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wild type parental u2os cells  (ATCC)


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    ATCC wild type parental u2os cells
    Figure 2. Expression of PGRMC1 potentiates NAADP-evoked Ca2+ release via TPC1. A, fluorescence traces from single <t>U2OS</t> cells microinjected with buffer (“mock injection,” blue) or NAADP (100 nM pipette concentration) in the absence (black) or presence (red) of RFP-PGRMC1. B–D, similar microinjection experiments in single U2OS cells expressing (B) TPC1, (C) TPC2 or (D) TPC2[1-31]-TPC1[32-816] in the absence (black) or presence (red) of RFP-PGRMC1. Traces report the fluorescence of GCaMP6M over time-averaged (mean ± sd) from n ≥3 injected cells, with only every third data point shown for clarity. E, area under the curve from NAADP-evoked Ca2+ mobilization responses, values represent mean area ± sd from independent fluorescence traces recorded from n ≥3 injected cells. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, ***p < 0.001.
    Wild Type Parental U2os Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 2500 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wild+type+parental+u2os+cells/pm37866635-168-0-8?v=ATCC
    Average 98 stars, based on 2500 article reviews
    wild type parental u2os cells - by Bioz Stars, 2026-08
    98/100 stars

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    1) Product Images from "Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum."

    Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum.

    Journal: The Journal of biological chemistry

    doi: 10.1016/j.jbc.2023.105378

    Figure 2. Expression of PGRMC1 potentiates NAADP-evoked Ca2+ release via TPC1. A, fluorescence traces from single U2OS cells microinjected with buffer (“mock injection,” blue) or NAADP (100 nM pipette concentration) in the absence (black) or presence (red) of RFP-PGRMC1. B–D, similar microinjection experiments in single U2OS cells expressing (B) TPC1, (C) TPC2 or (D) TPC2[1-31]-TPC1[32-816] in the absence (black) or presence (red) of RFP-PGRMC1. Traces report the fluorescence of GCaMP6M over time-averaged (mean ± sd) from n ≥3 injected cells, with only every third data point shown for clarity. E, area under the curve from NAADP-evoked Ca2+ mobilization responses, values represent mean area ± sd from independent fluorescence traces recorded from n ≥3 injected cells. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, ***p < 0.001.
    Figure Legend Snippet: Figure 2. Expression of PGRMC1 potentiates NAADP-evoked Ca2+ release via TPC1. A, fluorescence traces from single U2OS cells microinjected with buffer (“mock injection,” blue) or NAADP (100 nM pipette concentration) in the absence (black) or presence (red) of RFP-PGRMC1. B–D, similar microinjection experiments in single U2OS cells expressing (B) TPC1, (C) TPC2 or (D) TPC2[1-31]-TPC1[32-816] in the absence (black) or presence (red) of RFP-PGRMC1. Traces report the fluorescence of GCaMP6M over time-averaged (mean ± sd) from n ≥3 injected cells, with only every third data point shown for clarity. E, area under the curve from NAADP-evoked Ca2+ mobilization responses, values represent mean area ± sd from independent fluorescence traces recorded from n ≥3 injected cells. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, ***p < 0.001.

    Techniques Used: Expressing, Injection, Transferring, Concentration Assay, Microinjection, Two Tailed Test

    Figure 3. Molecular disruption of the PGRMC1-TPC1 interaction impairs PGRMC1 potentiation of NAADP-evoked Ca2+ release. A–C, heme binding is necessary for interaction with TPC1. A, Myc-tagged WT PGRMC1 and PGRMC1[Y113F] constructs were co-expressed in U2OS cells with GFP-tagged TPC1, with detection by Western blot. B, immunodetection of myc-tagged PGRMC1 after TPC1-GFP immunoprecipitation. C, densitometry quantification of immunodetection of myc-tagged PGRMC1 in TPC1-GFP immunoprecipitates. D–F, interaction of PGRMC1 with TPC1 is mediated by the COOH-terminal coiled-coil domain of PGRMC1. D, PGRMC1 mutants harboring point-mutations in predicted carboxyl-terminus helices co-expressed with TPC1-GFP in U2OS cells, detected by Western blot. E, immunodetection of myc-tagged PGRMC1 mutants after TPC1-GFP immunoprecipitation. F, densitometry quantification of immunodetected myc-tagged PGRMC1 mutants in TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 independent experiments. G, traces of Ca2+ flux in U2OS cells expressing the indicated PGRMC1 constructs microinjected with NAADP (100 nM pipette concentration) as
    Figure Legend Snippet: Figure 3. Molecular disruption of the PGRMC1-TPC1 interaction impairs PGRMC1 potentiation of NAADP-evoked Ca2+ release. A–C, heme binding is necessary for interaction with TPC1. A, Myc-tagged WT PGRMC1 and PGRMC1[Y113F] constructs were co-expressed in U2OS cells with GFP-tagged TPC1, with detection by Western blot. B, immunodetection of myc-tagged PGRMC1 after TPC1-GFP immunoprecipitation. C, densitometry quantification of immunodetection of myc-tagged PGRMC1 in TPC1-GFP immunoprecipitates. D–F, interaction of PGRMC1 with TPC1 is mediated by the COOH-terminal coiled-coil domain of PGRMC1. D, PGRMC1 mutants harboring point-mutations in predicted carboxyl-terminus helices co-expressed with TPC1-GFP in U2OS cells, detected by Western blot. E, immunodetection of myc-tagged PGRMC1 mutants after TPC1-GFP immunoprecipitation. F, densitometry quantification of immunodetected myc-tagged PGRMC1 mutants in TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 independent experiments. G, traces of Ca2+ flux in U2OS cells expressing the indicated PGRMC1 constructs microinjected with NAADP (100 nM pipette concentration) as

    Techniques Used: Disruption, Binding Assay, Construct, Western Blot, Immunodetection, Immunoprecipitation, Expressing, Transferring, Concentration Assay

    Figure 4. Pharmacological disruption of the PGRMC1-TPC1 interaction blocks PGRMC1 potentiation of NAADP-evoked Ca2+ release. A and D, immunodetection of PGRMC-myc and TPC1-GFP in lysates from U2OS cells pretreated for 1 h with the indicated concentration of CORM3 (A) or AG-205 (D). B and E, immunodetection of PGRMC1-myc after immunoprecipitation of TPC1-GFP from lysates shown in (A and D). C and F, densitometric quantification of myc-tagged PGRMC1 from TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 experiments. G–I, averaged fluorescence traces (mean ± sd from n ≥3 injections) showing NAADP responses in U2OS cells transfected with empty vector (black) or PGRMC1 (red, pretreated with (G) DMSO (0.1%), (H) CORM3 (30 μM), or (I) AG-205 (30 μM) for 1 h, detected by following GCaMP6M fluorescence. J, peak F/F0 values (mean ± sd) of independent NAADP responses summarized in (G–I). K, area under the curves (mean ± sd) of independent traces of NAADP-responses summarized in (G–I). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: Figure 4. Pharmacological disruption of the PGRMC1-TPC1 interaction blocks PGRMC1 potentiation of NAADP-evoked Ca2+ release. A and D, immunodetection of PGRMC-myc and TPC1-GFP in lysates from U2OS cells pretreated for 1 h with the indicated concentration of CORM3 (A) or AG-205 (D). B and E, immunodetection of PGRMC1-myc after immunoprecipitation of TPC1-GFP from lysates shown in (A and D). C and F, densitometric quantification of myc-tagged PGRMC1 from TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 experiments. G–I, averaged fluorescence traces (mean ± sd from n ≥3 injections) showing NAADP responses in U2OS cells transfected with empty vector (black) or PGRMC1 (red, pretreated with (G) DMSO (0.1%), (H) CORM3 (30 μM), or (I) AG-205 (30 μM) for 1 h, detected by following GCaMP6M fluorescence. J, peak F/F0 values (mean ± sd) of independent NAADP responses summarized in (G–I). K, area under the curves (mean ± sd) of independent traces of NAADP-responses summarized in (G–I). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01, ***p < 0.001.

    Techniques Used: Disruption, Immunodetection, Concentration Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Two Tailed Test

    Figure 5. PGRMC1 localizes to the endoplasmic reticulum in U2OS cells. A, U2OS cells imaged using various live cell markers (left) and co-transfected with PGRMC1-RFP (center). The fluorescence overlay of PGRMC1-RFP and the organelle marker channel is shown on the right. Markers were TPC1-GFP, TPC2- GFP, an endoplasmic reticulum marker (CYP2C9[1-27]-GCaMP-6M), a plasma membrane marker (GAP43[1-20]-GFP) and a mitochondrial marker (COX8A[1- 29]-CFP), Scale bar, 10 μm. Right, dashed line represents a line scan of fluorescence intensity from the green, blue or red fluorescence channels along the indicated axis. Gamma values were uniformly adjusted for clarity.
    Figure Legend Snippet: Figure 5. PGRMC1 localizes to the endoplasmic reticulum in U2OS cells. A, U2OS cells imaged using various live cell markers (left) and co-transfected with PGRMC1-RFP (center). The fluorescence overlay of PGRMC1-RFP and the organelle marker channel is shown on the right. Markers were TPC1-GFP, TPC2- GFP, an endoplasmic reticulum marker (CYP2C9[1-27]-GCaMP-6M), a plasma membrane marker (GAP43[1-20]-GFP) and a mitochondrial marker (COX8A[1- 29]-CFP), Scale bar, 10 μm. Right, dashed line represents a line scan of fluorescence intensity from the green, blue or red fluorescence channels along the indicated axis. Gamma values were uniformly adjusted for clarity.

    Techniques Used: Transfection, Marker, Clinical Proteomics, Membrane

    Figure 6. Expression of PGRMC1 increases NAADP-evoked Ca2+ release from the endoplasmic reticulum. A and B, averaged fluorescence traces as detected by GCaMP6M fluorescence changes reporting NAADP-evoked Ca2+ release in U2OS cells transfected with empty vector or in cells expressing PGRMC1. Microinjections were performed with NAADP (100 nM pipette concentration) in the presence of (A) thapsigargin (1 μM) or (B) bafilomycin A1 (100 nM). C, quantification of peak ΔF/F0 values from (A and B). D, quantification of the area under the curves from (A and B). E, left, Images of U2OS cell co- expressing GCaMP6M (top) and RCEPIA1er (bottom). Middle, pseudocolored images of GCaMP6M and RCEPIA1er fluorescence intensity before (middle image) and after (right image) microinjection of NAADP. Scale bar,10 μm. F, representative traces of GCaMP6M fluorescence (top) and RCEPIA1er fluo- rescence (bottom) in response to microinjection of NAADP (100 nM pipette concentration) in U2OS cells transfected with empty vector (grey), or from cells expressing PGRMC1 (red). G, quantification of ΔF/F0 values from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). H, quantification of area under the curves from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01.
    Figure Legend Snippet: Figure 6. Expression of PGRMC1 increases NAADP-evoked Ca2+ release from the endoplasmic reticulum. A and B, averaged fluorescence traces as detected by GCaMP6M fluorescence changes reporting NAADP-evoked Ca2+ release in U2OS cells transfected with empty vector or in cells expressing PGRMC1. Microinjections were performed with NAADP (100 nM pipette concentration) in the presence of (A) thapsigargin (1 μM) or (B) bafilomycin A1 (100 nM). C, quantification of peak ΔF/F0 values from (A and B). D, quantification of the area under the curves from (A and B). E, left, Images of U2OS cell co- expressing GCaMP6M (top) and RCEPIA1er (bottom). Middle, pseudocolored images of GCaMP6M and RCEPIA1er fluorescence intensity before (middle image) and after (right image) microinjection of NAADP. Scale bar,10 μm. F, representative traces of GCaMP6M fluorescence (top) and RCEPIA1er fluo- rescence (bottom) in response to microinjection of NAADP (100 nM pipette concentration) in U2OS cells transfected with empty vector (grey), or from cells expressing PGRMC1 (red). G, quantification of ΔF/F0 values from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). H, quantification of area under the curves from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Transferring, Concentration Assay, Microinjection, Two Tailed Test

    Figure 7. Expression of PGRMC1 inhibits EGF-evoked Ca2+ release. A, U2OS cells were transfected with vector encoding PGRMC1-RFP (top right) and loaded with fluo-4 dye (top left). Pseudocolored images of fluo-4 fluorescence intensity after addition of buffer (bottom, left) or EGF (32 PM, bottom, right). Kinetics of Ca2+ signals (fluo-4 fluorescence) from U2OS cells expressing PGRMC1-RFP (red boxes) and untransfected cells (white boxes) were compared. Scale bar, 20 μm. B, single-cell fluorescence traces from EGF responses in U2OS cells in the absence (black, top) or presence of PGRMC1-RFP (red, bottom). C and D, quantification of (C) peak F/F0 or (D) area under the curve (AUC) in response to EGF (32 PM) or acetylcholine (ACh, 100 μM) in control U2OS cells (black) or cells overexpressing PGRMC1 (red). Data are shown as average±sem. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05; ns, not significant.
    Figure Legend Snippet: Figure 7. Expression of PGRMC1 inhibits EGF-evoked Ca2+ release. A, U2OS cells were transfected with vector encoding PGRMC1-RFP (top right) and loaded with fluo-4 dye (top left). Pseudocolored images of fluo-4 fluorescence intensity after addition of buffer (bottom, left) or EGF (32 PM, bottom, right). Kinetics of Ca2+ signals (fluo-4 fluorescence) from U2OS cells expressing PGRMC1-RFP (red boxes) and untransfected cells (white boxes) were compared. Scale bar, 20 μm. B, single-cell fluorescence traces from EGF responses in U2OS cells in the absence (black, top) or presence of PGRMC1-RFP (red, bottom). C and D, quantification of (C) peak F/F0 or (D) area under the curve (AUC) in response to EGF (32 PM) or acetylcholine (ACh, 100 μM) in control U2OS cells (black) or cells overexpressing PGRMC1 (red). Data are shown as average±sem. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05; ns, not significant.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Control, Two Tailed Test



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    wild type parental u2os cells  (ATCC)
    98
    ATCC wild type parental u2os cells
    Figure 2. Expression of PGRMC1 potentiates NAADP-evoked Ca2+ release via TPC1. A, fluorescence traces from single <t>U2OS</t> cells microinjected with buffer (“mock injection,” blue) or NAADP (100 nM pipette concentration) in the absence (black) or presence (red) of RFP-PGRMC1. B–D, similar microinjection experiments in single U2OS cells expressing (B) TPC1, (C) TPC2 or (D) TPC2[1-31]-TPC1[32-816] in the absence (black) or presence (red) of RFP-PGRMC1. Traces report the fluorescence of GCaMP6M over time-averaged (mean ± sd) from n ≥3 injected cells, with only every third data point shown for clarity. E, area under the curve from NAADP-evoked Ca2+ mobilization responses, values represent mean area ± sd from independent fluorescence traces recorded from n ≥3 injected cells. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, ***p < 0.001.
    Wild Type Parental U2os Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wild+type+parental+u2os+cells/pm37866635-168-0-8?v=ATCC
    Average 98 stars, based on 1 article reviews
    wild type parental u2os cells - by Bioz Stars, 2026-08
    98/100 stars
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    Figure 2. Expression of PGRMC1 potentiates NAADP-evoked Ca2+ release via TPC1. A, fluorescence traces from single U2OS cells microinjected with buffer (“mock injection,” blue) or NAADP (100 nM pipette concentration) in the absence (black) or presence (red) of RFP-PGRMC1. B–D, similar microinjection experiments in single U2OS cells expressing (B) TPC1, (C) TPC2 or (D) TPC2[1-31]-TPC1[32-816] in the absence (black) or presence (red) of RFP-PGRMC1. Traces report the fluorescence of GCaMP6M over time-averaged (mean ± sd) from n ≥3 injected cells, with only every third data point shown for clarity. E, area under the curve from NAADP-evoked Ca2+ mobilization responses, values represent mean area ± sd from independent fluorescence traces recorded from n ≥3 injected cells. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, ***p < 0.001.

    Journal: The Journal of biological chemistry

    Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum.

    doi: 10.1016/j.jbc.2023.105378

    Figure Lengend Snippet: Figure 2. Expression of PGRMC1 potentiates NAADP-evoked Ca2+ release via TPC1. A, fluorescence traces from single U2OS cells microinjected with buffer (“mock injection,” blue) or NAADP (100 nM pipette concentration) in the absence (black) or presence (red) of RFP-PGRMC1. B–D, similar microinjection experiments in single U2OS cells expressing (B) TPC1, (C) TPC2 or (D) TPC2[1-31]-TPC1[32-816] in the absence (black) or presence (red) of RFP-PGRMC1. Traces report the fluorescence of GCaMP6M over time-averaged (mean ± sd) from n ≥3 injected cells, with only every third data point shown for clarity. E, area under the curve from NAADP-evoked Ca2+ mobilization responses, values represent mean area ± sd from independent fluorescence traces recorded from n ≥3 injected cells. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, ***p < 0.001.

    Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

    Techniques: Expressing, Injection, Transferring, Concentration Assay, Microinjection, Two Tailed Test

    Figure 3. Molecular disruption of the PGRMC1-TPC1 interaction impairs PGRMC1 potentiation of NAADP-evoked Ca2+ release. A–C, heme binding is necessary for interaction with TPC1. A, Myc-tagged WT PGRMC1 and PGRMC1[Y113F] constructs were co-expressed in U2OS cells with GFP-tagged TPC1, with detection by Western blot. B, immunodetection of myc-tagged PGRMC1 after TPC1-GFP immunoprecipitation. C, densitometry quantification of immunodetection of myc-tagged PGRMC1 in TPC1-GFP immunoprecipitates. D–F, interaction of PGRMC1 with TPC1 is mediated by the COOH-terminal coiled-coil domain of PGRMC1. D, PGRMC1 mutants harboring point-mutations in predicted carboxyl-terminus helices co-expressed with TPC1-GFP in U2OS cells, detected by Western blot. E, immunodetection of myc-tagged PGRMC1 mutants after TPC1-GFP immunoprecipitation. F, densitometry quantification of immunodetected myc-tagged PGRMC1 mutants in TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 independent experiments. G, traces of Ca2+ flux in U2OS cells expressing the indicated PGRMC1 constructs microinjected with NAADP (100 nM pipette concentration) as

    Journal: The Journal of biological chemistry

    Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum.

    doi: 10.1016/j.jbc.2023.105378

    Figure Lengend Snippet: Figure 3. Molecular disruption of the PGRMC1-TPC1 interaction impairs PGRMC1 potentiation of NAADP-evoked Ca2+ release. A–C, heme binding is necessary for interaction with TPC1. A, Myc-tagged WT PGRMC1 and PGRMC1[Y113F] constructs were co-expressed in U2OS cells with GFP-tagged TPC1, with detection by Western blot. B, immunodetection of myc-tagged PGRMC1 after TPC1-GFP immunoprecipitation. C, densitometry quantification of immunodetection of myc-tagged PGRMC1 in TPC1-GFP immunoprecipitates. D–F, interaction of PGRMC1 with TPC1 is mediated by the COOH-terminal coiled-coil domain of PGRMC1. D, PGRMC1 mutants harboring point-mutations in predicted carboxyl-terminus helices co-expressed with TPC1-GFP in U2OS cells, detected by Western blot. E, immunodetection of myc-tagged PGRMC1 mutants after TPC1-GFP immunoprecipitation. F, densitometry quantification of immunodetected myc-tagged PGRMC1 mutants in TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 independent experiments. G, traces of Ca2+ flux in U2OS cells expressing the indicated PGRMC1 constructs microinjected with NAADP (100 nM pipette concentration) as

    Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

    Techniques: Disruption, Binding Assay, Construct, Western Blot, Immunodetection, Immunoprecipitation, Expressing, Transferring, Concentration Assay

    Figure 4. Pharmacological disruption of the PGRMC1-TPC1 interaction blocks PGRMC1 potentiation of NAADP-evoked Ca2+ release. A and D, immunodetection of PGRMC-myc and TPC1-GFP in lysates from U2OS cells pretreated for 1 h with the indicated concentration of CORM3 (A) or AG-205 (D). B and E, immunodetection of PGRMC1-myc after immunoprecipitation of TPC1-GFP from lysates shown in (A and D). C and F, densitometric quantification of myc-tagged PGRMC1 from TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 experiments. G–I, averaged fluorescence traces (mean ± sd from n ≥3 injections) showing NAADP responses in U2OS cells transfected with empty vector (black) or PGRMC1 (red, pretreated with (G) DMSO (0.1%), (H) CORM3 (30 μM), or (I) AG-205 (30 μM) for 1 h, detected by following GCaMP6M fluorescence. J, peak F/F0 values (mean ± sd) of independent NAADP responses summarized in (G–I). K, area under the curves (mean ± sd) of independent traces of NAADP-responses summarized in (G–I). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01, ***p < 0.001.

    Journal: The Journal of biological chemistry

    Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum.

    doi: 10.1016/j.jbc.2023.105378

    Figure Lengend Snippet: Figure 4. Pharmacological disruption of the PGRMC1-TPC1 interaction blocks PGRMC1 potentiation of NAADP-evoked Ca2+ release. A and D, immunodetection of PGRMC-myc and TPC1-GFP in lysates from U2OS cells pretreated for 1 h with the indicated concentration of CORM3 (A) or AG-205 (D). B and E, immunodetection of PGRMC1-myc after immunoprecipitation of TPC1-GFP from lysates shown in (A and D). C and F, densitometric quantification of myc-tagged PGRMC1 from TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 experiments. G–I, averaged fluorescence traces (mean ± sd from n ≥3 injections) showing NAADP responses in U2OS cells transfected with empty vector (black) or PGRMC1 (red, pretreated with (G) DMSO (0.1%), (H) CORM3 (30 μM), or (I) AG-205 (30 μM) for 1 h, detected by following GCaMP6M fluorescence. J, peak F/F0 values (mean ± sd) of independent NAADP responses summarized in (G–I). K, area under the curves (mean ± sd) of independent traces of NAADP-responses summarized in (G–I). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01, ***p < 0.001.

    Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

    Techniques: Disruption, Immunodetection, Concentration Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Two Tailed Test

    Figure 5. PGRMC1 localizes to the endoplasmic reticulum in U2OS cells. A, U2OS cells imaged using various live cell markers (left) and co-transfected with PGRMC1-RFP (center). The fluorescence overlay of PGRMC1-RFP and the organelle marker channel is shown on the right. Markers were TPC1-GFP, TPC2- GFP, an endoplasmic reticulum marker (CYP2C9[1-27]-GCaMP-6M), a plasma membrane marker (GAP43[1-20]-GFP) and a mitochondrial marker (COX8A[1- 29]-CFP), Scale bar, 10 μm. Right, dashed line represents a line scan of fluorescence intensity from the green, blue or red fluorescence channels along the indicated axis. Gamma values were uniformly adjusted for clarity.

    Journal: The Journal of biological chemistry

    Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum.

    doi: 10.1016/j.jbc.2023.105378

    Figure Lengend Snippet: Figure 5. PGRMC1 localizes to the endoplasmic reticulum in U2OS cells. A, U2OS cells imaged using various live cell markers (left) and co-transfected with PGRMC1-RFP (center). The fluorescence overlay of PGRMC1-RFP and the organelle marker channel is shown on the right. Markers were TPC1-GFP, TPC2- GFP, an endoplasmic reticulum marker (CYP2C9[1-27]-GCaMP-6M), a plasma membrane marker (GAP43[1-20]-GFP) and a mitochondrial marker (COX8A[1- 29]-CFP), Scale bar, 10 μm. Right, dashed line represents a line scan of fluorescence intensity from the green, blue or red fluorescence channels along the indicated axis. Gamma values were uniformly adjusted for clarity.

    Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

    Techniques: Transfection, Marker, Clinical Proteomics, Membrane

    Figure 6. Expression of PGRMC1 increases NAADP-evoked Ca2+ release from the endoplasmic reticulum. A and B, averaged fluorescence traces as detected by GCaMP6M fluorescence changes reporting NAADP-evoked Ca2+ release in U2OS cells transfected with empty vector or in cells expressing PGRMC1. Microinjections were performed with NAADP (100 nM pipette concentration) in the presence of (A) thapsigargin (1 μM) or (B) bafilomycin A1 (100 nM). C, quantification of peak ΔF/F0 values from (A and B). D, quantification of the area under the curves from (A and B). E, left, Images of U2OS cell co- expressing GCaMP6M (top) and RCEPIA1er (bottom). Middle, pseudocolored images of GCaMP6M and RCEPIA1er fluorescence intensity before (middle image) and after (right image) microinjection of NAADP. Scale bar,10 μm. F, representative traces of GCaMP6M fluorescence (top) and RCEPIA1er fluo- rescence (bottom) in response to microinjection of NAADP (100 nM pipette concentration) in U2OS cells transfected with empty vector (grey), or from cells expressing PGRMC1 (red). G, quantification of ΔF/F0 values from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). H, quantification of area under the curves from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01.

    Journal: The Journal of biological chemistry

    Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum.

    doi: 10.1016/j.jbc.2023.105378

    Figure Lengend Snippet: Figure 6. Expression of PGRMC1 increases NAADP-evoked Ca2+ release from the endoplasmic reticulum. A and B, averaged fluorescence traces as detected by GCaMP6M fluorescence changes reporting NAADP-evoked Ca2+ release in U2OS cells transfected with empty vector or in cells expressing PGRMC1. Microinjections were performed with NAADP (100 nM pipette concentration) in the presence of (A) thapsigargin (1 μM) or (B) bafilomycin A1 (100 nM). C, quantification of peak ΔF/F0 values from (A and B). D, quantification of the area under the curves from (A and B). E, left, Images of U2OS cell co- expressing GCaMP6M (top) and RCEPIA1er (bottom). Middle, pseudocolored images of GCaMP6M and RCEPIA1er fluorescence intensity before (middle image) and after (right image) microinjection of NAADP. Scale bar,10 μm. F, representative traces of GCaMP6M fluorescence (top) and RCEPIA1er fluo- rescence (bottom) in response to microinjection of NAADP (100 nM pipette concentration) in U2OS cells transfected with empty vector (grey), or from cells expressing PGRMC1 (red). G, quantification of ΔF/F0 values from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). H, quantification of area under the curves from the cumulative dataset for GCaMP6M (top) and RCEPIA1er fluorescence (bottom). Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05, **p < 0.01.

    Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

    Techniques: Expressing, Transfection, Plasmid Preparation, Transferring, Concentration Assay, Microinjection, Two Tailed Test

    Figure 7. Expression of PGRMC1 inhibits EGF-evoked Ca2+ release. A, U2OS cells were transfected with vector encoding PGRMC1-RFP (top right) and loaded with fluo-4 dye (top left). Pseudocolored images of fluo-4 fluorescence intensity after addition of buffer (bottom, left) or EGF (32 PM, bottom, right). Kinetics of Ca2+ signals (fluo-4 fluorescence) from U2OS cells expressing PGRMC1-RFP (red boxes) and untransfected cells (white boxes) were compared. Scale bar, 20 μm. B, single-cell fluorescence traces from EGF responses in U2OS cells in the absence (black, top) or presence of PGRMC1-RFP (red, bottom). C and D, quantification of (C) peak F/F0 or (D) area under the curve (AUC) in response to EGF (32 PM) or acetylcholine (ACh, 100 μM) in control U2OS cells (black) or cells overexpressing PGRMC1 (red). Data are shown as average±sem. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05; ns, not significant.

    Journal: The Journal of biological chemistry

    Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum.

    doi: 10.1016/j.jbc.2023.105378

    Figure Lengend Snippet: Figure 7. Expression of PGRMC1 inhibits EGF-evoked Ca2+ release. A, U2OS cells were transfected with vector encoding PGRMC1-RFP (top right) and loaded with fluo-4 dye (top left). Pseudocolored images of fluo-4 fluorescence intensity after addition of buffer (bottom, left) or EGF (32 PM, bottom, right). Kinetics of Ca2+ signals (fluo-4 fluorescence) from U2OS cells expressing PGRMC1-RFP (red boxes) and untransfected cells (white boxes) were compared. Scale bar, 20 μm. B, single-cell fluorescence traces from EGF responses in U2OS cells in the absence (black, top) or presence of PGRMC1-RFP (red, bottom). C and D, quantification of (C) peak F/F0 or (D) area under the curve (AUC) in response to EGF (32 PM) or acetylcholine (ACh, 100 μM) in control U2OS cells (black) or cells overexpressing PGRMC1 (red). Data are shown as average±sem. Statistical significance was determined using paired two-tailed Student’s t test, p-values, *p < 0.05; ns, not significant.

    Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

    Techniques: Expressing, Transfection, Plasmid Preparation, Control, Two Tailed Test