Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Effect of rSLURP-1 on phosphorylation of the Akt/mTOR and MAP/ERK pathways participants in A549 cells. A549 cells were incubated for 48 h with 1 μM of rSLURP-1; and PTEN (S380), AKT (S473), mTOR (S2448), PDGFRβ (Y751), Src (Y416), ERK (T202/Y204; T185/Y187), and p38 (T180/Y182) phosphorylation was detected by Bio-Plex magnetic beads assay. Data are presented as mean ± SEM, n = 4; * ( p < 0.05) and ** ( p < 0.01) indicate the significant difference by a two-tailed t -test.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Phospho-proteomics, Incubation, Magnetic Beads, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Influence of rSLURP-1 on cell cycle progression and apoptosis in A549 cells. (A) Representative nuclei population distributions of cells after 48 h incubation in absence (control) or presence of 1 μM rSLURP-1. (B) Percentage of cells in each cell cycle phase determined by ModFitLT software. Data are presented as mean ± SEM, n = 4; * ( p < 0.05) indicates the significant difference from the control by two-tailed t -test. (C) Representative pictures of phosphatidylserine externalization analysis upon the rSLURP-1 treatment of A549 cells by flow cytometry with Annexin V-488 and Propidium iodide. The cells were incubated with 1 μM rSLURP-1 or without it (control) during 48 h. (D) Percentage of cells with externalized phosphatidylserine and bound propidium iodide. The data are presented as % of live, early apoptotic, late apoptotic and dead cells ± SEM ( n = 4). * ( p < 0.05) and ** ( p < 0.01) indicate the significant difference from the control by two-tailed t -test.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Incubation, Control, Software, Two Tailed Test, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Influence of rSLURP-1 and the “loop I” and “loop III” peptides on migration of A549 cells (A–H) and primary mouse lung fibroblasts (I,J) . The data obtained for knockdown of the CHRNA7 , EGFR , and PDGFRA genes are shown on the panels (C,D) , (E,F) , and (G,H) , respectively. (A,C,E,G,I) Representative pictures of scratch assay, obtained on CloneSelect Imager after 24 h incubation of the cells with 1 μM of rSLURP-1 or 10 μM of the “loop I” peptide. (B,D,F,H,J) Effect of different rSLURP-1 and the “loop I” and “loop III” peptides concentrations on the migration of the cells. Data are presented as a mean scratch surface, occupied by migrating cells (% normalized to control), ±SEM, n = 7–21; The parameters describing the dose-response curves (EC 50 , A 1 ) and results of statistical comparisons are given in . Control level (100%) and position of the migration inhibition curves for rSLURP-1 and the “loop I” peptide in A549 cells without knock-down are shown by dashed lines. (J) Control level (100%) and the migration inhibition effects for 1 μM of rSLURP-1 and 10 μM of the “loop I” peptide in A549 cells are shown by dashed lines. ## ( p < 0.01) indicates significant difference from control group by one-way ANOVA/Dunnett’s test.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Migration, Knockdown, Wound Healing Assay, Incubation, Control, Inhibition

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Parameters descripting the effect of CHRNA7 , EGFR , and PDGFRA knock-down on the anti-migration activity of rSLURP-1 and synthetic peptide “loop I” in A549 cells ^a .

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Knockdown, Activity Assay, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Design and study of antiproliferative activity of chimeric NTII/SL-1 and SL-1/NTII proteins. (A) Amino acid sequence alignment of SLURP-1, NTII, and chimeras. Cys residues are shown by yellow. Disulfide bonds are shown by brackets. The β-strands are designated by arrows. Sequences of the grafted loops are highlighted by color frames. (B) Schemes depicting the chimeric NTII/SL-1 and SL-1/NTII proteins. Color coding is the same as on the panel (A) . (C) Antiproliferative activity of the chimeric proteins in A549 cells according to WST-1 test. Cells were incubated during 48 h with 1 μM of the proteins. Data are presented as % of control (untreated cells, dashed line) ± SEM ( n = 4–22). # ( p < 0.05) and ### ( p < 0.001) indicate significant difference from the control by one-sample t -test. * ( p < 0.05) and *** ( p < 0.001) indicate significant difference from the “rSLURP-1” group by one-way ANOVA/Dunnett’s test.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Activity Assay, Sequencing, Incubation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Design and activity of peptides, mimicking the SLURP-1 loops I and III. (A) Amino acid sequence alignment of rSLURP-1 and the synthetic peptides “loop I” and “loop III.” Cys residues are shown by yellow. Disulfide bonds are shown by brackets. (B) The dose-response curves for the inhibition of ACh-evoked currents at α7-nAChRs expressed in X. laevis oocytes by rSLURP-1 and synthetic peptides. The data are normalized to the peak amplitude of current recorded without compounds application (100%) and presented as mean ± SEM ( n = 4–6 oocytes for each compound). The standard slope (nH = 1) inhibition equation was fitted to normalized data. (C) Representative responses to 100 ms pulses of 100 μM ACh recorded in absence and presence of rSLURP-1, “loop I” and “loop III” peptides. The period of oocyte pre-incubation with compounds is shown by horizontal bars above current traces (not in the timescale), the application of ACh is shown by arrow. (D,E) Antiproliferative activity of rSLURP-1, and “loop I” and “loop III” peptides in A549 cells upon 48 h incubation according to WST-1 test. Data are presented as % of control (untreated cells, dashed line) ± SEM ( n = 4–22). ## ( p < 0.01) and ### ( p < 0.001) indicate significant difference from the control by one-sample t -test. *** ( p < 0.001) indicates significant difference from the “rSLURP-1” group by one-way ANOVA/Dunnett’s test.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Activity Assay, Sequencing, Inhibition, Incubation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Analysis of rSLURP-1 targets in A549 cells. NHS-Sepharose coupled with rSLURP-1 or rSLURP-1[K29A] and rSLURP-1[D81A] mutants was incubated with a membrane fraction of A549 cells and extracted proteins were analyzed by Western blotting using antibodies against α7-nAChR (A) , EGFR (B) , and PDGFRα (C) . For detection of EGFR non-reducing SDS-PAGE was used. Lines: “Input”–the membrane fraction of A549 cells used for analysis; “empty”–proteins extracted from the membrane fraction by empty resin without rSLURP-1 or its mutant; “rSL-1”, “rSL-1[K29A]” or “rSL-1[D81A]”–proteins extracted from the membrane fraction by resin coupled with rSLURP-1 or its mutants. Bands corresponding to the α7-nAChR, EGFR, and PDGFRα receptors are shown by arrows.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Incubation, Membrane, Western Blot, SDS Page, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Influence of CHRNA7 , EGFR , and PDGFRA knock-down on the rSLURP-1 and “loop I” peptide antiproliferative activity in A549 cells. (A–C) Representative histograms of the cell distribution after transfection with scramble RNA (purple) or siRNA to CHRNA7 , EGFR , or PDGFRA (red) according to the intensity of TRITC-labeled α-Bgtx (A) , TRITC-labeled antibody against EGFR (B) , and TRITC-labeled antibody against PDGFRα (C) . (D) Median fluorescence intensities for TRITC-labeled α-Bgtx, TRITC-labeled antibody against EGFR, or TRITC-labeled antibody against PDGFRα in cells transfected with scramble RNA and in cells with the blocked α7-nAChR, EGFR, or PDGFRα expression ( n = 4). ∗∗∗ ( p < 0.001) indicates the significant difference between the groups by two-tailed t -test. (E) Influence of 1 μM rSLURP-1 or 10 μM “loop I” on proliferation of cells transfected with scramble RNA and in cells with blocked α7-nAChR, EGFR, or PDGFRα expression upon 48 h incubation. Data presented as % of control ± SEM ( n = 3–25). # ( p < 0.05), ## ( p < 0.01), and ### ( p < 0.001) indicate significant difference from control (untreated cells, dashed line) by one-sample t -test. ∗ ( p < 0.05) indicates significant difference from the cells transfected with scramble RNA by one-way ANOVA/Dunnett’s hoc test.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: Knockdown, Activity Assay, Transfection, Labeling, Fluorescence, Expressing, Two Tailed Test, Incubation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer

doi: 10.3389/fcell.2021.739391

Figure Lengend Snippet: Scheme illustrating the mechanism of SLURP-1 action in A549 cells.

Article Snippet: Human lung adenocarcinoma A549 cells (ATCC, United States) were grown (37°C, 5% CO 2 ) in a DME medium with phenol red (PanEco, Russia), 10% fetal calf serum (Thermo Fisher Scientific, United States) and 2 mM L-glutamine (PanEco), abbreviated below as the A549 complete medium.

Techniques: