Journal: iScience

Article Title: SLO2.1/NALCN a sodium signaling complex that regulates uterine activity

doi: 10.1016/j.isci.2021.103210

Figure Lengend Snippet: SLO2.1 channels are activated by an NALCN-dependent Na + leak current in human MSMCs (A) Schematic of whole-cell bath and pipette ionic concentrations. TEA, tetraethylammonium. (B) Representative whole-cell currents (V h = 0 mV, with step pulses from −80 to +150 mV) from human MSMCs recorded in 0 mM Na + or 80 mM external Na + . The Na + -dependent currents were calculated by subtracting traces (80 mM–0 mM Na + ). (C) Same as (B), in the presence of 10 μM Gd 3+ . (D and E) Graphs depicting the percentage of Na + -dependent currents at +80 mV and −60 mV, respectively, in 0 μM Gd 3+ or 10 μM Gd 3+ (n = 5 cells; data plotted as the mean and standard deviation). In (D), values are 94.75 ± 77.14 for 0 μM Gd 3+ and 11.20 ± 16.36 for 10 μM Gd 3+ . In (E), values are 89.24 ± 63.56 for 0 μM Gd 3+ and 3.51 ± 23.50 for 10 μM Gd 3+ . (F) Schematic of whole-cell bath and pipette ionic concentrations. (G) Representative whole-cell currents (V h = 0 mV, with step pulses from −80 to +150 mV) from hTERT cells recorded in 0 mM Na + or 80 mM external Na + . The Na + -dependent currents were calculated by subtracting traces (80 mM–0 mM Na + ). (H) Same as (G), in the presence of 50 μM CP96345 (CP) (NALCN inhibitor). (I and J) Graphs depicting the percentage of Na + -dependent currents at +80 mV and −60 mV, respectively, in 0 μM CP or 60 μM CP (n = 6 and 8 cells for 0 and 50 μM CP, respectively; data plotted as the mean and standard deviation). In (I), values are 83.09 ± 20.57 for 0 μM CP and 6.50 ± 13.48 for 50 μM CP. In (J), values are 91.59 ± 68.47 for 0 μM CP and 8.19 ± 56.77 for 50 μM CP. ∗p < 0.05 by unpaired t test. See Figure S2 .

Article Snippet: Duolink in situ proximity ligation assay (Sigma, St. Louis, MO) labeling was performed with the following antibodies: NALCN (mouse monoclonal, 1:100, StressMarq), SLO2.1 (rabbit polyclonal, 1:200, Alomone), and SLO1α (BD Biosceiences 611249).

Techniques: Transferring, Standard Deviation

Journal: iScience

Article Title: SLO2.1/NALCN a sodium signaling complex that regulates uterine activity

doi: 10.1016/j.isci.2021.103210

Figure Lengend Snippet: Activation of SLO2.1 by the NALCN-dependent Na + leak hyperpolarizes the membrane potential (A) Experimental schemes and representative images of relative shifts of DiSC3(5) fluorescence induced by 80 mM sodium, 80 mM choline, 80 mM lithium, 10 μM gadolinium (Gd 3+ ), and 50 μM CP96345 (NALCN Inhibitor) in hTERT-HM cells. (B) Quantification of shifts in cells by sodium (n = 26), choline (n = 15), lithium (n = 6), and sodium plus Gd 3+ (n = 5) or NALCN inhibitor (n = 6) normalized to changes in fluorescence in the presence of valinomycin. Data are presented as mean and standard deviation. ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired t test with Mann-Whitney corrections. See .

Article Snippet: Duolink in situ proximity ligation assay (Sigma, St. Louis, MO) labeling was performed with the following antibodies: NALCN (mouse monoclonal, 1:100, StressMarq), SLO2.1 (rabbit polyclonal, 1:200, Alomone), and SLO1α (BD Biosceiences 611249).

Techniques: Activation Assay, Fluorescence, Standard Deviation, MANN-WHITNEY

Journal: iScience

Article Title: SLO2.1/NALCN a sodium signaling complex that regulates uterine activity

doi: 10.1016/j.isci.2021.103210

Figure Lengend Snippet: NALCN and SLO2.1 are in proximity in human MSMCs cells (A) Representative proximity ligation assay (PLA) labeling of hTERT-HM and human MSMCs with the indicated single antibodies and antibody combinations. (Scale bar, 10 μm). (B and C) Average number of PLA punctae in (B) hTERT-HM cells (n = 4) and (C) human primary MSMCs (from n = 4 patients). Over 300 cells per condition were processed. Data are presented as mean and standard deviation. ∗p < 0.050 and ∗∗∗p < 0.001 by unpaired t test. See Figure S5 for negative controls (SLO1 and NALCN).

Article Snippet: Duolink in situ proximity ligation assay (Sigma, St. Louis, MO) labeling was performed with the following antibodies: NALCN (mouse monoclonal, 1:100, StressMarq), SLO2.1 (rabbit polyclonal, 1:200, Alomone), and SLO1α (BD Biosceiences 611249).

Techniques: Proximity Ligation Assay, Labeling, Standard Deviation

Journal: iScience

Article Title: SLO2.1/NALCN a sodium signaling complex that regulates uterine activity

doi: 10.1016/j.isci.2021.103210

Figure Lengend Snippet: Proposed model by which the NALCN/SLO2.1 complex regulates myometrial excitability During the quiescent state, progesterone binding to the progesterone receptor (PR A/B ) increases NALCN expression and activity ( Amazu et al., 2020 ). Sodium current through NALCN activates SLO2.1 channels, increasing K + efflux to maintain the cell in a hyperpolarized state. As a result, voltage-dependent Ca 2+ channels (VDCCs) are closed, and uterine contractions do not occur. In the contractile state, estrogen acting on ERα inhibits NALCN expression ( Amazu et al., 2020 ), leading to decreased SLO2.1 activity. The reduced K + efflux depolarizes the membrane, leading to VDCC activation, an increase in intracellular Ca 2+ , and uterine contractility. At labor, oxytocin (OXT) binds to the oxytocin receptor (OTR), leading to activation of phospholipase C (PLC), production of phosphatidyl inositol 4,5-bisphosphate (PIP 2 ), and production of inositol triphosphate (IP 3 ). IP 3 activates the release of Ca 2+ from intracellular stores, and PIP 2 activates protein kinase C (PKC), which inhibits SLO2.1 ( Ferreira et al., 2019 ). This SLO2.1 inhibition further depolarizes the membrane, thus opening more VDCCs, increasing intracellular Ca 2+ , and further activating myosin to cause muscle contraction. Figure designed and created by Anthony Bartley and Chrystie Tyler.

Article Snippet: Duolink in situ proximity ligation assay (Sigma, St. Louis, MO) labeling was performed with the following antibodies: NALCN (mouse monoclonal, 1:100, StressMarq), SLO2.1 (rabbit polyclonal, 1:200, Alomone), and SLO1α (BD Biosceiences 611249).

Techniques: Binding Assay, Expressing, Activity Assay, Activation Assay, Inhibition

Journal: iScience

Article Title: SLO2.1/NALCN a sodium signaling complex that regulates uterine activity

doi: 10.1016/j.isci.2021.103210

Figure Lengend Snippet:

Article Snippet: Duolink in situ proximity ligation assay (Sigma, St. Louis, MO) labeling was performed with the following antibodies: NALCN (mouse monoclonal, 1:100, StressMarq), SLO2.1 (rabbit polyclonal, 1:200, Alomone), and SLO1α (BD Biosceiences 611249).

Techniques: Transduction, Recombinant, In Situ, Proximity Ligation Assay, Software, Stripping Membranes, Inverted Microscopy, Cytometry

Journal: Scientific Reports

Article Title: Hyperosmolarity impedes the cross-priming competence of dendritic cells in a TRIF-dependent manner

doi: 10.1038/s41598-017-00434-y

Figure Lengend Snippet: BMDCs matured in NaCl-rich microenvironment have higher surface expression of MHCI and show surface clustering of MHCI-SIINFEKL complex upon OVA uptake. ( a ) Representative histogram plots displaying relative surface expression intensity of MHCI (upper graph) and MHCII (lower graph) by BMDCs from indicated media osmolarities (black contour represents BMDCs raised in LPS-free conditions; red contour shows receptor expression by BMDCs stimulated by LPS) with corresponding statistical graphs showing GeoMFI data for LPS-free samples (right). ( b ) MHCI was immunostained for stimulated emission depletion (STED) imaging in BMDCs cultured in medium with an osmolarity of 290 mOsm, 370 mOsm or 450 mOsm, without addition of OVA. Column 1 shows standard confocal micrographs of MHCI clusters (red) in cells with Fast DiO membrane counterstaining (green) with scale bars indicating 5 μm. In the areas marked with a white box, MHCI was imaged using STED superresolution microscopy (column 3) and for comparision with the same settings without STED (column 2). Scale bars indicate 1 μm (101–171 clusters from 13–15 cells per condition, one experiment). Analysis of the MHCI cluster size revealed a similar full width at half maximum (FWHM) of around 71 nm for all three culturing conditions. ( c ) BMDCs cultured in 290 mOsm (left column) and 450 mOsm (middle column) plated on cover slips were incubated with 0.5 mg/mL OVA for 4 hours, fixed and stained in a proximity ligation assay (PLA) with anti-MHCI antibody and 25D1.16.APC antibody. PLA-spots (visible only in the case of specific binding of both antibodies in each other’s proximity) and –clusters (defined as a convincing grouping of multiple spots) were detected using Keyence BZ-9000 microscope (enlarged in the areas marked with a white box). A weak red fluorescence background in the nuclear area is occasionally observed. Scale bar indicates 10 µm. The right column shows negative control, omitting the anti-MHCI antibody. Statistical analysis (lower graph) demonstrates distribution of spots and clusters in 290 mOsm and 450 mOsm groups, displayed as % of PLA-positive cells having either spots or clusters. The data are displayed as mean ± SEM from two pooled experiments (***p ≤ 0.001, n = 19–30 high power fields).

Article Snippet: STED imaging was performed on a four-channel easy3D STED microscope (Abberior Instruments, Göttingen, Germany) at the STED facility of the Life and Medical Sciences Institute (Bonn) as described previously .

Techniques: Expressing, Imaging, Cell Culture, Membrane, Microscopy, Incubation, Staining, Proximity Ligation Assay, Binding Assay, Fluorescence, Negative Control