Journal: Advanced Science

Article Title: IGFBP1 Sustains Cell Survival during Spatially‐Confined Migration and Promotes Tumor Metastasis

doi: 10.1002/advs.202206540

Figure Lengend Snippet: IGFBP1 enhances SOD2 activity by inhibiting AKT1‐dependent SOD2 pS27 and promotes tumor metastasis. A) Transwell migration assays of A549 cells with or without IGFBP1 depletion were performed. After 6 h of culture, the confined cells were collected and subjected to immunoblotting analyses with indicated antibodies. B) A549 cells with or without IGFBP1 depletion were treated with or without the inhibitor of MEK (PD0325901, 10 µ m ) or AKT1/2/3 (MK2206, 5 µ m ), followed by Transwell migration assays. After 6 h of culture, SOD2 activities in confined cells were measured by WST‐8 kit. Relative SOD2 activities were normalized to the cells without IGFBP1 depletion. C) In vitro kinase assay was performed by mixing bacterial‐ purified recombinant GST‐AKT1 and recombinant WT or S27A mutant SOD2. D) Transwell migration assays of SOD2‐depleted A549 cells rescued with Flag‐rSOD2 WT or S27A were performed. After 6 h of culture, SOD2 activities in confined cells were measured by WST‐8 kit. Relative SOD2 activities were normalized to rSOD2 WT. E) Transwell migration assays of A549 cells with or without IGFBP1 were performed. After 6 h of culture, SOD2 S27 phosphorylations of confined cells were detected. F) Mito‐roGFP‐expressing SOD2‐depleted A549 cells were rescued with WT or S27D mutant SOD2. Transwell migration assays were performed. Representative images of Mito‐roGFP in confined cells were presented (left panel). Mito‐roGFP fluorescence intensities of confined cells were normalized to those of the cells expressing rSOD2 WT (right panel). G) SOD2‐depleted A549 cells were rescued with WT or S27D mutant SOD2. Transwell migration assays were performed. After 6 h of culture, the cells were stained with Annexin‐V‐FITC and photographed in situ. Representative images of the apoptotic confined cells were presented on the left panel. The percentages of apoptosis of confined cells were shown on the right panel. H,I) Luciferase‐expressing SOD2‐depleted A549 cells rescued with WT or S27D mutant SOD2 were injected into randomized NOD/SCID mice by tail vein injection (five mice per group). After 30 days inoculation, bioluminescence imaging was performed and representative images of lung metastasis were presented (right panel). The statistical analysis of luciferase intensities was shown respectively on the left panel (H). Data represent the mean ± S.D. of five mice. After bioluminescence imaging, mice were euthanized. Representative images of H&E staining of lung sections from these mice were shown (I, right). Tumor areas in H&E‐stained sections were calculated and normalized to those of the mice injected with A549 cells expressing WT SOD2 (I, left). Data represent the mean ± S.D. of five mice. (B, D, F, G), Data represent the mean ± S.D. of three independent experiments.

Article Snippet: Recombinant full‐length human AKT1 was bought from SignalChem.

Techniques: Activity Assay, Migration, Western Blot, In Vitro, Kinase Assay, Purification, Recombinant, Mutagenesis, Expressing, Fluorescence, Staining, In Situ, Luciferase, Injection, Imaging

Journal: Advanced Science

Article Title: IGFBP1 Sustains Cell Survival during Spatially‐Confined Migration and Promotes Tumor Metastasis

doi: 10.1002/advs.202206540

Figure Lengend Snippet: Schematic model for IGFBP1‐promoted tumor metastasis by sustaining cell survival during confined migration. IGFBP1 promotes tumor metastasis by sustaining cell survival during confined migration. Mechanistically, IGFBP1 enhances SOD2 activity by preventing AKT1‐mediated SOD2 S27 phosphorylation to scavenge accumulated mitochondrial ROS, thereby sustaining cell survival in confined space.

Article Snippet: Recombinant full‐length human AKT1 was bought from SignalChem.

Techniques: Migration, Activity Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

doi: 10.1101/2023.11.21.568047

Figure Lengend Snippet: A ) Schematic shows upstream and downstream kinases in the AKT signaling network. B ) BMDMs were primed, or not, with LPS and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). Phosphorylation of upstream AKT regulators (TBK1, IKKε, PDK1, PTEN and PI3K) was analyzed by immunoblot. Images are representative of three independent experiments. C ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). The phosphorylation of the indicated mTORC1/2-associated proteins was analyzed after 1h by immunoblotting. Data are representative of three independent experiments. D ) WT and Nfe2l2 −/− BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h. The indicated transcripts were analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. Statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparisons test. E-F ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with 15d-PGJ 2 (20, 10 or 1 μM in E and 10 μM in F ) for 1h ( E ) or 24h ( F ). AKT phosphorylation and NRF2 accumulation were assessed by immunoblot ( E ). Data are representative of three independent experiments. IL-10 and TNF release was quantified by ELISA ( F ). n = 6, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Sidak’s test. G ) Expression of Akt isoforms (RNA-seq, Immgen.org database) in the indicated murine macrophage populations. H ) Normalized expression of Akt1 , Akt2 and Akt3 in BMDMs, analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. I, J ) Microscale thermophoresis (MST) analysis of oxPAPC interactions with AKT2 ( I ) and AKT3 ( J ). Traces of fluorescently labeled human recombinant AKT incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes. n = 3, graph shows means ± SD. Images are representative of three independent experiments. K, L ) Active human recombinant AKT2 ( K ) or AKT3 ( L ) were incubated with oxPAPC and a kinase-specific FRET-peptide substrate (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

Article Snippet: His-tagged recombinant human AKT1 (BPS Bioscience, Cat# 40003), AKT2 (BPS Bioscience, Cat# 40011) and AKT3 (BPS Bioscience, Cat# 40012) were fluorescently labeled using His-Tag Labeling Kit RED-Tris-NTA 2 nd Generation (NanoTemper, Cat# MO-L018).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing Assay, Microscale Thermophoresis, Labeling, Recombinant, Incubation, Binding Assay, Derivative Assay, Fluorescence, Inhibition, Blocking Assay

Journal: bioRxiv

Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

doi: 10.1101/2023.11.21.568047

Figure Lengend Snippet: A-E ) WT and Tlr4 −/− ( A ), Tlr2 −/− ( B ), Cd14 −/− ( C ), Cd36 −/− ( D ) or Nfe2l2 −/− ( E ) BMDMs were primed, or not, with R848 (1 μg ml −1 ) ( A, C ) or LPS (1 μg ml −1 ) ( B-E ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h ( left, center ) or 24h ( right ). AKT phosphorylation ( A-E ) or NRF2 accumulation ( E ) were assessed by immunoblot ( left ). Data are representative of three independent experiments. ECAR ( center ) was measured using a Seahorse analyzer. n = 6 ( A-C, E ), n = 5 ( D ), data are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. IL-10 and TNF release ( right ) was quantified by ELISA. n = 3, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. F ) oxPAPC binding capacity of AKT was determined by pull down assay. Cellular lysate of 293T cells expressing HA-tagged human AKT1 was incubated with oxPAPE-N-Biotin and the indicated doses of oxPAPC. AKT associated with biotinylated lipids was captured by streptavidin beads and revealed by immunoblotting using anti-HA antibody. β-actin was used as a negative control. Data are representative of three independent experiments. G ) Microscale thermophoresis (MST) analysis of oxPAPC and AKT interactions. Traces of fluorescently labeled human recombinant AKT1 incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) ( left and central ). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes ( right ). n = 3, graph shows means ± SD. Images are representative of three independent experiments. H ) Cellular lysate of 293T cells expressing the indicated HA-tagged AKT1 truncated forms were incubated with oxPAPE-N-Biotin. HA-proteins associated with biotinylated lipid were captured by streptavidin beads and revealed by immunoblotting. Data shown are representative of three independent experiments. I ) Active human recombinant AKT1 or PDK1 were incubated with oxPAPC or DPPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, and 1.9 μM) and kinase-specific FRET-peptide substrates (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

Article Snippet: His-tagged recombinant human AKT1 (BPS Bioscience, Cat# 40003), AKT2 (BPS Bioscience, Cat# 40011) and AKT3 (BPS Bioscience, Cat# 40012) were fluorescently labeled using His-Tag Labeling Kit RED-Tris-NTA 2 nd Generation (NanoTemper, Cat# MO-L018).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Pull Down Assay, Expressing, Incubation, Negative Control, Microscale Thermophoresis, Labeling, Recombinant, Derivative Assay, Fluorescence, Inhibition, Blocking Assay

Journal: bioRxiv

Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

doi: 10.1101/2023.11.21.568047

Figure Lengend Snippet: A ) Schematic shows upstream and downstream kinases in the AKT signaling network. B ) BMDMs were primed, or not, with LPS and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). Phosphorylation of upstream AKT regulators (TBK1, IKKε, PDK1, PTEN and PI3K) was analyzed by immunoblot. Images are representative of three independent experiments. C ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). The phosphorylation of the indicated mTORC1/2-associated proteins was analyzed after 1h by immunoblotting. Data are representative of three independent experiments. D ) WT and Nfe2l2 −/− BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h. The indicated transcripts were analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. Statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparisons test. E-F ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with 15d-PGJ 2 (20, 10 or 1 μM in E and 10 μM in F ) for 1h ( E ) or 24h ( F ). AKT phosphorylation and NRF2 accumulation were assessed by immunoblot ( E ). Data are representative of three independent experiments. IL-10 and TNF release was quantified by ELISA ( F ). n = 6, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Sidak’s test. G ) Expression of Akt isoforms (RNA-seq, Immgen.org database) in the indicated murine macrophage populations. H ) Normalized expression of Akt1 , Akt2 and Akt3 in BMDMs, analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. I, J ) Microscale thermophoresis (MST) analysis of oxPAPC interactions with AKT2 ( I ) and AKT3 ( J ). Traces of fluorescently labeled human recombinant AKT incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes. n = 3, graph shows means ± SD. Images are representative of three independent experiments. K, L ) Active human recombinant AKT2 ( K ) or AKT3 ( L ) were incubated with oxPAPC and a kinase-specific FRET-peptide substrate (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

Article Snippet: Briefly, recombinant human AKT1 (BPS Bioscience, Cat# 40003), AKT2 (BPS Bioscience, Cat# 40011) and AKT3 (BPS Bioscience, Cat# 40012) were used at 7.5 ng per reaction and recombinant human PDK1 (BPS Bioscience, Cat# 40080) was used at 50 ng per reaction.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing Assay, Microscale Thermophoresis, Labeling, Recombinant, Incubation, Binding Assay, Derivative Assay, Fluorescence, Inhibition, Blocking Assay

Journal: bioRxiv

Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

doi: 10.1101/2023.11.21.568047

Figure Lengend Snippet: A-E ) WT and Tlr4 −/− ( A ), Tlr2 −/− ( B ), Cd14 −/− ( C ), Cd36 −/− ( D ) or Nfe2l2 −/− ( E ) BMDMs were primed, or not, with R848 (1 μg ml −1 ) ( A, C ) or LPS (1 μg ml −1 ) ( B-E ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h ( left, center ) or 24h ( right ). AKT phosphorylation ( A-E ) or NRF2 accumulation ( E ) were assessed by immunoblot ( left ). Data are representative of three independent experiments. ECAR ( center ) was measured using a Seahorse analyzer. n = 6 ( A-C, E ), n = 5 ( D ), data are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. IL-10 and TNF release ( right ) was quantified by ELISA. n = 3, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. F ) oxPAPC binding capacity of AKT was determined by pull down assay. Cellular lysate of 293T cells expressing HA-tagged human AKT1 was incubated with oxPAPE-N-Biotin and the indicated doses of oxPAPC. AKT associated with biotinylated lipids was captured by streptavidin beads and revealed by immunoblotting using anti-HA antibody. β-actin was used as a negative control. Data are representative of three independent experiments. G ) Microscale thermophoresis (MST) analysis of oxPAPC and AKT interactions. Traces of fluorescently labeled human recombinant AKT1 incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) ( left and central ). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes ( right ). n = 3, graph shows means ± SD. Images are representative of three independent experiments. H ) Cellular lysate of 293T cells expressing the indicated HA-tagged AKT1 truncated forms were incubated with oxPAPE-N-Biotin. HA-proteins associated with biotinylated lipid were captured by streptavidin beads and revealed by immunoblotting. Data shown are representative of three independent experiments. I ) Active human recombinant AKT1 or PDK1 were incubated with oxPAPC or DPPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, and 1.9 μM) and kinase-specific FRET-peptide substrates (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

Article Snippet: Briefly, recombinant human AKT1 (BPS Bioscience, Cat# 40003), AKT2 (BPS Bioscience, Cat# 40011) and AKT3 (BPS Bioscience, Cat# 40012) were used at 7.5 ng per reaction and recombinant human PDK1 (BPS Bioscience, Cat# 40080) was used at 50 ng per reaction.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Pull Down Assay, Expressing, Incubation, Negative Control, Microscale Thermophoresis, Labeling, Recombinant, Derivative Assay, Fluorescence, Inhibition, Blocking Assay

Journal: iScience

Article Title: AKT2-mediated nuclear deformation leads to genome instability during epithelial-mesenchymal transition

doi: 10.1016/j.isci.2023.106992

Figure Lengend Snippet: AKT2 is responsible for lamin A Ser390 phosphorylation and nuclear deformation on TGFβ stimulation (A) A549 cells were pre-treated with MK2206 (AKT inhibitor; 5 μM), PD98059 (ERK inhibitor; 20 μM), SP600125 (JNK inhibitor; 10 μM), SB23580 (p38 inhibitor; 10 μM), or the solvent DMSO as the control for 1 h and then co-treated with TGFβ for 72 h. The cells were stained for lamin A (green), lamin B1 (red), and DNA (blue). Representative images are shown. Scale bars, 20 μm. The percentage of the cells with a crumpled or lobulated nucleus was measured (n = 300). To further confirm the inhibitory effect of MK2206 on TGFβ-induced nuclear deformation, A549 cells were treated with TGFβ in the presence of MK2206 at various concentrations as indicated for 72 h. The percentage of the cells with a crumpled or lobulated nucleus was measured (n = 300). (B) A549 cells were pre-treated with LY294002 (PI3K inhibitor; 20 μM) for 1 h and then co-treated with TGFβ for 72 h. The cells were stained for lamin A (red) and DNA (blue). Representative images are shown. Scale bars, 10 μm. The percentage of the cells with a crumpled or lobulated nucleus was measured (n ≥ 245). (C) A549 cells were pre-treated with or without MK2206 (5 μM) for 1 h and co-treated with TGFβ for 48 h. The cells were lysed and an equal amount of whole cell lysates was analyzed by immunoblotting with anti-lamin A or anti-lamin A pS390 antibody. The level of lamin A pS390 was measured and expressed as -fold relative to the level of the cells without MK2206 treatment. (D) A549 cells were infected with lentiviruses encoding shRNAs specific to AKT1 (sh-AKT1) or AKT2 (sh-AKT2). The cells were treated with TGFβ for 72 h and lysed. An equal amount of whole cell lysates was analyzed by immunoblotting with the antibodies as indicated. The level of lamin A pS390 was measured and expressed as -fold relative to the level of the control group without TGFβ treatment. (E) A549 cells as described in (D) were treated with TGFβ for 24 h and stained for lamin A (green) and DNA (blue). Representative images are shown. Scale bars, 10 μm. The percentage of the cells with a crumpled or lobulated nucleus was measured (n ≥ 600). (F) A549 cells were transiently transfected with the plasmid encoding HA-AKT1 or HA-AKT2 for 24 h and then stained for HA-AKT (green) and lamin A (white). The arrows indicate deformed nuclei. Scale bars, 20 μm. The percentage of the transfection-positive cells with a deformed nucleus was measured (n ≥ 457). (G) FLAG-lamin A was transiently co-expressed with HA-AKT1 or HA-AKT2 in HEK293T cells for 24 h. The cells were lysed and FLAG-lamin A was immunoprecipitated (IP) from an equal amount of whole cell lysates with anti-FLAG antibody. The immunocomplexes were fractionated by SDS-polyacrylamide gel electrophoresis and the gel was stained with Phos-Tag phosphoprotein gel stain, according to the manufacturer’s instructions. Besides, the immunocomplexes were analyzed by immunoblotting with the antibodies as indicated. (H) Recombinant His-lamin A proteins prepared in our laboratory as described in Materials and Methods were subjected to an in vitro kinase assay in the presence of purified recombinant active AKT1 or AKT2 from Merck Millipore. The kinase reaction was terminated with the SDS sample buffer and the proteins in the reaction were analyzed by immunoblotting with the antibodies as indicated. The phosphorylation of His-lamin A at Ser390 was measured and expressed as –fold relative to the level of the control without AKT. Data information: In A, B, E, F and H, values (means ± SD) were from three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S2 .

Article Snippet: Active recombinant human AKT1 , Merck Millipore , Cat#14-453.

Techniques: Phospho-proteomics, Solvent, Control, Staining, Western Blot, Infection, Transfection, Plasmid Preparation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Recombinant, In Vitro, Kinase Assay, Purification

Journal: iScience

Article Title: AKT2-mediated nuclear deformation leads to genome instability during epithelial-mesenchymal transition

doi: 10.1016/j.isci.2023.106992

Figure Lengend Snippet:

Article Snippet: Active recombinant human AKT1 , Merck Millipore , Cat#14-453.

Techniques: Recombinant, Staining, Plasmid Preparation, Software, Imaging