Journal: PLoS ONE

Article Title: Identification of a Receptor for Extracellular Renalase

doi: 10.1371/journal.pone.0122932

Figure Lengend Snippet: A, PMCA4b inhibition abrogates RP-220 mediated ERK and p38 signaling in HK-2 cells; caloxin1b = peptide inhibitor of PMCA4; left panel : RP-220 mediated ERK and p38 activation, phospho = phosphorylated, representative blot; middle panel : Inhibition of RP-220 mediated ERK and p38 activation by caloxin1b (100 μM); right panel : quantification of phosphorylated p38 (P-p38) and phosphorylated ERK (p-ERK), signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control; n = 3, * = P<0.05. B, siRNA mediated inhibition of PMCA4b expression downregulates RP-220 mediated MAPK signaling; left panel : RP-220 mediated ERK and p38 activation in HK-2 cells transfected with non-targeting siRNA, p = phosphorylated, representative immunoblot; middle panel : Inhibition of RP-220 mediated ERK and p38 activation in HK-2 cells transfected with PMCA4b siRNA, representative blot; right panel : quantification of phosphorylated ERK (p-ERK), signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control; n = 3, * = P<0.05. C, Lack of effect of siRNA mediated inhibition of PMCA4b expression on epidermal growth factor (EGF)- mediated MAPK signaling; left panel : EGF (100 ng/ml) mediated ERK, p38 activation and c-Jun N-Terminal Kinase (JNK) in HK-2 cells transfected with non-targeting siRNA, p = phosphorylated, representative blot; right panel : EGF-mediated ERK, p38 and JNK activation in HK-2 cells unaffected by transfection with PMCA4b siRNA and downregulation of PMCA4b expression; representative blot (n = 3). D, Inhibition of PMCA4b expression abrogates protective effect of renalase peptides for HK-2 cells exposed to cisplatin: HK-2 cells exposed to 20μM cisplatin for 24 hrs; cell survival is depicted as % change in survival compared to that in cisplatin-treated HK-2 cells without renalase peptides; cell survival measured by the WST-1 method, peptide concentration 15 μg/ml, indicated in top line; n = 4, * = p<0.05. E, Endogenous expression of PMCA4b in WT and renalase KO mice, western immunoblot using an anti-renalase monoclonal antibody; 10 μg protein loaded in each lane.

Article Snippet: PMCA4b protein expression was assessed by western immunoblot using an anti-PMCA4 mouse monoclonal antibody (#H00000493-M07, Novus Biologicals, Littleton, CO).

Techniques: Inhibition, Activation Assay, Control, Expressing, Transfection, Western Blot, Concentration Assay

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 2 Reduced expression of PMCA4 in RBCs of MM. (A, B) Indirect cellular immunofluorescence labeling of the surface of RBCs with PMCA4 (blue), RBC membrane with Did labeling (red), PMCA4 expression in MM RBCs is significantly reduced (P = 5.1E-03). The white arrow indicates the stacking of RBCs (rouleaux formation) in MM (magnification, 60×). (C) Relative mRNA expression of ATPase plasma membrane Ca2+ transport 4 (ATP2B4) in nucleated RBCs of MM bone marrow is significantly lower than that in the normal control group (GAPDH was used as an internal control) (P = 5.1E-03). (D, E) The expression of PMCA4 in MM RBCs was significantly reduced (P = 5.1E-03), and CD138 was expressed. (F) After the antibody-labeled RBCs reacted with the substrate BCIP/NBT, the dark blue NBT-formazan deposits in the erythrocyte membrane of MM reacting with PMCA4 protein content were significantly lower than those of the normal control group. (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Labeling, Membrane, Clinical Proteomics, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 3 MiRNA-4261 targeting the ATP2B4 gene is highly expressed in MM. (A) Screening miRNAs by Venn diagram. The conditions were the following: expression in myeloma cells; lack of expression in normal RBCs; and target gene of ATP2B4. (B) qRT–PCR confirms that miR-4261 is highly expressed in MM RBCs (P = 9.2E-03). (C–G) qRT–PCR was used to measure the content of miR-4261 in various components in blood samples (PRBC = 2.3E-02, PPBMC = 2.3E-02, Pplatelet = 0.66, Pplasma = 3.7E-02, Pexosome = 2.8E-02). (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 5 MiR-4261 can target and regulate the ATP2B4 gene and reduce the expression of PMCA4 protein. (A) Schematic diagram of the predicted miR- 4261 binding site in the 3’-UTR of ATP2B4 mRNA. (B) Dual-luciferase reporter assay. The 3’-UTR fragments (wild-type and mutant sequences) of ATP2B4 were cloned into the psiCHECK-2 vector and then cotransfected with miR-4261 mimics or NC. Comparison of relative firefly/Renilla luciferase activity between the wild-type (WT) and negative control (NC) groups (PWT+mimics vs. WT+NC = 1.9E-4, and PWT+mimics vs. MUT+mimics = 1.4E-3). (C) The content of miR-4261 in HEK293T cells 36, 48, and 72 hours after transfection of miR-4261 mimics (P = 2.7E-2) and the endogenous content of miR-4261 in H929 cells and RPMI8226 cells. (D) The mRNA level of ATP2B4 was reduced by miR-4261, and the relative expression decreased most significantly at 72 hours after transfection (GAPDH was used as an internal control) (P = 3.6E-2). (E, F) MiR-4261 reduced the protein level of ATP2B4 (GAPDH was used as an internal control) (P = 4.7E-2). (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Clone Assay, Plasmid Preparation, Comparison, Activity Assay, Negative Control, Transfection, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 7 Functional analysis of PMCA4 in maintaining calcium homeostasis in RBCs. RBCs were pretreated with different concentrations of Omega- Agatoxin IVA for 10 minutes and then treated with 0.10 mM or 0.25 mM t-BHP for 0, 1, 2, 4, 8 and 10 minutes prior to the detection of the ROS and free calcium levels in RBCs. (A) ROS levels in RBCs after treatment with 0.10 mM t-BHP. (B) Free calcium levels in RBCs after treatment with 0.10 mM t-BHP. (C) ROS levels in RBCs after treatment with 0.25 mM t-BHP. (D) Free calcium levels in RBCs after treatment with 0.25 mM t- BHP. (Error bars represent the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the 0 nM control group. Refer to Supplementary Tables 5-8 for P values. One-way ANOVA was used for statistical analysis).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Functional Assay, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 8 Schematic diagram of RBC calcium overload mediated by MM exosomal miR-4261. Exosomes secreted by MM cells transfer miR-4261 to erythroid precursor cells, resulting in RBC calcium overload by downregulating the expression of ATP2B4. Calcium overload can cause crosstalk that leads to an increase in intracellular ROS levels, forming a vicious cycle that causes further damage to the activity of PMCA4 in RBCs.

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Activity Assay

Journal: Science signaling

Article Title: Plasma membrane Ca²⁺-ATPases can shape the pattern of Ca²⁺ transients induced by store-operated Ca²⁺ entry.

doi: 10.1126/scisignal.2005672

Figure Lengend Snippet: Fig. 1. Generation and characterization of a PMCA4 knockdown HeLa cell line. (A) PMCA abundance in control HeLa (nontransfected) and sh-PMCA4 (sh4) HeLa cells grown for 3, 5, or 7 days. Western blot analysis was per- formed with equal amounts (20 mg) of whole-cell lysates using either the 5F10 antibody to detect both PMCA1 and PMCA4 (top) or JA3 antibody specific for PMCA4b (middle). Na+,K+-ATPase served as a loading control. (B) Representative experiment of the Ca2+ re-addition protocol for studying SOCE. The recordings are from sh-PMCA4 HeLa cells. Thapsigargin (Tg) inhibits SERCA, ATP stimulates release from the ER store, and addition of 2 mM Ca2+ initiates SOCE. (C) SOCE-mediated Ca2+ transients in individ- ual control HeLa cells expressing GCaMP2. (D) SOCE-mediated Ca2+

Article Snippet: The sh-PMCA4 HeLa cell line was generated by transfecting HeLa cells with a PMCA4 shRNA plasmid (Santa Cruz Biotechnology Inc.) as described (29).

Techniques: Knockdown, Control, Western Blot, Expressing

Journal: Science signaling

Article Title: Plasma membrane Ca²⁺-ATPases can shape the pattern of Ca²⁺ transients induced by store-operated Ca²⁺ entry.

doi: 10.1126/scisignal.2005672

Figure Lengend Snippet: Fig. 5. PMCA isoforms differentially affect SOCE induced at different extra- cellular Ca2+ concentrations. (A to D) After store depletion, SOCE was ini- tiated in HeLa cells expressing the indicated mCherry-PMCA constructs or in sh-PMCA4 HeLa cells by the stepwise addition of increasing [Ca2+]e, and intracellular [Ca2+]i was followed for 5 min after each addition. Average traces from about 20 cells (or 8 cells for PMCA4b-LA) are shown (means ± 95% CI). Ca2+ signals in all panels were detected by the fluorescence of GCaMP2.

Article Snippet: The sh-PMCA4 HeLa cell line was generated by transfecting HeLa cells with a PMCA4 shRNA plasmid (Santa Cruz Biotechnology Inc.) as described (29).

Techniques: Expressing, Construct, Fluorescence