Journal: Cell Communication and Signaling : CCS

Article Title: K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3

doi: 10.1186/s12964-024-01484-2

Figure Lengend Snippet: K-Ras(V12) interacts with PI3-Kα and activates Akt. A , B Interaction of K-Ras(V12) with PI3-K. A PANC-1 cells, transiently expressing TAP-EGFP, TAP-EGFP-K-Ras(V12), or TAP-EGFP-K-Ras(N17), and non-transfected control cells were lysed 24 h after transfection. TAP-containing proteins were precipitated from 5 mg of cell lysate (1 mg for TAP-EGFP) by binding to IgG sepharose beads and eluted by cleavage of the TAP tag with TEV protease. Lysates and eluates were analyzed by SDS PAGE and Western blotting using anti-p85α PI3-K or anti-GFP antibody and ECL. The left panel [lysate] shows the expression of the TAP-EGFP proteins and endogenous PI3-K p85α subunit in 8 μg (first lane) or 40 μg of lysate. The right panel [eluate] shows the precipitated, TEV-cleaved (cl.) TAP-proteins and demonstrates that PI3-K co-precipitated only with TAP-EGFP-K-Ras(V12). One representative assay ( n ≥ 3) is shown. B Purified, post-translationally modified HA-Ras proteins [HA-H-Ras(V12), HA-K-Ras(V12), HA-N-Ras(V12), 500 ng each], were immobilized on anti-HA μMacs beads and incubated with PANC-1 cell lysate [lanes 1–3]. As controls, anti-HA μMacs beads incubated with PANC-1 lysate [PANC-1 lysate] or purified HA-K-Ras(V12) [HA-K-Ras(V12)], and to determine endogenous PI3-K p85α an aliquot of PANC-1 lysate (50 µg) [Control] were used. The Western blot in the upper panel demonstrates the detection of the co-precipitated PI3-K whereas the lower panel shows a silver-stained gel with one third of the precipitated HA-Ras and IgG (light chain) proteins ( n = 3). C - F Activation of Akt by EGFP-K-Ras(V12). C PANC-1 cells and PANC-1 cell clones stably expressing EGFP, EGFP-K-Ras(V12) (clone 4.1 and 4.4), EGFP-N-Ras(V12), EGFP-H-Ras(G12), or EGFP-K-Ras(N17) were analyzed for expression of EGFP, EGFP-Ras proteins, and Akt by Western blotting. D The amount of pAkt Ser473 (upper blot) and total Akt (lower blot) was detected by Western blotting in serum starved EGFP and EGFP-Ras cell clones. pAkt Ser473 is increased in K-Ras(V12)-expressing PANC-1 cells ( n > 3). E Serum-starved PANC-1 cell clones were incubated with 10 μM LY294002 for 20 min, 30 ng/ml EGF for 10 min, or with EGF after preincubation with LY294002. Cell lysates were subjected to Western blotting for detection of pAkt Ser473 and Akt ( n > 3). Inhibition of PI3-K reduced basal as well as EGF-induced Akt phosphorylation. F Lysates of HEK293 and Colo-699 cells stably expressing EGFP or EGFP-K-Ras(V12) were subjected to Western blotting. The amount of pAkt Ser473 , Akt, EGFP-Ras, and Ras was detected by using appropriate antibodies demonstrating that pAkt is increased in K-Ras(V12)-expressing cells

Article Snippet: Packaging plasmids for third generation lentiviral transfection (pCMV-VSV-G, pMDLG/PRRE, pRSV-REV (Addgene, Watertown, USA) were used.

Techniques: Expressing, Transfection, Control, Binding Assay, SDS Page, Western Blot, Purification, Modification, Incubation, Staining, Activation Assay, Clone Assay, Stable Transfection, Inhibition, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3

doi: 10.1186/s12964-024-01484-2

Figure Lengend Snippet: PI3-K/Akt activity modulates cell migration. A , B EGFP- and EGFP-K-Ras(V12)-expressing PANC-1 cells were incubated until confluency and proliferation was inhibited by mitomycin C. A wound of 250–300 μm was scratched into the monolayer (time point t 0 ) and cells were incubated in serum-free medium with 10 μM LY294002 [LY] or solvent [Control, Co.]. Cell migration was documented after 24 h and 48 h at three randomly chosen positions. Images in A show representative phase contrast pictures at t 0 and t 48 . The Western blot on the right shows the detection of pAkt Ser473 in lysates 48 h after wounding. Detection of ERK2 served as loading control. B - D Quantification and statistics of independent wounding assays. The size of the wound was measured and calculated as percent wound closure at t 48 . The bar graph shows the mean ± SD, the dots represent individual data points (triplicate values (B + C) or 3–5 values (D), t-test (unpaired, two-tailed), *** p ≤ 0.001; **** p ≤ 0.0001). B Bar graph of the quantification of the wound closure of 3 independent experiments (triplicate values) as described above. C Bar graph of the quantification of 4–5 independent (triplicate values) in which cells were transfected with 66 nM siAkt targeting Akt1 and Akt2. Control cells [Co.] were treated with 66 nM scrambled siRNA (siControl). Wounding was started 48 h after transfection. Quantification was performed as described above. The Western blot on the right shows the detection of pAkt Ser473 , total Akt, and ERK2 as loading control in lysates 48 h after wounding. D Bar graph presenting the quantification of wounding assays with EGFP- and EGFP-K-Ras(V12)-expressing Colo-699 cell clones performed in DMEM with 5% FCS for 48 h. Quantification was conducted as described above

Article Snippet: Packaging plasmids for third generation lentiviral transfection (pCMV-VSV-G, pMDLG/PRRE, pRSV-REV (Addgene, Watertown, USA) were used.

Techniques: Activity Assay, Migration, Expressing, Incubation, Solvent, Control, Western Blot, Two Tailed Test, Transfection, Clone Assay

Journal: Cell Communication and Signaling : CCS

Article Title: K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3

doi: 10.1186/s12964-024-01484-2

Figure Lengend Snippet: Akt2 depletion upregulates Akt3 expression and activity. A Downregulation of individual Akt isoforms was performed by lentiviral transduction in PANC-1/K-Ras(V12) and PANC-1/EGFP pancreatic, as well as H23 lung carcinoma cells using Akt isoform-specific shRNAs. Expression of each Akt isoform was detected by Western blot analysis with Akt isoform-specific antibodies. B The bar graphs show the relative Akt isoform expression in relation to GAPDH or β-actin, normalized to the corresponding cell clone transduced with a scrambled shRNA [Scr] set to 1. Mean ± SD ( n = 5–8, one sample t-test, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01; * p ≤ 0.05) is shown. C Akt isoform-specific kinase activity. Akt isoforms were precipitated with isoform-specific antibodies and the precipitate was incubated with recombinant GST-GSK3α/β as Akt substrate. To precipitate equal amounts of Akt isoforms the amount of lysate was adjusted according to the expression, except for the depleted isoform. Phosphorylation of GST-GSK3α/β and amounts of immunoprecipitated Akt isoforms were detected by Western blotting procedure using antibodies reactive to pGSK3 and Akt1/2/3. Representative blots ( n = 3) are shown

Article Snippet: Packaging plasmids for third generation lentiviral transfection (pCMV-VSV-G, pMDLG/PRRE, pRSV-REV (Addgene, Watertown, USA) were used.

Techniques: Expressing, Activity Assay, Transduction, Western Blot, shRNA, Incubation, Recombinant, Phospho-proteomics, Immunoprecipitation