniques Used: Flow Cytometry, Mouse Assay, Two Tailed Test

4) Product Images from "The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles"

Article Title: The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles

Journal: eLife

doi: 10.7554/eLife.29887

Measurement of intracellular Ca2 2+ in follicle cells after ionomycin or octopamine stimulation. ( A–D ) Intracellular Ca 2+ depicted by GCaMP5G (green in A-C’) increase in response to ionomycin stimulation in both control ( A–A’ ) and hnt RNAi ( B–C’ ) egg chambers with FC2 Gal4, although hnt RNAi egg chambers show slightly weaker response. The frames with peak GCaMP5G signal after ionomycin stimulation are shown in A’-C’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in D and the number of egg chambers analyzed is noted at the end of each trace. ( E–H ) Intracellular Ca 2+ depicted by GCaMP5G (green in E-G’) increase in control egg chambers ( E–E’ ) after OA stimulation but does not in hnt RNAi egg chambers with FC2 Gal4 ( F–G’ ). The frames with peak GCaMP5G signal after OA stimulation are shown in E’-G’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in H and the number of egg chambers analyzed is noted at the end of each trace. Also see Videos 1 – 6 .
Figure Legend Snippet: Measurement of intracellular Ca2 2+ in follicle cells after ionomycin or octopamine stimulation. ( A–D ) Intracellular Ca 2+ depicted by GCaMP5G (green in A-C’) increase in response to ionomycin stimulation in both control ( A–A’ ) and hnt RNAi ( B–C’ ) egg chambers with FC2 Gal4, although hnt RNAi egg chambers show slightly weaker response. The frames with peak GCaMP5G signal after ionomycin stimulation are shown in A’-C’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in D and the number of egg chambers analyzed is noted at the end of each trace. ( E–H ) Intracellular Ca 2+ depicted by GCaMP5G (green in E-G’) increase in control egg chambers ( E–E’ ) after OA stimulation but does not in hnt RNAi egg chambers with FC2 Gal4 ( F–G’ ). The frames with peak GCaMP5G signal after OA stimulation are shown in E’-G’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in H and the number of egg chambers analyzed is noted at the end of each trace. Also see Videos 1 – 6 .

Techniques Used:

5) Product Images from "VacA generates a protective intracellular reservoir for Helicobacter pylori that is eliminated by activation of the lysosomal calcium channel TRPML1"

Article Title: VacA generates a protective intracellular reservoir for Helicobacter pylori that is eliminated by activation of the lysosomal calcium channel TRPML1

Journal: Nature microbiology

doi: 10.1038/s41564-019-0441-6

VacA impairs TRPML1 activity. a , Intraluminal lysosomal Ca 2+ levels in VacA − -or VacA + -treated AGS cells. AGS cells were co-loaded with Oregon Green 488 BAPTA-5N (OG BAPTA-5N), a membrane-impermeant Ca 2+ indicator with negligible sensitivity to pH, and Alexa-Fluor-586-conjugated dextran (Dextran Red, as a calcium-insensitive probe) by endocytosis. The fluorescence ratio between green and red signals was compared. A representative scatter plot (mean ± s.e.m.) of the Oregon Green/Dextran Red intensity ratio of 12 fields containing 97 VacA − -and 70 VacA + -treated cells is displayed on the right. b , c , Lysosomal calcium levels assessed by GPN (200 μM)-induced GECO-ML1 response (measured as change in fluorescence (∆ F ) over basal fluorescence ( F 0 ); ∆ F / F 0 ) in AGS cells expressing GECO-ML1) after 4 h VacA − or VacA + incubation. d , Estimation of GECO-ML1 expression by ionomycin (2 μM)-induced GECO-ML1 response in cells treated as in b . e , f , ML-SA1 (20 μM)-induced GECO-ML1 response (measured as change of fluorescence (∆ F ) over basal fluorescence ( F 0 ); ∆ F / F 0 ) in AGS cells expressing GECO-ML1) after 4 h VacA − or VacA + incubation. c , d , f , Representative scatter plots (mean ± s.e.m.) of the number of cells measured in each assay (indicated within parentheses). All experiments were repeated three times with similar results. Two-tailed unpaired Student’s t -test was utilized for data analysis.
Figure Legend Snippet: VacA impairs TRPML1 activity. a , Intraluminal lysosomal Ca 2+ levels in VacA − -or VacA + -treated AGS cells. AGS cells were co-loaded with Oregon Green 488 BAPTA-5N (OG BAPTA-5N), a membrane-impermeant Ca 2+ indicator with negligible sensitivity to pH, and Alexa-Fluor-586-conjugated dextran (Dextran Red, as a calcium-insensitive probe) by endocytosis. The fluorescence ratio between green and red signals was compared. A representative scatter plot (mean ± s.e.m.) of the Oregon Green/Dextran Red intensity ratio of 12 fields containing 97 VacA − -and 70 VacA + -treated cells is displayed on the right. b , c , Lysosomal calcium levels assessed by GPN (200 μM)-induced GECO-ML1 response (measured as change in fluorescence (∆ F ) over basal fluorescence ( F 0 ); ∆ F / F 0 ) in AGS cells expressing GECO-ML1) after 4 h VacA − or VacA + incubation. d , Estimation of GECO-ML1 expression by ionomycin (2 μM)-induced GECO-ML1 response in cells treated as in b . e , f , ML-SA1 (20 μM)-induced GECO-ML1 response (measured as change of fluorescence (∆ F ) over basal fluorescence ( F 0 ); ∆ F / F 0 ) in AGS cells expressing GECO-ML1) after 4 h VacA − or VacA + incubation. c , d , f , Representative scatter plots (mean ± s.e.m.) of the number of cells measured in each assay (indicated within parentheses). All experiments were repeated three times with similar results. Two-tailed unpaired Student’s t -test was utilized for data analysis.

Techniques Used: Activity Assay, Fluorescence, Expressing, Incubation, Two Tailed Test

6) Product Images from "A clinical and mechanistic study of topical borneol‐induced analgesia"

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201607300

Borneol activates TRPM 8 Quantification of consecutively applied 100 μM menthol‐ and 600 μM borneol‐induced hTRPM8 currents. Currents were normalized to 100 μM menthol‐induced currents at +80 mV ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing mouse TRPM8 in response to different concentrations of borneol. Dose–response curves of borneol‐induced increase in intracellular Ca 2+ in mouse TRPM8‐expressing HEK 293 cells. The smooth curve is a fit to the Hill equation with an EC 50 of 116 μM ( n = 12). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in mouse TRPM8‐expressing HEK 293 cells ( n = 5). Data information: All the data are presented as the mean ± standard error of the mean (SEM).
Figure Legend Snippet: Borneol activates TRPM 8 Quantification of consecutively applied 100 μM menthol‐ and 600 μM borneol‐induced hTRPM8 currents. Currents were normalized to 100 μM menthol‐induced currents at +80 mV ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing mouse TRPM8 in response to different concentrations of borneol. Dose–response curves of borneol‐induced increase in intracellular Ca 2+ in mouse TRPM8‐expressing HEK 293 cells. The smooth curve is a fit to the Hill equation with an EC 50 of 116 μM ( n = 12). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in mouse TRPM8‐expressing HEK 293 cells ( n = 5). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Techniques Used: Expressing

The borneol effect on mock‐transfected HEK 293 cells and the menthol effect on TRPM 8‐expressing cells Representative intracellular Ca 2+ signals in HEK 293 cells transfected with empty vector in response to borneol and subsequent applied Ca 2+ ionophore ionomycin ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing hTRPM8 in response to different concentrations of menthol and the subsequently applied Ca 2+ ionophore ionomycin.
Figure Legend Snippet: The borneol effect on mock‐transfected HEK 293 cells and the menthol effect on TRPM 8‐expressing cells Representative intracellular Ca 2+ signals in HEK 293 cells transfected with empty vector in response to borneol and subsequent applied Ca 2+ ionophore ionomycin ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing hTRPM8 in response to different concentrations of menthol and the subsequently applied Ca 2+ ionophore ionomycin.

Techniques Used: Transfection, Expressing, Plasmid Preparation

The effects of borneol on common peripheral molecular targets in pain sensation Representative whole‐cell currents in HEK 293 cells expressing TRPV1 (A), ASIC3 (B), P2X2 (C), or P2X4 (D) in response to capsaicin (A), acidic pH (B), or ATP (C, D) in the absence of presence of borneol ( n = 5 for each channel). The effect of different concentrations of borneol on the enzymatic activity of human COX‐2. The number of independent measurements is marked on top of each bar. Averaged intracellular Ca 2+ increases in cultured mouse DRG neurons in response to consecutive applications of 200 μM borneol, 200 μM menthol, and 67 mM KCl. A total of 81 in 1,689 neurons from four mice were found to be borneol‐sensitive and were included in the analysis. Representative intracellular Ca 2+ signals in HEK 293 cells expressing human TRPM8 (hTRPM8) in response to different concentrations of borneol. After each application of borneol, Ca 2+ ionophore ionomycin was applied to calibrate Ca 2+ response. RFU: relative fluorescence unit. Dose–response curves of the borneol‐ or menthol‐induced increase in intracellular Ca 2+ in hTRPM8‐expressing HEK 293 cells. Smooth curves are fit to the Hill equation with an EC 50 of 65 μM and a Hill coefficient of 2.0 for borneol ( n = 15) and an EC 50 of 13 μM and a Hill coefficient of 2.0 for menthol ( n = 6 at concentrations of 0.1, 0.3, and 1 μM; n = 9 at concentration of 3 μM; n = 13 at concentrations of 10, 30, 100, and 300 μM). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in hTRPM8‐expressing HEK 293 cells ( n = 6). Data information: All the data are presented as the mean ± standard error of the mean (SEM).
Figure Legend Snippet: The effects of borneol on common peripheral molecular targets in pain sensation Representative whole‐cell currents in HEK 293 cells expressing TRPV1 (A), ASIC3 (B), P2X2 (C), or P2X4 (D) in response to capsaicin (A), acidic pH (B), or ATP (C, D) in the absence of presence of borneol ( n = 5 for each channel). The effect of different concentrations of borneol on the enzymatic activity of human COX‐2. The number of independent measurements is marked on top of each bar. Averaged intracellular Ca 2+ increases in cultured mouse DRG neurons in response to consecutive applications of 200 μM borneol, 200 μM menthol, and 67 mM KCl. A total of 81 in 1,689 neurons from four mice were found to be borneol‐sensitive and were included in the analysis. Representative intracellular Ca 2+ signals in HEK 293 cells expressing human TRPM8 (hTRPM8) in response to different concentrations of borneol. After each application of borneol, Ca 2+ ionophore ionomycin was applied to calibrate Ca 2+ response. RFU: relative fluorescence unit. Dose–response curves of the borneol‐ or menthol‐induced increase in intracellular Ca 2+ in hTRPM8‐expressing HEK 293 cells. Smooth curves are fit to the Hill equation with an EC 50 of 65 μM and a Hill coefficient of 2.0 for borneol ( n = 15) and an EC 50 of 13 μM and a Hill coefficient of 2.0 for menthol ( n = 6 at concentrations of 0.1, 0.3, and 1 μM; n = 9 at concentration of 3 μM; n = 13 at concentrations of 10, 30, 100, and 300 μM). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in hTRPM8‐expressing HEK 293 cells ( n = 6). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Techniques Used: Expressing, Activity Assay, Cell Culture, Mouse Assay, Fluorescence, Concentration Assay

7) Product Images from "Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization"

Article Title: Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020605

Evaluation of the NanoLuc reporter system by expressing murine NFAT1. ( A ) HEK293 cells were co-transfected with an expression vector of murine NFAT1, a firefly luciferase (Fluc) expression plasmid (pGL4.53[ luc2/ PGK]), and pNL3.2[ NlucP/ minP] containing different numbers (none, 3, 6 and 9) of the IL8 NFAT-RE. One day after transfection, cells were stimulated with ionomycin (IM, 1 μM; vehicle, 0.007% ethanol) for 6 h. Cell lysates were used to measure luminescent signals of NanoLuc (Nluc) and Fluc using a Nano-Glo Dual-Luciferase Reporter Assay System. The ratio of Nluc to Fluc, Nluc/Fluc, is expressed as normalized relative luciferase activity (RLA). Dots and bars represent individual and averaged RLA values obtained from triplicate assays, respectively. ( B ) HEK293 cells were co-transfected with expression plasmids for NanoLuc reporter and Fluc together with either wild type (WT), constitutively active type (CA) murine NFAT1 or empty vector (pcDNA3). One day after transfection, cells were pre-treated with FK506 (10 μM) or vehicle (0.16% ethanol) for 1 h and then subjected to ionomycin (IM) stimulation for 6 h, followed by luciferase assays.
Figure Legend Snippet: Evaluation of the NanoLuc reporter system by expressing murine NFAT1. ( A ) HEK293 cells were co-transfected with an expression vector of murine NFAT1, a firefly luciferase (Fluc) expression plasmid (pGL4.53[ luc2/ PGK]), and pNL3.2[ NlucP/ minP] containing different numbers (none, 3, 6 and 9) of the IL8 NFAT-RE. One day after transfection, cells were stimulated with ionomycin (IM, 1 μM; vehicle, 0.007% ethanol) for 6 h. Cell lysates were used to measure luminescent signals of NanoLuc (Nluc) and Fluc using a Nano-Glo Dual-Luciferase Reporter Assay System. The ratio of Nluc to Fluc, Nluc/Fluc, is expressed as normalized relative luciferase activity (RLA). Dots and bars represent individual and averaged RLA values obtained from triplicate assays, respectively. ( B ) HEK293 cells were co-transfected with expression plasmids for NanoLuc reporter and Fluc together with either wild type (WT), constitutively active type (CA) murine NFAT1 or empty vector (pcDNA3). One day after transfection, cells were pre-treated with FK506 (10 μM) or vehicle (0.16% ethanol) for 1 h and then subjected to ionomycin (IM) stimulation for 6 h, followed by luciferase assays.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

8) Product Images from "Neural Cell Adhesion Molecule 2 Promotes the Formation of Filopodia and Neurite Branching by Inducing Submembrane Increases in Ca2+ Levels"

Article Title: Neural Cell Adhesion Molecule 2 Promotes the Formation of Filopodia and Neurite Branching by Inducing Submembrane Increases in Ca2+ Levels

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1714-14.2015

LCK-GCaMP5 and Red-GECO detect ionomycin-induced increases in submembrane and cytosolic [Ca 2+ ] in somata and neurites of cortical neurons. A , Pseudocolored images of a 2-d-old cortical neuron cotransfected with LCK-GCaMP5 and Red-GECO are shown. Images were taken before and after application of ionomycin. Note an increase in submembrane and cytosolic [Ca 2+ ] after application of ionomycin. Scale bar, 10 μm. B , Graphs show changes in fluorescence intensities of LCK-GCaMP5 and Red-GECO in neurites of cultured 1- to 3-d-old cortical neurons before and after application of ionomycin. An individual recording (top) and the average of n = 11 recordings (mean ± SEM) are shown. Fluorescence intensities were normalized to the mean fluorescence intensities before application of ionomycin, set to 0. C , Graphs show mean + SEM amplitudes of LCK-GCaMP5 and Red-GECO fluorescence intensity increases in response to ionomycin in somata and neurites of neurons ( n = 11 neurons in each group were analyzed).
Figure Legend Snippet: LCK-GCaMP5 and Red-GECO detect ionomycin-induced increases in submembrane and cytosolic [Ca 2+ ] in somata and neurites of cortical neurons. A , Pseudocolored images of a 2-d-old cortical neuron cotransfected with LCK-GCaMP5 and Red-GECO are shown. Images were taken before and after application of ionomycin. Note an increase in submembrane and cytosolic [Ca 2+ ] after application of ionomycin. Scale bar, 10 μm. B , Graphs show changes in fluorescence intensities of LCK-GCaMP5 and Red-GECO in neurites of cultured 1- to 3-d-old cortical neurons before and after application of ionomycin. An individual recording (top) and the average of n = 11 recordings (mean ± SEM) are shown. Fluorescence intensities were normalized to the mean fluorescence intensities before application of ionomycin, set to 0. C , Graphs show mean + SEM amplitudes of LCK-GCaMP5 and Red-GECO fluorescence intensity increases in response to ionomycin in somata and neurites of neurons ( n = 11 neurons in each group were analyzed).

Techniques Used: Fluorescence, Cell Culture

9) Product Images from "CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice"

Article Title: CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00775

The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P
Figure Legend Snippet: The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P

Techniques Used: Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Injection

10) Product Images from "Transbilayer phospholipid movement facilitates the translocation of annexin across membranes"

Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

Journal: Journal of Cell Science

doi: 10.1242/jcs.217034

TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P
Figure Legend Snippet: TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P

Techniques Used: Knock-Out, Incubation, Western Blot, Expressing, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Transfection

TMEM16F regulates lipid movement in HeLa cells. (A) TMEM16F-knockout (KO) cells do not externalise PS in response to ionomycin stimulation. Wild-type (WT), matched controls and TMEM16F-knockout cells were treated with ionomycin for 10 min at 37°C in the presence of recombinant annexin-A5–Cy5 and PI. Recombinant annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of recombinant annexin-A5–Cy5 binding to live cells are shown ( n =4). A quantification of the geometric mean±s.d. fluorescence intensity of annexin-A5–Cy5 binding from four separate experiments are shown. * P
Figure Legend Snippet: TMEM16F regulates lipid movement in HeLa cells. (A) TMEM16F-knockout (KO) cells do not externalise PS in response to ionomycin stimulation. Wild-type (WT), matched controls and TMEM16F-knockout cells were treated with ionomycin for 10 min at 37°C in the presence of recombinant annexin-A5–Cy5 and PI. Recombinant annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of recombinant annexin-A5–Cy5 binding to live cells are shown ( n =4). A quantification of the geometric mean±s.d. fluorescence intensity of annexin-A5–Cy5 binding from four separate experiments are shown. * P

Techniques Used: Knock-Out, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Fluorescence

11) Product Images from "Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis"

Article Title: Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis

Journal: ACS Pharmacology & Translational Science

doi: 10.1021/acsptsci.9b00052

Proteasome inhibition suppresses ionomycin-induced necrosis in primary cardiomyocytes. (A) NRCMs were transfected with control (siControl) or PSMB5 siRNA (siPSMB5). Knockdown efficiency was assessed by Western blotting. (B) NRCMs transfected with siControl or siPSMB5 were incubated with ionomycin (1 μM) for 1 h ( n = 3). Cell viability and LDH release were analyzed by MTT and LDH assays, respectively. **, p
Figure Legend Snippet: Proteasome inhibition suppresses ionomycin-induced necrosis in primary cardiomyocytes. (A) NRCMs were transfected with control (siControl) or PSMB5 siRNA (siPSMB5). Knockdown efficiency was assessed by Western blotting. (B) NRCMs transfected with siControl or siPSMB5 were incubated with ionomycin (1 μM) for 1 h ( n = 3). Cell viability and LDH release were analyzed by MTT and LDH assays, respectively. **, p

Techniques Used: Inhibition, Transfection, Western Blot, Incubation, MTT Assay

Enrichment of functional groups, biological processes and pathways in ionomycin-induced necrosis. (A) Candidate genes from the primary screen were grouped by molecular function using the PANTHER classification system. Top molecular functions related with necrosis were catalytic activity and binding. (B) Detailed protein categorization of the catalytic activity group in panel (A). Hydrolase and transferase activities were the primary catalytic activities involved in necrosis. (C) Candidate genes from the primary screen were grouped by biological process using the PANTHER classification system. More than half of all hits were associated with cellular and metabolic processes. (D) Detailed protein categorization of the cellular process group in panel (C). Cell communication and cell cycle were the predominant cellular processes identified. (E) Highly scored pathways in ionomycin-induced necrosis based on PANTHER pathway analysis.
Figure Legend Snippet: Enrichment of functional groups, biological processes and pathways in ionomycin-induced necrosis. (A) Candidate genes from the primary screen were grouped by molecular function using the PANTHER classification system. Top molecular functions related with necrosis were catalytic activity and binding. (B) Detailed protein categorization of the catalytic activity group in panel (A). Hydrolase and transferase activities were the primary catalytic activities involved in necrosis. (C) Candidate genes from the primary screen were grouped by biological process using the PANTHER classification system. More than half of all hits were associated with cellular and metabolic processes. (D) Detailed protein categorization of the cellular process group in panel (C). Cell communication and cell cycle were the predominant cellular processes identified. (E) Highly scored pathways in ionomycin-induced necrosis based on PANTHER pathway analysis.

Techniques Used: Functional Assay, Activity Assay, Binding Assay

12) Product Images from "Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes"

Article Title: Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02949

Effect of activation on T2R38 expression in PBMC. (A) PBMC were stimulated with different concentrations of PMA/ionomycin (1 μg/ml), PHA or anti-CD3/CD28 mAbs for 72 h ( n ≥ 5) (B) Isolated PBMC from young and elderly individuals were stimulated with 1 ng/ml CD3/CD28 for 72 h ( n ≥ 5) (C) PBMC were stimulated with 1 ng/ml CD3/CD28 for the indicated time points, activated cells were determined by the percentage of blast cells ( n ≥ 3) (D) PBMC were stimulated with 1 ng/ml CD3/CD28 and T2R38 expression on CD69+/CD25+ CD3+T lymphocytes determined at the indicated time points ( n = 5). A representative staining of surface markers CD69+/CD25+ at day 2 from one subject is shown as scattergram. T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control; bars are means + SD (A,B) or means ± SD (C,D) . Significance of difference was calculated relatively to the respective control, * p
Figure Legend Snippet: Effect of activation on T2R38 expression in PBMC. (A) PBMC were stimulated with different concentrations of PMA/ionomycin (1 μg/ml), PHA or anti-CD3/CD28 mAbs for 72 h ( n ≥ 5) (B) Isolated PBMC from young and elderly individuals were stimulated with 1 ng/ml CD3/CD28 for 72 h ( n ≥ 5) (C) PBMC were stimulated with 1 ng/ml CD3/CD28 for the indicated time points, activated cells were determined by the percentage of blast cells ( n ≥ 3) (D) PBMC were stimulated with 1 ng/ml CD3/CD28 and T2R38 expression on CD69+/CD25+ CD3+T lymphocytes determined at the indicated time points ( n = 5). A representative staining of surface markers CD69+/CD25+ at day 2 from one subject is shown as scattergram. T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control; bars are means + SD (A,B) or means ± SD (C,D) . Significance of difference was calculated relatively to the respective control, * p

Techniques Used: Activation Assay, Expressing, Isolation, Staining

13) Product Images from "Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes"

Article Title: Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02949

Effect of activation on T2R38 expression in PBMC. (A) PBMC were stimulated with different concentrations of PMA/ionomycin (1 μg/ml), PHA or anti-CD3/CD28 mAbs for 72 h ( n ≥ 5) (B) Isolated PBMC from young and elderly individuals were stimulated with 1 ng/ml CD3/CD28 for 72 h ( n ≥ 5) (C) PBMC were stimulated with 1 ng/ml CD3/CD28 for the indicated time points, activated cells were determined by the percentage of blast cells ( n ≥ 3) (D) PBMC were stimulated with 1 ng/ml CD3/CD28 and T2R38 expression on CD69+/CD25+ CD3+T lymphocytes determined at the indicated time points ( n = 5). A representative staining of surface markers CD69+/CD25+ at day 2 from one subject is shown as scattergram. T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control; bars are means + SD (A,B) or means ± SD (C,D) . Significance of difference was calculated relatively to the respective control, * p
Figure Legend Snippet: Effect of activation on T2R38 expression in PBMC. (A) PBMC were stimulated with different concentrations of PMA/ionomycin (1 μg/ml), PHA or anti-CD3/CD28 mAbs for 72 h ( n ≥ 5) (B) Isolated PBMC from young and elderly individuals were stimulated with 1 ng/ml CD3/CD28 for 72 h ( n ≥ 5) (C) PBMC were stimulated with 1 ng/ml CD3/CD28 for the indicated time points, activated cells were determined by the percentage of blast cells ( n ≥ 3) (D) PBMC were stimulated with 1 ng/ml CD3/CD28 and T2R38 expression on CD69+/CD25+ CD3+T lymphocytes determined at the indicated time points ( n = 5). A representative staining of surface markers CD69+/CD25+ at day 2 from one subject is shown as scattergram. T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control; bars are means + SD (A,B) or means ± SD (C,D) . Significance of difference was calculated relatively to the respective control, * p

Techniques Used: Activation Assay, Expressing, Isolation, Staining

14) Product Images from "Characterization of the Two CART Genes (CART1 and CART2) in Chickens (Gallus gallus)"

Article Title: Characterization of the Two CART Genes (CART1 and CART2) in Chickens (Gallus gallus)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0127107

Expression of CART1 in chicken tissues. RT-PCR detection of cCART1 mRNA expression in ( A ) adult chicken tissues including the whole brain (Br), heart (He), duodenum (Du), kidneys (Ki), liver (Li), lung (Lu), muscle (Mu), ovary (Ov), testes (Te), pituitary (Pi), spleen (Sp) and pancreas (Pa); ( B ) various adult chicken brain regions including the telencephalon (Tc), midbrain (Mb), cerebellum (Cb), hindbrain (Hb) and hypothalamus (Hp). Numbers in brackets indicate the numbers of PCR cycles used. Quantitative RT-PCR assay of cCART1 mRNA expression in ( C ) adult chicken tissues; ( D ) various adult brain regions and the pituitary. In ( C ) and ( D ), the mRNA levels of CART1 genes were normalized to that of β-actin and expressed as the fold difference compared with that of the whole brain (Br) or hypothalamus (Hp). Each data point represents the mean ± SEM of 6 individual adult chickens ( N = 6). ND indicates that CART1 mRNA is undetectable in this tissue. ( E ) Amplification plot of cCART1 and β-actin genes using the same amount of adult pituitary cDNA template. ( F ) Western blot detection of the 4 major CART1-ir bands (named CART1-I, II, III, IV) in adult chicken anterior pituitary tissue lysate (iCART1 means intracellular CART1), or in the medium (sCART1 means secreted CART1) of adult pituitaries incubated in vitro for 4 h. ( G ) Western blot detection of the effect of pharmacological drugs (500 nM ionomycin, 100 nM PMA, and 5 μM forskolin) on the secretion of CART1 from 1-week-old chick anterior pituitaries incubated in vitro . Multiple CART1-ir bands, including the two large CART1 bands (cCART1-I and cCART1-II, indicated by arrows) and a small band (cCART1-III/cCART1-IV, denoted by arrow heads) were identified in pituitary tissue lysate (iCART1), whereas only the small band (CART1-III or CART1-IV) was identified in the incubation medium of pituitaries treated with ionomycin and forskolin.
Figure Legend Snippet: Expression of CART1 in chicken tissues. RT-PCR detection of cCART1 mRNA expression in ( A ) adult chicken tissues including the whole brain (Br), heart (He), duodenum (Du), kidneys (Ki), liver (Li), lung (Lu), muscle (Mu), ovary (Ov), testes (Te), pituitary (Pi), spleen (Sp) and pancreas (Pa); ( B ) various adult chicken brain regions including the telencephalon (Tc), midbrain (Mb), cerebellum (Cb), hindbrain (Hb) and hypothalamus (Hp). Numbers in brackets indicate the numbers of PCR cycles used. Quantitative RT-PCR assay of cCART1 mRNA expression in ( C ) adult chicken tissues; ( D ) various adult brain regions and the pituitary. In ( C ) and ( D ), the mRNA levels of CART1 genes were normalized to that of β-actin and expressed as the fold difference compared with that of the whole brain (Br) or hypothalamus (Hp). Each data point represents the mean ± SEM of 6 individual adult chickens ( N = 6). ND indicates that CART1 mRNA is undetectable in this tissue. ( E ) Amplification plot of cCART1 and β-actin genes using the same amount of adult pituitary cDNA template. ( F ) Western blot detection of the 4 major CART1-ir bands (named CART1-I, II, III, IV) in adult chicken anterior pituitary tissue lysate (iCART1 means intracellular CART1), or in the medium (sCART1 means secreted CART1) of adult pituitaries incubated in vitro for 4 h. ( G ) Western blot detection of the effect of pharmacological drugs (500 nM ionomycin, 100 nM PMA, and 5 μM forskolin) on the secretion of CART1 from 1-week-old chick anterior pituitaries incubated in vitro . Multiple CART1-ir bands, including the two large CART1 bands (cCART1-I and cCART1-II, indicated by arrows) and a small band (cCART1-III/cCART1-IV, denoted by arrow heads) were identified in pituitary tissue lysate (iCART1), whereas only the small band (CART1-III or CART1-IV) was identified in the incubation medium of pituitaries treated with ionomycin and forskolin.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Western Blot, Incubation, In Vitro

15) Product Images from "Histone deacetylase 1 reduces NO production in endothelial cells via lysine deacetylation of NO synthase 3"

Article Title: Histone deacetylase 1 reduces NO production in endothelial cells via lysine deacetylation of NO synthase 3

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00243.2014

HDAC1 small-interfering RNA (siRNA) knockdown in BAECs resulted in a 50% reduction in HDAC1 protein expression ( A ) compared with scramble siRNA control BAECs ( B ). C : HDAC1 knockdown resulted in an increase in nitrite production under basal and ET-1 (100 nM, 1 h)-stimulated states, whereas ionomycin (3 μM, 1 h)-stimulated nitrite production was not significantly affected by HDAC1 knockdown. D : NOS3 acetylation was similar between scramble and HDAC1 siRNA cells as quantified in E . F : NOS3 phosphorylation levels were not significantly different from scramble controls. G : densitometry of the phosphorylation sites of NOS3. N = 3–5. * P
Figure Legend Snippet: HDAC1 small-interfering RNA (siRNA) knockdown in BAECs resulted in a 50% reduction in HDAC1 protein expression ( A ) compared with scramble siRNA control BAECs ( B ). C : HDAC1 knockdown resulted in an increase in nitrite production under basal and ET-1 (100 nM, 1 h)-stimulated states, whereas ionomycin (3 μM, 1 h)-stimulated nitrite production was not significantly affected by HDAC1 knockdown. D : NOS3 acetylation was similar between scramble and HDAC1 siRNA cells as quantified in E . F : NOS3 phosphorylation levels were not significantly different from scramble controls. G : densitometry of the phosphorylation sites of NOS3. N = 3–5. * P

Techniques Used: Small Interfering RNA, Expressing

Overexpression of HDAC1 in BAECs reduced nitrite production under basal and endothelin-1 (ET-1; 100 nM, 1 h)-stimulated states ( A ), whereas ionomycin (3 μM, 1 h)-stimulated nitrite production was not significantly affected by overexpression of HDAC1 ( B ). Ionomycin did not significantly affect NOS3 acetylation ( C ), but ionomycin significantly reduced NOS3 T497 phosphorylation ( D ) after 5 or 60 min of treatment ( E ). N = 3 to 4. * P
Figure Legend Snippet: Overexpression of HDAC1 in BAECs reduced nitrite production under basal and endothelin-1 (ET-1; 100 nM, 1 h)-stimulated states ( A ), whereas ionomycin (3 μM, 1 h)-stimulated nitrite production was not significantly affected by overexpression of HDAC1 ( B ). Ionomycin did not significantly affect NOS3 acetylation ( C ), but ionomycin significantly reduced NOS3 T497 phosphorylation ( D ) after 5 or 60 min of treatment ( E ). N = 3 to 4. * P

Techniques Used: Over Expression

16) Product Images from "Transbilayer phospholipid movement facilitates the translocation of annexin across membranes"

Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

Journal: Journal of Cell Science

doi: 10.1242/jcs.217034

TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P
Figure Legend Snippet: TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P

Techniques Used: Knock-Out, Incubation, Western Blot, Expressing, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Transfection

TMEM16F regulates lipid movement in HeLa cells. (A) TMEM16F-knockout (KO) cells do not externalise PS in response to ionomycin stimulation. Wild-type (WT), matched controls and TMEM16F-knockout cells were treated with ionomycin for 10 min at 37°C in the presence of recombinant annexin-A5–Cy5 and PI. Recombinant annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of recombinant annexin-A5–Cy5 binding to live cells are shown ( n =4). A quantification of the geometric mean±s.d. fluorescence intensity of annexin-A5–Cy5 binding from four separate experiments are shown. * P
Figure Legend Snippet: TMEM16F regulates lipid movement in HeLa cells. (A) TMEM16F-knockout (KO) cells do not externalise PS in response to ionomycin stimulation. Wild-type (WT), matched controls and TMEM16F-knockout cells were treated with ionomycin for 10 min at 37°C in the presence of recombinant annexin-A5–Cy5 and PI. Recombinant annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of recombinant annexin-A5–Cy5 binding to live cells are shown ( n =4). A quantification of the geometric mean±s.d. fluorescence intensity of annexin-A5–Cy5 binding from four separate experiments are shown. * P

Techniques Used: Knock-Out, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Fluorescence

17) Product Images from "Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease"

Article Title: Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.00039

Etrolizumab-s does not elicit agonistic activity and cytokine production. (A) Left panels: Representative flow cytometry showing expression of CD25 (upper panels) and CD69 (lower panels) after cell treatment with or without PMA/ionomycin and etrolizumab-s for 6 or 24 h. Right panels: Quantitative flow cytometry ( n = 11–26). (B) Left panels: Representative flow cytometry showing expression of pro-inflammatory cytokines after cell treatment with or without PMA/ionomycin and etrolizumab-s for 6 h. Right panels: Quantitative flow cytometry ( n = 21–23 per group). ∗∗∗ p
Figure Legend Snippet: Etrolizumab-s does not elicit agonistic activity and cytokine production. (A) Left panels: Representative flow cytometry showing expression of CD25 (upper panels) and CD69 (lower panels) after cell treatment with or without PMA/ionomycin and etrolizumab-s for 6 or 24 h. Right panels: Quantitative flow cytometry ( n = 11–26). (B) Left panels: Representative flow cytometry showing expression of pro-inflammatory cytokines after cell treatment with or without PMA/ionomycin and etrolizumab-s for 6 h. Right panels: Quantitative flow cytometry ( n = 21–23 per group). ∗∗∗ p

Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Expressing

18) Product Images from "Disease-associated mutations in TRPM3 render the channel overactive via two distinct mechanisms"

Article Title: Disease-associated mutations in TRPM3 render the channel overactive via two distinct mechanisms

Journal: bioRxiv

doi: 10.1101/2020.04.20.052167

Disease-associated mutations increase agonist sensitivity and basal activity of TRPM3. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the methods section. A-C fluorescence traces for TRPM3 (A), V992M (B) and P1092Q (C); the applications of various concentrations of PregS and 2 μM ionomycin are indicated by the arrows. Basal fluorescence before the application of PregS was first subtracted, then the traces were normalized to the fluorescence after the application of ionomycin. Each trace shows the average of 4 replicates from the same 96-well plate. Measurements were performed at 21 °C. D : Hill fits of the concentration dependence of the fluorescence signals evoked by PregS. Symbols represent individual wells from 3 independent transfections. The EC 50 values were 7.01 ± 0.69 μM for wild type channels, 0.32 ± 0.03 μM for V992M and 1.97 ± 0.08 μM for P1092Q. E-G fluorescence traces for TRPM3 (E), V992M (F) and P1092Q (G), the applications of various concentrations of CIM0216 and 2 μM ionomycin are indicated by the arrows. Basal fluorescence before the application of CIM0216 was first subtracted, then the traces were normalized to the fluorescence after the application of ionomycin. Measurements were performed at 21 °C. H: Hill fits of the concentration dependence of the fluorescence signals evoked by CIM0216. Symbols represent individual wells from 2 independent transfections. The EC 50 values were 2.72 ± 0.17 μM for wild type channels, 0.17 ± 0.02 μM for V992M and 0.88 ± 0.08 μM for P1092Q. I-K: fluorescence traces for TRPM3 (I), V992M (J) and P1092Q (K), the applications of various concentrations of primidone are indicated by the arrows; traces were not normalized and shown as arbitrary fluorescence units (A.U.). Measurements were performed at 21 °C. L : Hill1 fits of the concentration dependence of the inhibition evoked by primidone. Symbols represent mean ± SEM from two independent transfections, 5 or 6 wells in each. The IC 50 values were 2.41 ± 0.74 μM for V992M and 0.64 ± 0.09 μM for P1092Q. M-O: fluorescence traces for TRPM3 (M), V992M (N) and P1092Q (O), the applications of 50 μM primidone are indicated by the arrows; traces show the average of 16 wells from two independent transfections, normalized to the effect of ionomycin. Measurements were performed at 37 °C. P: Summary of the data, Mean ± SEM and scatter plots. Statistical significance was calculated with one-way analysis of variance with Bonferroni post hoc comparison for differences of basal fluorescence values between mutant and wild-type channels. The effect of primidone in wild type and mutant channel was evaluated with paired t-test; the p values for significance are shown above the bars.
Figure Legend Snippet: Disease-associated mutations increase agonist sensitivity and basal activity of TRPM3. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the methods section. A-C fluorescence traces for TRPM3 (A), V992M (B) and P1092Q (C); the applications of various concentrations of PregS and 2 μM ionomycin are indicated by the arrows. Basal fluorescence before the application of PregS was first subtracted, then the traces were normalized to the fluorescence after the application of ionomycin. Each trace shows the average of 4 replicates from the same 96-well plate. Measurements were performed at 21 °C. D : Hill fits of the concentration dependence of the fluorescence signals evoked by PregS. Symbols represent individual wells from 3 independent transfections. The EC 50 values were 7.01 ± 0.69 μM for wild type channels, 0.32 ± 0.03 μM for V992M and 1.97 ± 0.08 μM for P1092Q. E-G fluorescence traces for TRPM3 (E), V992M (F) and P1092Q (G), the applications of various concentrations of CIM0216 and 2 μM ionomycin are indicated by the arrows. Basal fluorescence before the application of CIM0216 was first subtracted, then the traces were normalized to the fluorescence after the application of ionomycin. Measurements were performed at 21 °C. H: Hill fits of the concentration dependence of the fluorescence signals evoked by CIM0216. Symbols represent individual wells from 2 independent transfections. The EC 50 values were 2.72 ± 0.17 μM for wild type channels, 0.17 ± 0.02 μM for V992M and 0.88 ± 0.08 μM for P1092Q. I-K: fluorescence traces for TRPM3 (I), V992M (J) and P1092Q (K), the applications of various concentrations of primidone are indicated by the arrows; traces were not normalized and shown as arbitrary fluorescence units (A.U.). Measurements were performed at 21 °C. L : Hill1 fits of the concentration dependence of the inhibition evoked by primidone. Symbols represent mean ± SEM from two independent transfections, 5 or 6 wells in each. The IC 50 values were 2.41 ± 0.74 μM for V992M and 0.64 ± 0.09 μM for P1092Q. M-O: fluorescence traces for TRPM3 (M), V992M (N) and P1092Q (O), the applications of 50 μM primidone are indicated by the arrows; traces show the average of 16 wells from two independent transfections, normalized to the effect of ionomycin. Measurements were performed at 37 °C. P: Summary of the data, Mean ± SEM and scatter plots. Statistical significance was calculated with one-way analysis of variance with Bonferroni post hoc comparison for differences of basal fluorescence values between mutant and wild-type channels. The effect of primidone in wild type and mutant channel was evaluated with paired t-test; the p values for significance are shown above the bars.

Techniques Used: Activity Assay, Transfection, Fluorescence, Concentration Assay, Inhibition, Mutagenesis

PregS responses of wild type and mutant TRPM3 at 37 °C. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the method section. A: average traces from one plate (4 wells for each condition) for stimulation with different concentrations of PregS for cells expressing wild type TRPM3; at the end of the experiment 2 μM ionomycin was applied as a normalizing stimulus. B: Hill fit of the PregS concentration response relationship, symbols show values obtained in individual wells n=8 from two independent transfections. The dashed line shows the fit for the concentration response curve obtained at 21 °C from Figure 1D . C-D: identical experiments to those shown in panel A, on cells expressing the V992M (C) and the P1092Q (D) mutants.
Figure Legend Snippet: PregS responses of wild type and mutant TRPM3 at 37 °C. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the method section. A: average traces from one plate (4 wells for each condition) for stimulation with different concentrations of PregS for cells expressing wild type TRPM3; at the end of the experiment 2 μM ionomycin was applied as a normalizing stimulus. B: Hill fit of the PregS concentration response relationship, symbols show values obtained in individual wells n=8 from two independent transfections. The dashed line shows the fit for the concentration response curve obtained at 21 °C from Figure 1D . C-D: identical experiments to those shown in panel A, on cells expressing the V992M (C) and the P1092Q (D) mutants.

Techniques Used: Mutagenesis, Transfection, Fluorescence, Expressing, Concentration Assay

19) Product Images from "HIV LTR-Driven Antisense RNA by Itself Has Regulatory Function and May Curtail Virus Reactivation From Latency"

Article Title: HIV LTR-Driven Antisense RNA by Itself Has Regulatory Function and May Curtail Virus Reactivation From Latency

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01066

Sense RNA reactivation is influenced by antisense RNA transcription. (A) Representative results of sense (mCherry) and antisense RNA expression (Venus) after PMA/Ionomycin treatment of 2 individual cell clones (AS+2G8 and DN12D7c). Dot plots from flow cytometry at 24 h post stimulation are shown. (B–E) Increase of sense and antisense RNA expression in cloned cells after activation with PMA/Ionomycin (B,C) or SAHA (D,E) . The fold increase of the MFI of mCherry and Venus expression is shown for all individual cell clones. Fold increase was calculated from mCherry or Venus MFI of activated cells using that of non-activated (DMSO treated) cells as reference. Means ± SD of three to five independent experiments are given. P -values were calculated by Mann–Whitney’s tests. (F) Fold sense RNA increase after cell clone activation with PMA/Ionomycin or SAHA. Relative levels of mCherry RNA were measured by sense-strand-specific RT-qPCR. Dots represent PMA/Ionomycin-treated clones; triangles represent SAHA-treated clones. The p value was calculated by Mann–Whitney’s test.
Figure Legend Snippet: Sense RNA reactivation is influenced by antisense RNA transcription. (A) Representative results of sense (mCherry) and antisense RNA expression (Venus) after PMA/Ionomycin treatment of 2 individual cell clones (AS+2G8 and DN12D7c). Dot plots from flow cytometry at 24 h post stimulation are shown. (B–E) Increase of sense and antisense RNA expression in cloned cells after activation with PMA/Ionomycin (B,C) or SAHA (D,E) . The fold increase of the MFI of mCherry and Venus expression is shown for all individual cell clones. Fold increase was calculated from mCherry or Venus MFI of activated cells using that of non-activated (DMSO treated) cells as reference. Means ± SD of three to five independent experiments are given. P -values were calculated by Mann–Whitney’s tests. (F) Fold sense RNA increase after cell clone activation with PMA/Ionomycin or SAHA. Relative levels of mCherry RNA were measured by sense-strand-specific RT-qPCR. Dots represent PMA/Ionomycin-treated clones; triangles represent SAHA-treated clones. The p value was calculated by Mann–Whitney’s test.

Techniques Used: RNA Expression, Clone Assay, Flow Cytometry, Cytometry, Activation Assay, Expressing, MANN-WHITNEY, Quantitative RT-PCR

20) Product Images from "High-throughput screening identifies inhibitors of DUX4-induced myoblast toxicity"

Article Title: High-throughput screening identifies inhibitors of DUX4-induced myoblast toxicity

Journal: Skeletal Muscle

doi: 10.1186/2044-5040-4-4

Activity of repurchased compounds on cell death induced by DUX4 or other cytotoxic pathways. (A) Viability of DUX4-expressing C2C12 cells exposed to various concentrations of compounds, from 0.01 μM to 7.44 μM. Compounds are arranged in order of greatest viability at any concentration. Four Prestwich compounds are also shown: α-tocopherol, vitamin K2, ethoxyquin, and ethopropazine HCl. B-G : Viability of C2C12 cells exposed to various cell death-inducing compounds in the presence of 5 μM compounds 1 to 52 (the active compounds of the 54 that were purchased). The Y axis represents viability (ATP content). The first point in each series represents untreated cells, the second represents cells treated with toxic agent alone, and the remaining points represent cells treated with toxic agent plus inhibitory compounds, in order from 1 to 52 ( n = 3), error bars = SEM. The dashed red line represents 3 standard deviations above the control sample without compound. (B) Protection from ABT-263. (C) Protection from Staurosporine. (D) Protection from Etoposide. (E) Protection from Ionomycin. (F) Protection from tBHP. (G) Protection from Tunicamycin. (H) Viability of 3T3 cells exposed to tBHP in the presence of compounds 1 to 52. (I) Protection from tBHP in C2C12 cells vs. 3T3 cells. R = Spearman’s correlation coefficient. The strong correlation indicates that compounds tend to protect equally well in both cell types. (J) Activity of 52 repurchased compounds on cell death induced by DUX4 in 293 T cells. Viability of 293T cells transfected with vector control (EV), DUX4 plus carrier (DMSO) alone, or DUX4 and treated with of 5 μM compounds 1 to 52 ( n = 6). The Y axis represents improvement in viability over DUX4 transfection alone. Red dots represent compounds that gave a statistically significant ( P
Figure Legend Snippet: Activity of repurchased compounds on cell death induced by DUX4 or other cytotoxic pathways. (A) Viability of DUX4-expressing C2C12 cells exposed to various concentrations of compounds, from 0.01 μM to 7.44 μM. Compounds are arranged in order of greatest viability at any concentration. Four Prestwich compounds are also shown: α-tocopherol, vitamin K2, ethoxyquin, and ethopropazine HCl. B-G : Viability of C2C12 cells exposed to various cell death-inducing compounds in the presence of 5 μM compounds 1 to 52 (the active compounds of the 54 that were purchased). The Y axis represents viability (ATP content). The first point in each series represents untreated cells, the second represents cells treated with toxic agent alone, and the remaining points represent cells treated with toxic agent plus inhibitory compounds, in order from 1 to 52 ( n = 3), error bars = SEM. The dashed red line represents 3 standard deviations above the control sample without compound. (B) Protection from ABT-263. (C) Protection from Staurosporine. (D) Protection from Etoposide. (E) Protection from Ionomycin. (F) Protection from tBHP. (G) Protection from Tunicamycin. (H) Viability of 3T3 cells exposed to tBHP in the presence of compounds 1 to 52. (I) Protection from tBHP in C2C12 cells vs. 3T3 cells. R = Spearman’s correlation coefficient. The strong correlation indicates that compounds tend to protect equally well in both cell types. (J) Activity of 52 repurchased compounds on cell death induced by DUX4 in 293 T cells. Viability of 293T cells transfected with vector control (EV), DUX4 plus carrier (DMSO) alone, or DUX4 and treated with of 5 μM compounds 1 to 52 ( n = 6). The Y axis represents improvement in viability over DUX4 transfection alone. Red dots represent compounds that gave a statistically significant ( P

Techniques Used: Activity Assay, Expressing, Concentration Assay, Transfection, Plasmid Preparation

21) Product Images from "Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis"

Article Title: Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis

Journal: ACS Pharmacology & Translational Science

doi: 10.1021/acsptsci.9b00052

Proteasome inhibition suppresses ionomycin-induced necrosis in primary cardiomyocytes. (A) NRCMs were transfected with control (siControl) or PSMB5 siRNA (siPSMB5). Knockdown efficiency was assessed by Western blotting. (B) NRCMs transfected with siControl or siPSMB5 were incubated with ionomycin (1 μM) for 1 h ( n = 3). Cell viability and LDH release were analyzed by MTT and LDH assays, respectively. **, p
Figure Legend Snippet: Proteasome inhibition suppresses ionomycin-induced necrosis in primary cardiomyocytes. (A) NRCMs were transfected with control (siControl) or PSMB5 siRNA (siPSMB5). Knockdown efficiency was assessed by Western blotting. (B) NRCMs transfected with siControl or siPSMB5 were incubated with ionomycin (1 μM) for 1 h ( n = 3). Cell viability and LDH release were analyzed by MTT and LDH assays, respectively. **, p

Techniques Used: Inhibition, Transfection, Western Blot, Incubation, MTT Assay

Enrichment of functional groups, biological processes and pathways in ionomycin-induced necrosis. (A) Candidate genes from the primary screen were grouped by molecular function using the PANTHER classification system. Top molecular functions related with necrosis were catalytic activity and binding. (B) Detailed protein categorization of the catalytic activity group in panel (A). Hydrolase and transferase activities were the primary catalytic activities involved in necrosis. (C) Candidate genes from the primary screen were grouped by biological process using the PANTHER classification system. More than half of all hits were associated with cellular and metabolic processes. (D) Detailed protein categorization of the cellular process group in panel (C). Cell communication and cell cycle were the predominant cellular processes identified. (E) Highly scored pathways in ionomycin-induced necrosis based on PANTHER pathway analysis.
Figure Legend Snippet: Enrichment of functional groups, biological processes and pathways in ionomycin-induced necrosis. (A) Candidate genes from the primary screen were grouped by molecular function using the PANTHER classification system. Top molecular functions related with necrosis were catalytic activity and binding. (B) Detailed protein categorization of the catalytic activity group in panel (A). Hydrolase and transferase activities were the primary catalytic activities involved in necrosis. (C) Candidate genes from the primary screen were grouped by biological process using the PANTHER classification system. More than half of all hits were associated with cellular and metabolic processes. (D) Detailed protein categorization of the cellular process group in panel (C). Cell communication and cell cycle were the predominant cellular processes identified. (E) Highly scored pathways in ionomycin-induced necrosis based on PANTHER pathway analysis.

Techniques Used: Functional Assay, Activity Assay, Binding Assay

22) Product Images from "A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation"

Article Title: A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005604

Intracellular Ca 2+ is the second messenger downstream of follicular adrenergic signaling. (A-C) Pretreatment of BAPTA-AM blocks OA-induced follicle rupture. Representative images show mature follicles treated with DMSO (A) or BAPTA-AM (B) followed a three-hour stimulation with 20 μM of OA. Ruptured follicles were quantified in C. Three replicates are used for each condition. (D-F) Ionomycin is sufficient to induced follicle rupture. Representative images show follicles after three-hour culture with ethanol (D) or 5 μM of ionomycin. Ruptured follicles after different doses of ionomycin treatment are quantified in F. All conditions have three replicates except in 5 μM, which has four replicates. (G-H) Representative images of Mmp2 -knockdown (G) and Timp -overexpressing (H) follicles treated with 5 μM of ionomycin for three hours. (I) Quantification of ruptured follicles with Mmp2 knockdown or Timp overexpression in mature follicle cells in response to 20 μM of OA or 5 μM of ionomycin stimulation. All conditions have three replicates except for Timp overexpression with ionomycin treatment, which has six replicates. (J-L) Ionomycin, but not OA, is sufficient to induce rupture in Oamb mutant follicles. Representative images show Oamb +/- (J) and Oamb -/- (K) follicles after three-hour culture with ionomycin. (L) Quantification of ruptured follicles after three-hour culture with 20 μM of OA or 5 μM of ionomycin. The number of replicates for each condition is 4, 4, 3, and 5. (M) A cartoon showing the model of follicular adrenergic signaling in Mmp activity and follicle rupture. Octopaminergic neurons are shown in green.
Figure Legend Snippet: Intracellular Ca 2+ is the second messenger downstream of follicular adrenergic signaling. (A-C) Pretreatment of BAPTA-AM blocks OA-induced follicle rupture. Representative images show mature follicles treated with DMSO (A) or BAPTA-AM (B) followed a three-hour stimulation with 20 μM of OA. Ruptured follicles were quantified in C. Three replicates are used for each condition. (D-F) Ionomycin is sufficient to induced follicle rupture. Representative images show follicles after three-hour culture with ethanol (D) or 5 μM of ionomycin. Ruptured follicles after different doses of ionomycin treatment are quantified in F. All conditions have three replicates except in 5 μM, which has four replicates. (G-H) Representative images of Mmp2 -knockdown (G) and Timp -overexpressing (H) follicles treated with 5 μM of ionomycin for three hours. (I) Quantification of ruptured follicles with Mmp2 knockdown or Timp overexpression in mature follicle cells in response to 20 μM of OA or 5 μM of ionomycin stimulation. All conditions have three replicates except for Timp overexpression with ionomycin treatment, which has six replicates. (J-L) Ionomycin, but not OA, is sufficient to induce rupture in Oamb mutant follicles. Representative images show Oamb +/- (J) and Oamb -/- (K) follicles after three-hour culture with ionomycin. (L) Quantification of ruptured follicles after three-hour culture with 20 μM of OA or 5 μM of ionomycin. The number of replicates for each condition is 4, 4, 3, and 5. (M) A cartoon showing the model of follicular adrenergic signaling in Mmp activity and follicle rupture. Octopaminergic neurons are shown in green.

Techniques Used: Over Expression, Mutagenesis, Activity Assay

23) Product Images from "A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism"

Article Title: A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism

Journal: Autophagy

doi: 10.1080/15548627.2017.1389822

MTORC1-dependent regulation of MCOLN1-mediated lysosomal Ca 2+ release. ( A ) Starvation increased MCOLN1 activity as indicated by elevated GECO-MCOLN1 responses to ML-SA1 in HEK293T cells expressing GECO-MCOLN1. Cells were kept in normal culture medium, starved for 50 min or followed by nutrient refeeding for 15 min prior to the measurement. ( B ) Summary of ML-SA1-induced GECO-MCOLN1 responses as in ( A ). ( C, D ) Starvation or refeeding did not affect GECO-MCOLN1 response to GPN (200 µM) ( C ) or ionomycin (Iono, 2 µM) ( D ). ( E ) RRAGB GTP decreased GECO-MCOLN1 responses in starved cells, whereas RRAGB GDP increased GECO-MCOLN1 responses in refeeding cells. HEK293T cells expressing GECO-MCOLN1 and RRAGB WT together with RRAGB GTP or RRAGB GDP were subjected to starvation, or nutrient refeeding (DMEM + 10% FBS, 15 min). ( F, G ) GECO-MCOLN1 response to GPN (200 µM) ( F ) and Ionomycin (2 µM) ( G ) was comparable to conditions in ( E ). ( H-J ) Inhibition of MTOR with AZD8055 (1 µM) induced GECO responses in HEK293T cells expressing GECO-MCOLN1 but not GECO-MCOLN1-DDKK, a nonconducting mutant of MCOLN1. GECO signals were measured in the absence of external Ca 2+ . GECO responses to ionomycin (2 µM, with 2 mM Ca 2+ in the bath) was used to compare the expression levels of GECO-MCOLN1 and GECO-MCOLN1-DDKK. ( K ) S571,576E phosphomimetic mutation of GECO-MCOLN1 (GECO-MCOLN1-SSEE) decreased ML-SA1-induced GECO responses upon starvation compared to GECO-MCOLN1. HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSEE, respectively, were subjected to starvation (50 min) prior to the measurement. ( L, M ) GECO-MCOLN1 response to GPN (200 µM) ( I ) or ionomycin (2 µM) ( M ) was comparable in conditions as in ( H ). ( N ) WT MCOLN1 but not MCOLN1-SSEE and MCOLN1-DDKK increased MTORC1 activity. HEK293T cells expressing LAMP1-GFP, MCOLN1-EGFP, MCOLN1 S51E -EGFP, MCOLN1-SSEE-EGFP and MCOLN1-DDKK-GFP, respectively, were starved for 30 min. MCOLN1-DDKK-GFP (MCOLN1 D471,472K MCOLN1 S51E Cell extracts were analyzed by western blotting using anti-p-RPS6KB (T389) and anti-RPS6KB antibodies, and anti-GAPDH antibody was used as a loading control. Histograms represent the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments) in the indicated conditions, relative to that of cells expressing LAMP1. ( O-Q ) Summary of ML-SA1-induced GECO responses in HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSAA under normal fed conditions. MCOLN1-SSAA displayed a higher activity in normal fed conditions compared to MCOLN1. However, MCOLN1-SSAA did not have an effect on lysosomal Ca 2+ content. These findings suggest that the lysosome must have a mechanism to maintain its Ca 2+ homeostasis. In this case, although MCOLN1-SSAA increases Ca 2+ release, a compensational mechanism exists to increase Ca 2+ uptake that depends on the endoplasmic reticulum Ca 2+ NS, not significant; *, P
Figure Legend Snippet: MTORC1-dependent regulation of MCOLN1-mediated lysosomal Ca 2+ release. ( A ) Starvation increased MCOLN1 activity as indicated by elevated GECO-MCOLN1 responses to ML-SA1 in HEK293T cells expressing GECO-MCOLN1. Cells were kept in normal culture medium, starved for 50 min or followed by nutrient refeeding for 15 min prior to the measurement. ( B ) Summary of ML-SA1-induced GECO-MCOLN1 responses as in ( A ). ( C, D ) Starvation or refeeding did not affect GECO-MCOLN1 response to GPN (200 µM) ( C ) or ionomycin (Iono, 2 µM) ( D ). ( E ) RRAGB GTP decreased GECO-MCOLN1 responses in starved cells, whereas RRAGB GDP increased GECO-MCOLN1 responses in refeeding cells. HEK293T cells expressing GECO-MCOLN1 and RRAGB WT together with RRAGB GTP or RRAGB GDP were subjected to starvation, or nutrient refeeding (DMEM + 10% FBS, 15 min). ( F, G ) GECO-MCOLN1 response to GPN (200 µM) ( F ) and Ionomycin (2 µM) ( G ) was comparable to conditions in ( E ). ( H-J ) Inhibition of MTOR with AZD8055 (1 µM) induced GECO responses in HEK293T cells expressing GECO-MCOLN1 but not GECO-MCOLN1-DDKK, a nonconducting mutant of MCOLN1. GECO signals were measured in the absence of external Ca 2+ . GECO responses to ionomycin (2 µM, with 2 mM Ca 2+ in the bath) was used to compare the expression levels of GECO-MCOLN1 and GECO-MCOLN1-DDKK. ( K ) S571,576E phosphomimetic mutation of GECO-MCOLN1 (GECO-MCOLN1-SSEE) decreased ML-SA1-induced GECO responses upon starvation compared to GECO-MCOLN1. HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSEE, respectively, were subjected to starvation (50 min) prior to the measurement. ( L, M ) GECO-MCOLN1 response to GPN (200 µM) ( I ) or ionomycin (2 µM) ( M ) was comparable in conditions as in ( H ). ( N ) WT MCOLN1 but not MCOLN1-SSEE and MCOLN1-DDKK increased MTORC1 activity. HEK293T cells expressing LAMP1-GFP, MCOLN1-EGFP, MCOLN1 S51E -EGFP, MCOLN1-SSEE-EGFP and MCOLN1-DDKK-GFP, respectively, were starved for 30 min. MCOLN1-DDKK-GFP (MCOLN1 D471,472K MCOLN1 S51E Cell extracts were analyzed by western blotting using anti-p-RPS6KB (T389) and anti-RPS6KB antibodies, and anti-GAPDH antibody was used as a loading control. Histograms represent the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments) in the indicated conditions, relative to that of cells expressing LAMP1. ( O-Q ) Summary of ML-SA1-induced GECO responses in HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSAA under normal fed conditions. MCOLN1-SSAA displayed a higher activity in normal fed conditions compared to MCOLN1. However, MCOLN1-SSAA did not have an effect on lysosomal Ca 2+ content. These findings suggest that the lysosome must have a mechanism to maintain its Ca 2+ homeostasis. In this case, although MCOLN1-SSAA increases Ca 2+ release, a compensational mechanism exists to increase Ca 2+ uptake that depends on the endoplasmic reticulum Ca 2+ NS, not significant; *, P

Techniques Used: Activity Assay, Expressing, Inhibition, Mutagenesis, Western Blot

24) Product Images from "Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization"

Article Title: Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020605

Evaluation of the NanoLuc reporter system by expressing murine NFAT1. ( A ) HEK293 cells were co-transfected with an expression vector of murine NFAT1, a firefly luciferase (Fluc) expression plasmid (pGL4.53[ luc2/ PGK]), and pNL3.2[ NlucP/ minP] containing different numbers (none, 3, 6 and 9) of the IL8 NFAT-RE. One day after transfection, cells were stimulated with ionomycin (IM, 1 μM; vehicle, 0.007% ethanol) for 6 h. Cell lysates were used to measure luminescent signals of NanoLuc (Nluc) and Fluc using a Nano-Glo Dual-Luciferase Reporter Assay System. The ratio of Nluc to Fluc, Nluc/Fluc, is expressed as normalized relative luciferase activity (RLA). Dots and bars represent individual and averaged RLA values obtained from triplicate assays, respectively. ( B ) HEK293 cells were co-transfected with expression plasmids for NanoLuc reporter and Fluc together with either wild type (WT), constitutively active type (CA) murine NFAT1 or empty vector (pcDNA3). One day after transfection, cells were pre-treated with FK506 (10 μM) or vehicle (0.16% ethanol) for 1 h and then subjected to ionomycin (IM) stimulation for 6 h, followed by luciferase assays.
Figure Legend Snippet: Evaluation of the NanoLuc reporter system by expressing murine NFAT1. ( A ) HEK293 cells were co-transfected with an expression vector of murine NFAT1, a firefly luciferase (Fluc) expression plasmid (pGL4.53[ luc2/ PGK]), and pNL3.2[ NlucP/ minP] containing different numbers (none, 3, 6 and 9) of the IL8 NFAT-RE. One day after transfection, cells were stimulated with ionomycin (IM, 1 μM; vehicle, 0.007% ethanol) for 6 h. Cell lysates were used to measure luminescent signals of NanoLuc (Nluc) and Fluc using a Nano-Glo Dual-Luciferase Reporter Assay System. The ratio of Nluc to Fluc, Nluc/Fluc, is expressed as normalized relative luciferase activity (RLA). Dots and bars represent individual and averaged RLA values obtained from triplicate assays, respectively. ( B ) HEK293 cells were co-transfected with expression plasmids for NanoLuc reporter and Fluc together with either wild type (WT), constitutively active type (CA) murine NFAT1 or empty vector (pcDNA3). One day after transfection, cells were pre-treated with FK506 (10 μM) or vehicle (0.16% ethanol) for 1 h and then subjected to ionomycin (IM) stimulation for 6 h, followed by luciferase assays.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

25) Product Images from "Transcriptional inhibitors identified in a 160,000-compound small molecule DUX4-viability screen"

Article Title: Transcriptional inhibitors identified in a 160,000-compound small molecule DUX4-viability screen

Journal: Journal of biomolecular screening

doi: 10.1177/1087057116651868

Protection from other cell death-inducing pathways WT C2C12 cells were exposed to cell death-inducing drugs acting on various pathways in the presence of 5 μM of purchased compounds. Viability (ATP content) is shown on the Y axis. The first two points in each series represents controls: cells not treated with toxic agent followed by cells treated with toxic agent plus DMSO alone. Compounds tested for protection are indicated from 1–46 on the X axis, error bars = SEM. A cutoff of 3 standard deviations above control (toxic agent + DMSO alone) is shown as a dotted red line. (A) Protection from 12.5 μM ABT-263. (B) Protection from 0.0125 μM Staurosporine. (C) Protection from 12.5 μM Etoposide. (D) Protection from 12.5 μM Ionomycin. (E) Protection from 25 μM tBHP. (F) Protection from 2.5 μM Tunicamycin.
Figure Legend Snippet: Protection from other cell death-inducing pathways WT C2C12 cells were exposed to cell death-inducing drugs acting on various pathways in the presence of 5 μM of purchased compounds. Viability (ATP content) is shown on the Y axis. The first two points in each series represents controls: cells not treated with toxic agent followed by cells treated with toxic agent plus DMSO alone. Compounds tested for protection are indicated from 1–46 on the X axis, error bars = SEM. A cutoff of 3 standard deviations above control (toxic agent + DMSO alone) is shown as a dotted red line. (A) Protection from 12.5 μM ABT-263. (B) Protection from 0.0125 μM Staurosporine. (C) Protection from 12.5 μM Etoposide. (D) Protection from 12.5 μM Ionomycin. (E) Protection from 25 μM tBHP. (F) Protection from 2.5 μM Tunicamycin.

Techniques Used:

26) Product Images from "TRPA1 and TRPV1 contribute to iodine antiseptics‐associated pain and allergy"

Article Title: TRPA1 and TRPV1 contribute to iodine antiseptics‐associated pain and allergy

Journal: EMBO Reports

doi: 10.15252/embr.201642349

Iodine specifically activates TRPA1 in heterologous expression systems and native neurons A, B Representative intracellular Ca 2+ signals in HEK 293 cells transfected with the empty vector (A) or in cells expressing TRPV1 (B) in response to iodine and subsequently applied a Ca 2+ ionophore ionomycin (A) or TRPV1 agonist capsaicin (B), respectively ( n ≥ 3). C Time course of currents in hTRPA1‐expressing Xenopus oocytes in response to iodine and subsequently applied non‐specific TRP channel antagonist ruthenium red (RR) ( n = 4). D Time course of iodine‐induced whole‐cell currents in HEK 293 cell expressing mouse TRPA1 ( n = 3). E, F Iodine does not elicit membrane currents in HEK 293 cells (E) and Xenopus oocytes (F) without TRPA1 expression ( n ≥ 3). G, H Representative whole‐cell currents induced by iodine ( n = 4) (G) or capsaicin ( n = 6) (H) in cultured DRG neurons. The holding potential was −60 mV.
Figure Legend Snippet: Iodine specifically activates TRPA1 in heterologous expression systems and native neurons A, B Representative intracellular Ca 2+ signals in HEK 293 cells transfected with the empty vector (A) or in cells expressing TRPV1 (B) in response to iodine and subsequently applied a Ca 2+ ionophore ionomycin (A) or TRPV1 agonist capsaicin (B), respectively ( n ≥ 3). C Time course of currents in hTRPA1‐expressing Xenopus oocytes in response to iodine and subsequently applied non‐specific TRP channel antagonist ruthenium red (RR) ( n = 4). D Time course of iodine‐induced whole‐cell currents in HEK 293 cell expressing mouse TRPA1 ( n = 3). E, F Iodine does not elicit membrane currents in HEK 293 cells (E) and Xenopus oocytes (F) without TRPA1 expression ( n ≥ 3). G, H Representative whole‐cell currents induced by iodine ( n = 4) (G) or capsaicin ( n = 6) (H) in cultured DRG neurons. The holding potential was −60 mV.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Cell Culture

27) Product Images from "Rapid flow cytometric measurement of protein inclusions and nuclear trafficking"

Article Title: Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

Journal: Scientific Reports

doi: 10.1038/srep31138

FloIT can quantify nuclear flux of fluorescently tagged proteins. ( a ) Time-dependent efflux of TDP-43 M337V -tGFP from the nuclei of MG132 treated N2a cells, shown as % change in tGFP-fluorescing nuclei (at t = 0, the % value represents the transfection efficiency). ( b ) Dose-dependent influx of NFAT-eGFP into 1 µM ionomycin-treated HEK293 cell nuclei, shown as % NFAT-eGFP-positive nuclei. ( c ) Confocal microscopy images of NFAT-eGFP-expressing HEK293 cells with and without 1 µM ionomycin treatment. Scale bar is 10 μm. Values are means (n = 3) ± SEM; each result is representative of two or more independent experiments.
Figure Legend Snippet: FloIT can quantify nuclear flux of fluorescently tagged proteins. ( a ) Time-dependent efflux of TDP-43 M337V -tGFP from the nuclei of MG132 treated N2a cells, shown as % change in tGFP-fluorescing nuclei (at t = 0, the % value represents the transfection efficiency). ( b ) Dose-dependent influx of NFAT-eGFP into 1 µM ionomycin-treated HEK293 cell nuclei, shown as % NFAT-eGFP-positive nuclei. ( c ) Confocal microscopy images of NFAT-eGFP-expressing HEK293 cells with and without 1 µM ionomycin treatment. Scale bar is 10 μm. Values are means (n = 3) ± SEM; each result is representative of two or more independent experiments.

Techniques Used: Transfection, Confocal Microscopy, Expressing

28) Product Images from "Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation"

Article Title: Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02481

PAD4 is essential for ionophore-induced chromatin decondensation and nuclear protein degradation in murine granulocytes. Murine peritoneal neutrophils were stimulated with ionomycin for 120 min or left unstimulated. (A) H3cit immunocytochemistry reveals PAD4 activity in WT cells and its absence in PAD4-deficient neutrophils ( n = 3 independent experiments). (B) Identical cultures were subjected to Lamin-B1 and (C) HMGB1 immune cytochemistry. (D) The morphology and spread area of the chromatin was analyzed after 2 h as assessed via fluorescence microscopy after SYTOX Green-mediated DNA staining ( n = 4 independent experiments with at least 100 nuclei per analysis, *** p
Figure Legend Snippet: PAD4 is essential for ionophore-induced chromatin decondensation and nuclear protein degradation in murine granulocytes. Murine peritoneal neutrophils were stimulated with ionomycin for 120 min or left unstimulated. (A) H3cit immunocytochemistry reveals PAD4 activity in WT cells and its absence in PAD4-deficient neutrophils ( n = 3 independent experiments). (B) Identical cultures were subjected to Lamin-B1 and (C) HMGB1 immune cytochemistry. (D) The morphology and spread area of the chromatin was analyzed after 2 h as assessed via fluorescence microscopy after SYTOX Green-mediated DNA staining ( n = 4 independent experiments with at least 100 nuclei per analysis, *** p

Techniques Used: Immunocytochemistry, Activity Assay, Fluorescence, Microscopy, Staining

Differential proteolytic cleavage events in NET formation. (A ) Lamin B1, HMGB1, and citrullinated Histone H3 were assessed by western blotting in lysates from human neutrophils after stimulation for 120 min with PMA or ionomycin ( n = 3 independent healthy donors). (B) Band intensity calculations of western blots as in (A) were performed using Image J-assisted density quantification of at least 4 lanes ( ** p
Figure Legend Snippet: Differential proteolytic cleavage events in NET formation. (A ) Lamin B1, HMGB1, and citrullinated Histone H3 were assessed by western blotting in lysates from human neutrophils after stimulation for 120 min with PMA or ionomycin ( n = 3 independent healthy donors). (B) Band intensity calculations of western blots as in (A) were performed using Image J-assisted density quantification of at least 4 lanes ( ** p

Techniques Used: Western Blot

Ionophore-induced chromatin decondensation of human neutrophil granulocytes requires both PAD and calpain activity. Human neutrophils from the peripheral blood of healthy donors were stimulated with PMA (50 nM), Ionomycin (5 μM) or left unstimulated after pre-treatment with DMSO control, the specific calpain inhibitor PD150606 (50 μM) or the PAD-inhibitor Cl-amidine (200 μM), respectively. (A) The morphology and spread area of the chromatin was analyzed after 2 h as assessed via fluorescence microscopy after SYTOX Green-mediated DNA staining. Representative images of at least four independent experiments are shown (scale bar = 20 μm). (B) Mean + SEM of the median chromatin area of at least 100 single cells of four independent experiments is depicted ( *** p
Figure Legend Snippet: Ionophore-induced chromatin decondensation of human neutrophil granulocytes requires both PAD and calpain activity. Human neutrophils from the peripheral blood of healthy donors were stimulated with PMA (50 nM), Ionomycin (5 μM) or left unstimulated after pre-treatment with DMSO control, the specific calpain inhibitor PD150606 (50 μM) or the PAD-inhibitor Cl-amidine (200 μM), respectively. (A) The morphology and spread area of the chromatin was analyzed after 2 h as assessed via fluorescence microscopy after SYTOX Green-mediated DNA staining. Representative images of at least four independent experiments are shown (scale bar = 20 μm). (B) Mean + SEM of the median chromatin area of at least 100 single cells of four independent experiments is depicted ( *** p

Techniques Used: Activity Assay, Fluorescence, Microscopy, Staining

Independent pathways induce chromatin decondensation in neutrophils. (A) Isolated neutrophils were stimulated in HBSS media with phorbol myristyl acetate (PMA, 50 nM) or ionomycin (5 μM). Imaging was performed 3 h after stimulation. The reproducibly observed response of peripheral blood neutrophils derived from healthy donors and a patient suffering from chronic granulomatous disease (CGD) is depicted. DNA of neutrophils is stained by SYTOX Green (in green), once the plasma membrane is disrupted and/or DNA has been expulsed. Decondensation of chromatin results in increased area and decreased intensity of SYTOX staining (scale bar = 20 μm). (B) SYTOX Green fluorescence intensity of microscopic images from equal numbers of cells as presented in (A) was quantified and represented as relative signal intensity ( *** p
Figure Legend Snippet: Independent pathways induce chromatin decondensation in neutrophils. (A) Isolated neutrophils were stimulated in HBSS media with phorbol myristyl acetate (PMA, 50 nM) or ionomycin (5 μM). Imaging was performed 3 h after stimulation. The reproducibly observed response of peripheral blood neutrophils derived from healthy donors and a patient suffering from chronic granulomatous disease (CGD) is depicted. DNA of neutrophils is stained by SYTOX Green (in green), once the plasma membrane is disrupted and/or DNA has been expulsed. Decondensation of chromatin results in increased area and decreased intensity of SYTOX staining (scale bar = 20 μm). (B) SYTOX Green fluorescence intensity of microscopic images from equal numbers of cells as presented in (A) was quantified and represented as relative signal intensity ( *** p

Techniques Used: Isolation, Imaging, Derivative Assay, Staining, Fluorescence

29) Product Images from "Nuclear receptor Ftz-f1 promotes follicle maturation and ovulation partly via bHLH/PAS transcription factor Sim"

Article Title: Nuclear receptor Ftz-f1 promotes follicle maturation and ovulation partly via bHLH/PAS transcription factor Sim

Journal: eLife

doi: 10.7554/eLife.54568

ftz-f1 knockdown causes defects in ovulation and egg morphology. ( A–C ) Ftz-f1 protein (green in A-C) in stage-12 egg chambers from control ( A ), ftz-f1 RNAi1 ( B ), and ftz-f1R NAi2 ( C ) females with Vm26Aa-Gal4, UAS-dcr2; Oamb-RFP . The insets are higher magnification of Ftz-f1 expression in squared areas. ( D–E ) Quantification of egg laying ( D ) and mature follicles in females post egg laying ( E ) in control or ftz-f1 RNAi females with Vm26Aa-Gal4, UAS-dcr2; 47A04-LexA, lexAop2-6XGFP . The number of females is noted above each bar. ( F ) Quantification of OA- and Ionomycin-induced follicle rupture using mature follicles isolated from control or ftz-f1 RNAi females. Mature follicles were isolated according to 47A04-lexA > 6 XGFP expression. The number of mature follicles analyzed is noted above each bar. ( G–I ) Representative images show follicles from control ( G ), ftz-f1 RNA1i ( H ), and ftz-f1 RNAi2 ( I ) females after 3 hr culture with OA. Follicles were isolated according to Oamb-RFP expression (red). Ruptured follicles are marked by arrowheads. ( J–L ) Representative images show follicles from control ( J ), ftz-f1 RNA1i ( K ), and ftz-f1 RNAi2 ( L ) females after 3 hr culture with OA. Follicles were isolated according to 47A04-lexA > 6 XGFP expression (green). Ruptured follicles are marked by arrowheads. 6XGFP forms puncta inside follicle cells. ( M–O ) Representative DIC images show dorsal appendage morphology in control ( M ), ftz-f1 RNA1i ( N ), and ftz-f1 RNAi2 ( O ) stage-14 egg chambers. Blue arrowheads indicate stunted dorsal appendage formation. ***p
Figure Legend Snippet: ftz-f1 knockdown causes defects in ovulation and egg morphology. ( A–C ) Ftz-f1 protein (green in A-C) in stage-12 egg chambers from control ( A ), ftz-f1 RNAi1 ( B ), and ftz-f1R NAi2 ( C ) females with Vm26Aa-Gal4, UAS-dcr2; Oamb-RFP . The insets are higher magnification of Ftz-f1 expression in squared areas. ( D–E ) Quantification of egg laying ( D ) and mature follicles in females post egg laying ( E ) in control or ftz-f1 RNAi females with Vm26Aa-Gal4, UAS-dcr2; 47A04-LexA, lexAop2-6XGFP . The number of females is noted above each bar. ( F ) Quantification of OA- and Ionomycin-induced follicle rupture using mature follicles isolated from control or ftz-f1 RNAi females. Mature follicles were isolated according to 47A04-lexA > 6 XGFP expression. The number of mature follicles analyzed is noted above each bar. ( G–I ) Representative images show follicles from control ( G ), ftz-f1 RNA1i ( H ), and ftz-f1 RNAi2 ( I ) females after 3 hr culture with OA. Follicles were isolated according to Oamb-RFP expression (red). Ruptured follicles are marked by arrowheads. ( J–L ) Representative images show follicles from control ( J ), ftz-f1 RNA1i ( K ), and ftz-f1 RNAi2 ( L ) females after 3 hr culture with OA. Follicles were isolated according to 47A04-lexA > 6 XGFP expression (green). Ruptured follicles are marked by arrowheads. 6XGFP forms puncta inside follicle cells. ( M–O ) Representative DIC images show dorsal appendage morphology in control ( M ), ftz-f1 RNA1i ( N ), and ftz-f1 RNAi2 ( O ) stage-14 egg chambers. Blue arrowheads indicate stunted dorsal appendage formation. ***p

Techniques Used: Expressing, Isolation

30) Product Images from "HIV LTR-Driven Antisense RNA by Itself Has Regulatory Function and May Curtail Virus Reactivation From Latency"

Article Title: HIV LTR-Driven Antisense RNA by Itself Has Regulatory Function and May Curtail Virus Reactivation From Latency

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01066

Sense RNA reactivation is influenced by antisense RNA transcription. (A) Representative results of sense (mCherry) and antisense RNA expression (Venus) after PMA/Ionomycin treatment of 2 individual cell clones (AS+2G8 and DN12D7c). Dot plots from flow cytometry at 24 h post stimulation are shown. (B–E) Increase of sense and antisense RNA expression in cloned cells after activation with PMA/Ionomycin (B,C) or SAHA (D,E) . The fold increase of the MFI of mCherry and Venus expression is shown for all individual cell clones. Fold increase was calculated from mCherry or Venus MFI of activated cells using that of non-activated (DMSO treated) cells as reference. Means ± SD of three to five independent experiments are given. P -values were calculated by Mann–Whitney’s tests. (F) Fold sense RNA increase after cell clone activation with PMA/Ionomycin or SAHA. Relative levels of mCherry RNA were measured by sense-strand-specific RT-qPCR. Dots represent PMA/Ionomycin-treated clones; triangles represent SAHA-treated clones. The p value was calculated by Mann–Whitney’s test.
Figure Legend Snippet: Sense RNA reactivation is influenced by antisense RNA transcription. (A) Representative results of sense (mCherry) and antisense RNA expression (Venus) after PMA/Ionomycin treatment of 2 individual cell clones (AS+2G8 and DN12D7c). Dot plots from flow cytometry at 24 h post stimulation are shown. (B–E) Increase of sense and antisense RNA expression in cloned cells after activation with PMA/Ionomycin (B,C) or SAHA (D,E) . The fold increase of the MFI of mCherry and Venus expression is shown for all individual cell clones. Fold increase was calculated from mCherry or Venus MFI of activated cells using that of non-activated (DMSO treated) cells as reference. Means ± SD of three to five independent experiments are given. P -values were calculated by Mann–Whitney’s tests. (F) Fold sense RNA increase after cell clone activation with PMA/Ionomycin or SAHA. Relative levels of mCherry RNA were measured by sense-strand-specific RT-qPCR. Dots represent PMA/Ionomycin-treated clones; triangles represent SAHA-treated clones. The p value was calculated by Mann–Whitney’s test.

Techniques Used: RNA Expression, Clone Assay, Flow Cytometry, Cytometry, Activation Assay, Expressing, MANN-WHITNEY, Quantitative RT-PCR

31) Product Images from "Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms"

Article Title: Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms

Journal: eLife

doi: 10.7554/eLife.55634

PregS responses of wild type and mutant TRPM3 at 37°C. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the Materials and method section. ( A ) Average traces from one plate (four wells for each condition) for stimulation with different concentrations of PregS for cells expressing wild type TRPM3; at the end of the experiment, 2 μM ionomycin was applied as a normalizing stimulus. ( B ) Hill fit of the PregS concentration response relationship, symbols show values obtained in individual wells n = 8 from two independent transfections. The dashed line shows the fit for the concentration response curve obtained at 21°C from Figure 1D . ( C-D ) Identical experiments to those shown in panel A, on cells expressing the V992M (C) and the P1092Q (D) mutants.
Figure Legend Snippet: PregS responses of wild type and mutant TRPM3 at 37°C. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the Materials and method section. ( A ) Average traces from one plate (four wells for each condition) for stimulation with different concentrations of PregS for cells expressing wild type TRPM3; at the end of the experiment, 2 μM ionomycin was applied as a normalizing stimulus. ( B ) Hill fit of the PregS concentration response relationship, symbols show values obtained in individual wells n = 8 from two independent transfections. The dashed line shows the fit for the concentration response curve obtained at 21°C from Figure 1D . ( C-D ) Identical experiments to those shown in panel A, on cells expressing the V992M (C) and the P1092Q (D) mutants.

Techniques Used: Mutagenesis, Transfection, Fluorescence, Expressing, Concentration Assay

32) Product Images from "A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left–right patterning"

Article Title: A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left–right patterning

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1916858117

Disruption of Ca 2+ oscillation abrogates Bmp.a expression. ( A ) Eight-cell embryos were treated with cytochalasin B with ionomycin or the indicated inhibitors at the eight-cell stage. Bmp.a ( Left ) and Brachyury ( Right ) mRNA levels were detected at the hatching period (3 hpf). Asterisks indicate in situ hybridization signals. ( B ) Proportions of positive embryos in A (Fisher’s exact test). ( C and D ) qPCR analyses of Bmp.a ( C ) and Brachyury ( D ) expression. Expression levels were normalized to that of cytoplasmic actin (Student’s t test). ( E – G ) Bmp.a expression was abrogated by treatment with low concentrations (0.1 to 0.3 µM) of ionomycin. Embryos developed into normal-shaped tailbud embryos without cleavage arrest. ( E ) Anterior view. Arrowheads indicate Bmp.a expression on the right side ( Left images) or Brachyury expression in the notochord ( Center images). Insets represent lateral views. Right images highlight the nerve cord (NC) on the left side of the notochord (No). Numbers indicate the ratio of tailbud embryos with this morphological asymmetry of the NC. ( F and G ) The proportion of normal-shaped tailbud embryos ( F ) and positive embryos ( G ) (Fisher’s exact test). ( H – J ) Low steady-state Ca 2+ levels did not affect Bmp.a expression. ( H ) Embryos were treated with BAPTA-AM. Anterior views. Arrowheads indicate Bmp.a expression on the right side ( Left images) or Brachyury expression in the notochord ( Right images). Insets represent lateral views. ( I and J ) Proportion of normal-shaped tailbud embryos ( I ) and positive embryos ( J ) (Fisher’s exact test). (Scale bars, 20 μm.) * P
Figure Legend Snippet: Disruption of Ca 2+ oscillation abrogates Bmp.a expression. ( A ) Eight-cell embryos were treated with cytochalasin B with ionomycin or the indicated inhibitors at the eight-cell stage. Bmp.a ( Left ) and Brachyury ( Right ) mRNA levels were detected at the hatching period (3 hpf). Asterisks indicate in situ hybridization signals. ( B ) Proportions of positive embryos in A (Fisher’s exact test). ( C and D ) qPCR analyses of Bmp.a ( C ) and Brachyury ( D ) expression. Expression levels were normalized to that of cytoplasmic actin (Student’s t test). ( E – G ) Bmp.a expression was abrogated by treatment with low concentrations (0.1 to 0.3 µM) of ionomycin. Embryos developed into normal-shaped tailbud embryos without cleavage arrest. ( E ) Anterior view. Arrowheads indicate Bmp.a expression on the right side ( Left images) or Brachyury expression in the notochord ( Center images). Insets represent lateral views. Right images highlight the nerve cord (NC) on the left side of the notochord (No). Numbers indicate the ratio of tailbud embryos with this morphological asymmetry of the NC. ( F and G ) The proportion of normal-shaped tailbud embryos ( F ) and positive embryos ( G ) (Fisher’s exact test). ( H – J ) Low steady-state Ca 2+ levels did not affect Bmp.a expression. ( H ) Embryos were treated with BAPTA-AM. Anterior views. Arrowheads indicate Bmp.a expression on the right side ( Left images) or Brachyury expression in the notochord ( Right images). Insets represent lateral views. ( I and J ) Proportion of normal-shaped tailbud embryos ( I ) and positive embryos ( J ) (Fisher’s exact test). (Scale bars, 20 μm.) * P

Techniques Used: Expressing, In Situ Hybridization, Real-time Polymerase Chain Reaction

Repetitive Ca 2+ waves run asymmetrically along the L–R axis. ( A ) Time-course of intracellular Ca 2+ levels, visualized using G-Camp8. Note that Ca 2+ oscillation was observed before fertilization (red arrow) and after cleavage (blue arrow). ( B and C ) Sequential images of a Ca 2+ wave in the two-cell embryo and low ( B ) and high ( C ) time-resolution. The wave was transmitted from one cell to another via the center of the cleavage plane (red arrows). ( D and E ) The firing point of the Ca 2+ wave (red arrows) was not from the first cleavage plane (blue arrows). F-actin in the cleavage furrow was visualized using Lifeact-mCherry. Ca 2+ waves before and after cleavage plane formation are shown in D and E , respectively. ( F – L ) Ca 2+ oscillation is dependent on intracellular Ca 2+ levels. Embryos were recorded beginning 10 min after fertilization. ( F – J ) Ca 2+ oscillation was monitored for 20 min after the application of DMSO ( F ), 2-APB ( G ), Ruthenium red ( H ), ionomycin ( I ), or BAPTA-AM ( J ). ( K and L ) Time-lapse recordings were carried out immediately after transferring embryos into artificial seawater ( K ) or Ca 2+ -free seawater ( L ). Cell-to-cell contacts became loose in Ca 2+ -free seawater ( Inset , red arrow). (Scale bars, 20 μm.)
Figure Legend Snippet: Repetitive Ca 2+ waves run asymmetrically along the L–R axis. ( A ) Time-course of intracellular Ca 2+ levels, visualized using G-Camp8. Note that Ca 2+ oscillation was observed before fertilization (red arrow) and after cleavage (blue arrow). ( B and C ) Sequential images of a Ca 2+ wave in the two-cell embryo and low ( B ) and high ( C ) time-resolution. The wave was transmitted from one cell to another via the center of the cleavage plane (red arrows). ( D and E ) The firing point of the Ca 2+ wave (red arrows) was not from the first cleavage plane (blue arrows). F-actin in the cleavage furrow was visualized using Lifeact-mCherry. Ca 2+ waves before and after cleavage plane formation are shown in D and E , respectively. ( F – L ) Ca 2+ oscillation is dependent on intracellular Ca 2+ levels. Embryos were recorded beginning 10 min after fertilization. ( F – J ) Ca 2+ oscillation was monitored for 20 min after the application of DMSO ( F ), 2-APB ( G ), Ruthenium red ( H ), ionomycin ( I ), or BAPTA-AM ( J ). ( K and L ) Time-lapse recordings were carried out immediately after transferring embryos into artificial seawater ( K ) or Ca 2+ -free seawater ( L ). Cell-to-cell contacts became loose in Ca 2+ -free seawater ( Inset , red arrow). (Scale bars, 20 μm.)

Techniques Used: Transferring

33) Product Images from "Tregitopes regulate the tolerogenic immune response and decrease the foetal death rate in abortion-prone mouse matings"

Article Title: Tregitopes regulate the tolerogenic immune response and decrease the foetal death rate in abortion-prone mouse matings

Journal: Scientific Reports

doi: 10.1038/s41598-020-66957-z

Effect of tregitope treatment on regulatory T lymphocytes in a murine abortion-prone pregnancy model. Cells were stimulated with PMA and ionomycin in the presence of brefeldin A and monensin and the frequencies of CD4 + CD25 + Foxp3 + (a) splenocytes and (b) uterine-draining lymph node cells within CD4 + (c) splenocytes and (d) lymph node cells and the frequencies of CD4 + CD25 + FOXP3 + IL-10 + splenocytes (e) and (f) lymph node cells within CD4 + (g) splenocytes and (h) lymph nodes and the median fluorescence intensity (MFI) of IL-10 within CD4 + CD25 + FOXP3 + (i) splenocytes and (j) uterine-draining lymph node cells were measured. The data were analysed by one-way ANOVA (normal distribution) or the Kruskal-Wallis test (non-normal distribution) with Dunn’s multiple comparison post hoc test (P
Figure Legend Snippet: Effect of tregitope treatment on regulatory T lymphocytes in a murine abortion-prone pregnancy model. Cells were stimulated with PMA and ionomycin in the presence of brefeldin A and monensin and the frequencies of CD4 + CD25 + Foxp3 + (a) splenocytes and (b) uterine-draining lymph node cells within CD4 + (c) splenocytes and (d) lymph node cells and the frequencies of CD4 + CD25 + FOXP3 + IL-10 + splenocytes (e) and (f) lymph node cells within CD4 + (g) splenocytes and (h) lymph nodes and the median fluorescence intensity (MFI) of IL-10 within CD4 + CD25 + FOXP3 + (i) splenocytes and (j) uterine-draining lymph node cells were measured. The data were analysed by one-way ANOVA (normal distribution) or the Kruskal-Wallis test (non-normal distribution) with Dunn’s multiple comparison post hoc test (P

Techniques Used: Fluorescence

Effect of tregitope treatment on regulatory B lymphocytes in a murine abortion-prone pregnancy model. Cells were stimulated with PMA and ionomycin in the presence of brefeldin A and monensin and the frequencies of CD19 + CD1d + CD5 + IL-10 + ( a) splenocytes and (b) uterine-draining lymph node cells, and the production of IL-10 by CD19 + CD1d + CD5 + ( c) splenocytes and (d) lymph node cells were analysed by intracellular staining. The data were analysed by one-way ANOVA (normal distribution) or the Kruskal-Wallis test (non-normal distribution) with Dunn’s multiple comparison post hoc test (P
Figure Legend Snippet: Effect of tregitope treatment on regulatory B lymphocytes in a murine abortion-prone pregnancy model. Cells were stimulated with PMA and ionomycin in the presence of brefeldin A and monensin and the frequencies of CD19 + CD1d + CD5 + IL-10 + ( a) splenocytes and (b) uterine-draining lymph node cells, and the production of IL-10 by CD19 + CD1d + CD5 + ( c) splenocytes and (d) lymph node cells were analysed by intracellular staining. The data were analysed by one-way ANOVA (normal distribution) or the Kruskal-Wallis test (non-normal distribution) with Dunn’s multiple comparison post hoc test (P

Techniques Used: Staining

34) Product Images from "Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis"

Article Title: Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis

Journal: ACS Pharmacology & Translational Science

doi: 10.1021/acsptsci.9b00052

Proteasome inhibition suppresses ionomycin-induced necrosis in primary cardiomyocytes. (A) NRCMs were transfected with control (siControl) or PSMB5 siRNA (siPSMB5). Knockdown efficiency was assessed by Western blotting. (B) NRCMs transfected with siControl or siPSMB5 were incubated with ionomycin (1 μM) for 1 h ( n = 3). Cell viability and LDH release were analyzed by MTT and LDH assays, respectively. **, p
Figure Legend Snippet: Proteasome inhibition suppresses ionomycin-induced necrosis in primary cardiomyocytes. (A) NRCMs were transfected with control (siControl) or PSMB5 siRNA (siPSMB5). Knockdown efficiency was assessed by Western blotting. (B) NRCMs transfected with siControl or siPSMB5 were incubated with ionomycin (1 μM) for 1 h ( n = 3). Cell viability and LDH release were analyzed by MTT and LDH assays, respectively. **, p

Techniques Used: Inhibition, Transfection, Western Blot, Incubation, MTT Assay

Enrichment of functional groups, biological processes and pathways in ionomycin-induced necrosis. (A) Candidate genes from the primary screen were grouped by molecular function using the PANTHER classification system. Top molecular functions related with necrosis were catalytic activity and binding. (B) Detailed protein categorization of the catalytic activity group in panel (A). Hydrolase and transferase activities were the primary catalytic activities involved in necrosis. (C) Candidate genes from the primary screen were grouped by biological process using the PANTHER classification system. More than half of all hits were associated with cellular and metabolic processes. (D) Detailed protein categorization of the cellular process group in panel (C). Cell communication and cell cycle were the predominant cellular processes identified. (E) Highly scored pathways in ionomycin-induced necrosis based on PANTHER pathway analysis.
Figure Legend Snippet: Enrichment of functional groups, biological processes and pathways in ionomycin-induced necrosis. (A) Candidate genes from the primary screen were grouped by molecular function using the PANTHER classification system. Top molecular functions related with necrosis were catalytic activity and binding. (B) Detailed protein categorization of the catalytic activity group in panel (A). Hydrolase and transferase activities were the primary catalytic activities involved in necrosis. (C) Candidate genes from the primary screen were grouped by biological process using the PANTHER classification system. More than half of all hits were associated with cellular and metabolic processes. (D) Detailed protein categorization of the cellular process group in panel (C). Cell communication and cell cycle were the predominant cellular processes identified. (E) Highly scored pathways in ionomycin-induced necrosis based on PANTHER pathway analysis.

Techniques Used: Functional Assay, Activity Assay, Binding Assay

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Article Title: BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases
Article Snippet: Reagents The following chemicals were used: Texas Red 10 kD dextran (Invitrogen, 1 mg/ml); NS1619 (Sigma); NS11021 (Sigma); GPN (Santa Cruz Biotechnology); Paxilline (Cayman Chemical Company); ML-SA1 (Tocris); Ionomycin (Cayman), ML-SI1 (Enzo Life Sciences Inc).

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    Cayman Chemical ionomycin
    TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or <t>Ionomycin</t> ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).
    Ionomycin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).

    Journal: bioRxiv

    Article Title: A Novel PHOX/CD38/MCOLN1/TFEB Axis Important For Macrophage Activation During Bacterial Phagocytosis

    doi: 10.1101/669325

    Figure Lengend Snippet: TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).

    Article Snippet: Drugs and reagents Lipopolysaccharides (LPS) from S. enterica serotype Typhimurium (Sigma-Aldrich, L6143-1MG, 10ug/ml): Peptidoglycan (PGN) from S. aureus (Invivogen, tlrl-pgns2): Monophosphoryl Lipid A (MPLA-SM) (Invivogen, tlrl-mpla): ML-SA1 (Sigma Aldrich, SML0627) Ionomycin (Cayman Chemical Company, 10004974): FK-506 (Cayman Chemical Company, 10007965): BAPTA AM (Cayman Chemical Company, 15551): Ned-19 (Cayman Chemical Company, 17527): Kuromanin (Cayman Chemical Company, 16406): Apigenin (Cayman Chemical Company, 10010275): CCCP (Cayman Chemical Company, 25458): N-acetyl-L-Cysteine (NAC) (Cayman Chemical Company, 20261): N-acetyl-L-Cysteine amide (NACA) (Cayman Chemical Company, 25866): Apocynin (Cayman Chemical Company, 11976): D609 (Cayman Chemical, 13307, 50 µM): kb NB 142-70 (Cayman Chemical Company, 18002).

    Techniques: Activation Assay, Infection

    Activation of BK by NS1619 facilitates ML1-mediated Ca 2+ release. ( A,B ) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. ( C ) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca 2+ content was not affected. ( D,E ) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). ( F ) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca 2+ content. ( G ) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.

    Journal: Scientific Reports

    Article Title: BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

    doi: 10.1038/srep33684

    Figure Lengend Snippet: Activation of BK by NS1619 facilitates ML1-mediated Ca 2+ release. ( A,B ) NS1619 (15 μM) treatment increased ML-SA1 (10 μM)-mediated GECO-TRPML1 response, which was inhibited by co-applying Paxilline (PAX, 3 μM) in HEK293T cells. ( C ) NS1619 (15 μM) treatment did not alter GECO-TRPML1 response to GPN (200 μM), suggesting lysosomal Ca 2+ content was not affected. ( D,E ) NPC1 human fibroblasts exhibited impaired GECO-TRPML1 response to ML-SA1 (10 μM). NS1619 (15 μM) treatment increased GECO-TRPML1 response to ML-SA1, and this was inhibited by co-applying Paxilline (PAX, 3 μM). ( F ) GECO-TRPML1 responses to GPN (200 μM) was not altered by NS1619 (15 μM) in NPC1 cells, suggesting that NS1619 does not change lysosomal Ca 2+ content. ( G ) GECO-TRPML1 responses to Ionomycin (1 μM) were not altered, indicating a similar level of GECO-TRPML1 expression.

    Article Snippet: Reagents The following chemicals were used: Texas Red 10 kD dextran (Invitrogen, 1 mg/ml); NS1619 (Sigma); NS11021 (Sigma); GPN (Santa Cruz Biotechnology); Paxilline (Cayman Chemical Company); ML-SA1 (Tocris); Ionomycin (Cayman), ML-SI1 (Enzo Life Sciences Inc).

    Techniques: Activation Assay, Expressing

    BK upregulation promotes lysosomal Ca 2+ release in ML4 fibroblasts carrying F408Δ mutation. ( A ) BK overexpression increased ML-SA1 (10 μM)-induced GECO-TRPML1-F408Δ response in HEK293T cells. ( B ) BK overexpression did not alter ML-SA1 (10 μM)-induced GECO-TRPML1-R403C response in HEK293T cells. ( C ) Statistical analysis of GECO response to ML-SA1 (10 μM) showing that BK overexpression significantly increased GECO-TRPML1-F408Δ signal but not others. ( D ) BK overexpression did not alter GPN (200 μM)-induced GECO responses. ( E ) BK overexpression did not alter Ionomycin (1 μM)-induced GECO responses. ( F,G ) BK overexpression enhanced GECO-TRPML1-F408Δ response to ML-SA1 (10 μM) in TRPML1-F408Δ human fibroblasts, suggesting that BK upregulation facilitates TRPML1-F408Δ activity. ( H,I ) GECO-TRPML1-F408Δ responses to GPN (200 μM) ( H ) and Ionomycin (1 μM) ( I ) were not altered in TRPML1-F408Δ human fibroblasts by BK overexpession, suggesting BK overexpression did not alter lysosomal Ca 2+ content and GECO- TRPML1-F408Δ expression level in TRPML1-F408Δ human fibroblasts, respectively.

    Journal: Scientific Reports

    Article Title: BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

    doi: 10.1038/srep33684

    Figure Lengend Snippet: BK upregulation promotes lysosomal Ca 2+ release in ML4 fibroblasts carrying F408Δ mutation. ( A ) BK overexpression increased ML-SA1 (10 μM)-induced GECO-TRPML1-F408Δ response in HEK293T cells. ( B ) BK overexpression did not alter ML-SA1 (10 μM)-induced GECO-TRPML1-R403C response in HEK293T cells. ( C ) Statistical analysis of GECO response to ML-SA1 (10 μM) showing that BK overexpression significantly increased GECO-TRPML1-F408Δ signal but not others. ( D ) BK overexpression did not alter GPN (200 μM)-induced GECO responses. ( E ) BK overexpression did not alter Ionomycin (1 μM)-induced GECO responses. ( F,G ) BK overexpression enhanced GECO-TRPML1-F408Δ response to ML-SA1 (10 μM) in TRPML1-F408Δ human fibroblasts, suggesting that BK upregulation facilitates TRPML1-F408Δ activity. ( H,I ) GECO-TRPML1-F408Δ responses to GPN (200 μM) ( H ) and Ionomycin (1 μM) ( I ) were not altered in TRPML1-F408Δ human fibroblasts by BK overexpession, suggesting BK overexpression did not alter lysosomal Ca 2+ content and GECO- TRPML1-F408Δ expression level in TRPML1-F408Δ human fibroblasts, respectively.

    Article Snippet: Reagents The following chemicals were used: Texas Red 10 kD dextran (Invitrogen, 1 mg/ml); NS1619 (Sigma); NS11021 (Sigma); GPN (Santa Cruz Biotechnology); Paxilline (Cayman Chemical Company); ML-SA1 (Tocris); Ionomycin (Cayman), ML-SI1 (Enzo Life Sciences Inc).

    Techniques: Mutagenesis, Over Expression, Activity Assay, Expressing

    BK upregulation reduces lipofuscin accumulation in NPA cells by promoting TRPML1-mediated Ca 2+ release. ( A,B ) Abnormal lipofuscin accumulation in NPA human fibroblasts and its rescue by BK overexpression or NS1619 (15 μM, 16 h) treatment. Expression of TRPML1-DD/KK or Syt VII DN reversed the rescue effect of BK upregulation. More than 35 cells were analyzed for each condition. ( C–E ) Impaired ML-SA1 (10 μM)-mediated GECO-TRPML1 response in NPA human fibroblasts and its rescue by NS1619 (15 μM, ~60 s) pretreatment ( C ) or BK overexpression ( D ). Paxilline (PAX, 3 μM) treatment reversed the rescue effect of NS1619 or BK overexpression. ( F,G ) GECO-TRPML1 responses to GPN (200 μM) (F) and Ionomycin (1 μM) ( G ) were not altered in all the treatments, suggesting lysosomal Ca 2+ content, or GECO-TRPML1 expression level was not affected.

    Journal: Scientific Reports

    Article Title: BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

    doi: 10.1038/srep33684

    Figure Lengend Snippet: BK upregulation reduces lipofuscin accumulation in NPA cells by promoting TRPML1-mediated Ca 2+ release. ( A,B ) Abnormal lipofuscin accumulation in NPA human fibroblasts and its rescue by BK overexpression or NS1619 (15 μM, 16 h) treatment. Expression of TRPML1-DD/KK or Syt VII DN reversed the rescue effect of BK upregulation. More than 35 cells were analyzed for each condition. ( C–E ) Impaired ML-SA1 (10 μM)-mediated GECO-TRPML1 response in NPA human fibroblasts and its rescue by NS1619 (15 μM, ~60 s) pretreatment ( C ) or BK overexpression ( D ). Paxilline (PAX, 3 μM) treatment reversed the rescue effect of NS1619 or BK overexpression. ( F,G ) GECO-TRPML1 responses to GPN (200 μM) (F) and Ionomycin (1 μM) ( G ) were not altered in all the treatments, suggesting lysosomal Ca 2+ content, or GECO-TRPML1 expression level was not affected.

    Article Snippet: Reagents The following chemicals were used: Texas Red 10 kD dextran (Invitrogen, 1 mg/ml); NS1619 (Sigma); NS11021 (Sigma); GPN (Santa Cruz Biotechnology); Paxilline (Cayman Chemical Company); ML-SA1 (Tocris); Ionomycin (Cayman), ML-SI1 (Enzo Life Sciences Inc).

    Techniques: Over Expression, Expressing

    The long-term non-adjustive ADV condition accelerates generation of senescence-associated (SA) T cells and germinal center B cells. ( A , B ) Flow cytometry of senescence-associated T (SA-T) and follicular helper T (Tfh) cells in CD4 + TCRβ + -gated spleen cells ( A ), and percentages and cell numbers of SA-T (CD4 + TCRβ + CD44 + PD-1 + ), CD153 + SA-T, Tfh (CD4 + TCRβ + CXCR5 + PD-1 + ), and regulatory T (Treg, CD4 + TCRβ + CD25 + ) cells (B, n = 12) in spleens of LD- and ADV-conditioned mice. ( C , D ) Flow cytometry of SA-T and Tfh cells in CD4 + TCRβ + -gated mLN cells ( C ) and percentages and cell numbers of SA-T, CD153 + SA-T, Tfh, and Treg (D, n = 12–16) in mLNs of LD- and ADV-conditioned mice. ( E ) Whole mLN cells from LD- and ADV-conditioned mice were stimulated with PMA and ionomycin for 3 hours. Percentages of IFN-γ–, IL-4–, and IL-17A–producing helper T cells in CD4 T cells were shown (n = 12). ( F ) Flow cytometry of germinal center B cell (GC-B) and IgG1 + and IgA + class-switched B cells in mLNs of LD- and ADV-conditioned mice. ( G ) Cell numbers of GC-B (CD19 + B220 + CD95 + GL7 + ), IgG1 B cells (CD19 + B220 + IgG1 + ), and IgA B cells (CD19 + B220 + IgA + ) in mLN from LD- and ADV-conditioned mice (n = 12). Data are means ± SD. Two-tailed Student’s t -test, * P

    Journal: Scientific Reports

    Article Title: Chronic circadian misalignment accelerates immune senescence and abbreviates lifespan in mice

    doi: 10.1038/s41598-020-59541-y

    Figure Lengend Snippet: The long-term non-adjustive ADV condition accelerates generation of senescence-associated (SA) T cells and germinal center B cells. ( A , B ) Flow cytometry of senescence-associated T (SA-T) and follicular helper T (Tfh) cells in CD4 + TCRβ + -gated spleen cells ( A ), and percentages and cell numbers of SA-T (CD4 + TCRβ + CD44 + PD-1 + ), CD153 + SA-T, Tfh (CD4 + TCRβ + CXCR5 + PD-1 + ), and regulatory T (Treg, CD4 + TCRβ + CD25 + ) cells (B, n = 12) in spleens of LD- and ADV-conditioned mice. ( C , D ) Flow cytometry of SA-T and Tfh cells in CD4 + TCRβ + -gated mLN cells ( C ) and percentages and cell numbers of SA-T, CD153 + SA-T, Tfh, and Treg (D, n = 12–16) in mLNs of LD- and ADV-conditioned mice. ( E ) Whole mLN cells from LD- and ADV-conditioned mice were stimulated with PMA and ionomycin for 3 hours. Percentages of IFN-γ–, IL-4–, and IL-17A–producing helper T cells in CD4 T cells were shown (n = 12). ( F ) Flow cytometry of germinal center B cell (GC-B) and IgG1 + and IgA + class-switched B cells in mLNs of LD- and ADV-conditioned mice. ( G ) Cell numbers of GC-B (CD19 + B220 + CD95 + GL7 + ), IgG1 B cells (CD19 + B220 + IgG1 + ), and IgA B cells (CD19 + B220 + IgA + ) in mLN from LD- and ADV-conditioned mice (n = 12). Data are means ± SD. Two-tailed Student’s t -test, * P

    Article Snippet: For detection of cytokine production, cells were stimulated with PMA (50 ng/ml, Cayman) and ionomycin (2 μg/ml, Cayman) for 3 hours in the presence of Brefeldin A (5 μg/ml, Cayman).

    Techniques: Flow Cytometry, Mouse Assay, Two Tailed Test

    Measurement of intracellular Ca2 2+ in follicle cells after ionomycin or octopamine stimulation. ( A–D ) Intracellular Ca 2+ depicted by GCaMP5G (green in A-C’) increase in response to ionomycin stimulation in both control ( A–A’ ) and hnt RNAi ( B–C’ ) egg chambers with FC2 Gal4, although hnt RNAi egg chambers show slightly weaker response. The frames with peak GCaMP5G signal after ionomycin stimulation are shown in A’-C’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in D and the number of egg chambers analyzed is noted at the end of each trace. ( E–H ) Intracellular Ca 2+ depicted by GCaMP5G (green in E-G’) increase in control egg chambers ( E–E’ ) after OA stimulation but does not in hnt RNAi egg chambers with FC2 Gal4 ( F–G’ ). The frames with peak GCaMP5G signal after OA stimulation are shown in E’-G’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in H and the number of egg chambers analyzed is noted at the end of each trace. Also see Videos 1 – 6 .

    Journal: eLife

    Article Title: The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles

    doi: 10.7554/eLife.29887

    Figure Lengend Snippet: Measurement of intracellular Ca2 2+ in follicle cells after ionomycin or octopamine stimulation. ( A–D ) Intracellular Ca 2+ depicted by GCaMP5G (green in A-C’) increase in response to ionomycin stimulation in both control ( A–A’ ) and hnt RNAi ( B–C’ ) egg chambers with FC2 Gal4, although hnt RNAi egg chambers show slightly weaker response. The frames with peak GCaMP5G signal after ionomycin stimulation are shown in A’-C’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in D and the number of egg chambers analyzed is noted at the end of each trace. ( E–H ) Intracellular Ca 2+ depicted by GCaMP5G (green in E-G’) increase in control egg chambers ( E–E’ ) after OA stimulation but does not in hnt RNAi egg chambers with FC2 Gal4 ( F–G’ ). The frames with peak GCaMP5G signal after OA stimulation are shown in E’-G’. Quantification of intracellular Ca 2+ level (ΔF/F0) is shown in H and the number of egg chambers analyzed is noted at the end of each trace. Also see Videos 1 – 6 .

    Article Snippet: Within one hour, isolated mature follicles from ~10 females were separated into groups of ~30 egg chambers, then cultured in culture media (Grace’s medium, 10% fetal bovine serum, and 1X penicillin/streptomycin) supplemented with 20 μM OA (Sigma, St. Louis, MO), or 5 μM ionomycin (Cayman Chemical Co., Ann Arbor, MI).

    Techniques: