Bychkov M , Shulepko M , Osmakov D , Andreev Y , Sudarikova A , Vasileva V , Pavlyukov MS , Latyshev YA , Potapov AA , Kirpichnikov M , Shenkarev ZO , Lyukmanova E .

18-34-00497 Russian Foundation for Basic Research, 18-29-01050 Russian Foundation for Basic Research, sp-4316.2018.4 Council on grants of the President of the Russian Federation, Molecular and Cell Biology Program of Presidium of the Russian Academy of Sciences

	Figure 1. qPCR analysis of Acid-sensing Ion Channels (ASICs) and epithelial Na+ channels (ENaCs) mRNA expression in U251 MG and A172 cells and normal astrocytes. Gene expression was normalized to the β-ACTIN, GPDH and RPL13a housekeeping genes and presented as lg of relative mRNA level ± standard error of mean (SEM) (n = 3–5).
	Figure 2. Effect of recombinant mambalgin-2 on rat ASIC1a expressed in Xenopus laevis oocytes: (a) Representative responses recorded in absence of mambalgin-2 (control) or presence of different mambalgin-2 concentrations, induced by buffer pH change from 7.4 to 5.5; (b) Dose–response inhibitory curves for mambalgin-2 at rat ASIC1a were fitted using Hill equation with IC50 142 ± 12 nM and 79 ± 9 nM for pH 5.5 stimulus (n = 6) and pH 6.6 stimulus (n = 8), respectively. The Hill coefficient was assumed equal to 1.0. Data are presented as % of control (without mambalgin-2) ± SEM; (c) Comparison of the peak amplitude of the transient currents at ASIC1a at pH 5.5 in presence of 1 μM mambalgin-2 and its variants with L32A and L34A substitutions. Data are presented as normalized peak current amplitude, % of control ± SEM (n = 6). Control level (100%) is shown by dashed line. ** (p < 0.01) and *** (p < 0.0001) indicate significant difference between data groups according to One-way ANOVA followed by Dunnett’s test.
	Figure 3. Mambalgin-2 effect on ASIC1a expressed in U251 MG glioma cells: (a) Desensitization time constant τdes of ASIC1a. Data are presented as mean ± SEM (n = 6); (b) Whole-cell configuration of the patch clamp technique and protocol of experiment; (c) Activation of the ASIC1a currents in U251 MG cells caused by rapid change of the extracellular solution pH from 7.4 to 5.5 (control, on the left), in the solution with pH 7.4 (in the middle), and upon application of 10 µM benzamil to the extracellular solution with pH 5.5 (on the right); (d) Activation of the ASIC1a currents in U251 MG cells caused by rapid change of the extracellular solution pH from 7.4 to 5.5 (control, on the left), in the solution with pH 7.4 (in the middle), and upon application of 1.5 µM mambalgin-2 to the extracellular solution with pH 5.5 (on the right); (e) Influence of 1.5 µM mambalgin-2 application to the extracellular solution with pH 5.5 on the peak amplitude of the ASIC1a currents in U251 MG cells. Data are presented as normalized peak current amplitude, % of control ± SEM (n = 3). Control level (100%, untreated cells) is shown by dashed line. *** (p < 0.001) indicates significant difference from the control according to two-sided t-test.
	Figure 4. Mambalgin-2 influence on growth of U251 MG and A172 glioma cells and normal astrocytes: (a) 1 µM mambalgin-2 effect on U251 MG, A172 cells and astrocytes proliferation during different incubation times. Data are presented as % of control (untreated cells, dashed line) ± SEM (n = 4). # (p < 0.05), ## (p < 0.01), and ### (p < 0.001) indicate significant difference from the control according to One-way ANOVA followed by Dunnett’s test; (b) Influence of 1 μM mambalgin-2 and its Leu32Ala and Leu34Ala mutants on proliferation of U251 MG and A172 cells during 72-h incubation. Data are presented as % of control (untreated cells, dashed line) ± SEM (n = 4). ### (p < 0.001) indicates significant difference from the control according to One-way ANOVA followed Dunnett’s hoc test. *** (p < 0.001) indicates significant difference in activity of mutants and mambalgin-2 according to One-way ANOVA followed by Dunnett’s test; (c,d) effect of different mambalgin-2 and amiloride concentrations on U251 MG (c) and A172 (d) cell growth during 72-h incubation. The parameters describing concentration-effect curves (EC50, A0) are given in the Table 1. Data are presented as % of control (untreated cells) ± SEM (n = 3–11); (e) analysis of the pH value of culture medium during prolonged cultivation of U251 MG and A172 cells (n = 4). ** (p < 0.01) and *** (p < 0.001) indicate significant difference from the pH value of the culture media in an initial time point according two-tailed t-test.
	Figure 5. Mambalgin-2 influence on the cell cycle and activity of cell cycle regulators in U251 MG cells: (a) Representative nuclei population distributions of cells after 72-h incubation in absence (control) or presence of 1 µM mambalgin-2; (b) % of cells in each cell cycle phase determined by ModFitLT Software. Data are presented as % of cells in each cell cycle phase ± SEM, n = 4; * (p < 0.05) and *** (p < 0.001) indicate the significant difference from the control by two-tailed t-test; (c) Representative Western blots, showing the mambalgin-2 influence on phosphorylation of Cyclin D1 (pSer90), cyclin-dependent kinases (CDK)4 (pThr172), and CDK6 (pTyr24); (d) The optical density (OD) of blot bands for comparison of the Cyclin D1 (pSer90), CDK4 (pThr172), CDK6 (pTyr24) expression upon 72-h incubation of cells in absence (control) or presence of 1 µM mambalgin-2. Data are presented as normalized to the β-actin band intensity, where untreated cells are taken as the control (100%, dashed line) ± SEM, n = 6; * (p < 0.05) and ** (p < 0.01) indicate the significant difference between control and mambalgin-2 treated cells by two-tailed t-test.
	Figure 6. Mambalgin-2 influence on the cell cycle and activity of cell cycle regulators in A172 cells: (a) Representative nuclei population distributions of cells after 72-h incubation in absence (control) or presence of 1 µM mambalgin-2; (b) % of cells in each cell cycle phase determined by ModFitLT Software. Data are presented as % of cells in each cell cycle phase ± SEM, n = 4; **** (p < 0.0001) indicates the significant difference from the control by two-tailed t-test; (c) Representative Western blots, showing the mambalgin-2 influence on phosphorylation of Cyclin D1 (pSer90), CDK4 (pThr172), and CDK6 (pTyr24); (d) The optical density (OD) of blot bands for comparison of the Cyclin D1 (pSer90), CDK4 (pThr172), CDK6 (pTyr24) expression upon 72-h incubation of the cells in absence (control) or presence of 1 µM mambalgin-2. Data are presented as normalized to the β-actin band intensity, where untreated cells are taken as the control (100%, dashed line) ± SEM, n = 6; * (p < 0.05) indicates the significant difference between the control and mambalgin-2 treated cells by two-tailed t-test.
	Figure 7. Apoptosis induction by mambalgin-2 in U251 MG and A172 cells: (a) Microscopic examination of nuclei morphology of U251 MG cells after their incubation with 1 µM mambalgin-2 in absence or presence of pan-caspase inhibitor z-VAD-FMK, scale bar = 10 µm; (b) Influence of mambalgin-2 and z-VAD-FMK on U251 MG cell proliferation. Data are presented as % of control (untreated cells, dashed line) ± SEM (n = 4). ### (p < 0.001) indicates significant difference between treated and untreated cells according to One-way ANOVA followed by Dunnett’s test. ** (p < 0.01) indicates significant difference from the mambalgin-2 treated cells according to One-way ANOVA followed by Dunnett’s test; (c,e) representative pictures of phosphatidylserine externalization analysis upon the mambalgin-2 treatment of U251 MG and A172 cells by flow cytometry with Annexin V-488 and Propidium iodide; (d,f) percentage of U251 MG and A172 cells with externalized phosphatidylserine and bound propidium iodide upon 72-h incubation in absence (control) or presence of 1 µM mambalgin-2. The data are presented as % of live, early apoptotic, late apoptotic and dead cells ± SEM (n = 4). *** (p < 0.001) and **** (p < 0.0001) indicate the significant difference of the groups from each other by a two-tailed t-test.
	Figure 8. Mambalgin-2 effect on cells of primary culture obtained from a patient with glioblastoma (GBM). (a) Analysis of ASICs and ENaCs gene expression in primary GBM cells by qPCR. Gene expression was normalized to the β-ACTIN, GPDH and RPL13a housekeeping genes and presented as lg of relative mRNA level ± SEM (n = 3–5); (b) Influence of different mambalgin-2 concentrations on viability of primary GBM cells. Cells were incubated for five days with mambalgin-2 and their viability was accessed by Alamar Blue assay. Data are presented as % of control (untreated cells) ± SEM (n = 3); (c) Comparison of the 50 µM mambalgin-2 inhibitory effect on viability of normal astrocytes, U251 MG, A172, and primary GBM cells by Alamar Blue assay. Data are presented as % of control (untreated cells, dashed line) ± SEM (n = 3). ## (p < 0.01) and ### (p < 0.001) indicate significant difference between treated and untreated cells according to One-way ANOVA followed by Dunnett’s test. *** (p < 0.001) indicates significant difference of mambalgin-2 action on cells from the effect on astrocytes according to One-way ANOVA followed by Dunnett’s test.
	Figure S1. Characterization of the refolded mambalgin-2: (a) HPLC analysis of mambalgin-2 homogeneity and purity; (b) Mass-spectrometry analysis of mambalgin-2; (c) 1H-NMR spectra of mambalgin-2.
	Figure S2. Western blots from 6 independent portions of U251MG cells, showing the mambalgin-2 influence on phosphorylation of Cyclin D1 (pSer90), CDK4 (pThr172), and CDK6 (pTyr24) expression. Cells were incubated with 1 μM mambalgin-2 or 0,1% DMSO for 72 h (see methods) and protein phosphorylation/expression was analyzed by western blot. Cell portions of cells are shown as #1-#6. Portions #1-#3 were analyzed on separate nitrocellulose membranes, portions #4-6 were analyzed on the same membrane. Protein ladder (Thermo Fisher, 26619) from optical channel is shown on the left of each membrane. Optical density ratio of the protein bands corresponded to the untreated cells (control) and mambalgin-2 treated cells (Mamb-2) is presented below the lanes.
	Figure S3. Western blots from 6 independent portions of A172 cells, showing the mambalgin-2 influence on phosphorylation of Cyclin D1 (pSer90), CDK4 (pThr172), and CDK6 (pTyr24) expression. Cells were incubated with 1 μM mambalgin-2 or 0,1% DMSO for 72 h (see methods) and protein phosphorylation/expression was analyzed by western blot. Cell portions of cells are shown as #1-#6 and were analyzed on the same membrane. Protein ladder (Thermo Fisher, 26619) from optical channel is shown on the left of each membrane. Optical density ratio of the protein bands corresponded to the untreated cells (control) and mambalgin-2 treated cells (Mamb-2) is presented below the lanes.

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