Journal: The Journal of Biological Chemistry

Article Title: Protein Kinase A Rescues Microtubule Affinity-regulating Kinase 2-induced Microtubule Instability and Neurite Disruption by Phosphorylating Serine 409 *

doi: 10.1074/jbc.M114.629873

Figure Lengend Snippet: PKA phosphorylates MARK2 at a novel site, Ser-409. A, Western blot (IB) analysis directed against phospho-PKA substrates (RRX(S*/T*)) in endogenous MARK2 or CREB immunoprecipitated (IP) from HEK293 cells pretreated with 10 μm H89 (a PKA inhibitor) for 0.5 h before treatment with 10 μm forskolin for 30 min. B, sequence alignment of MARK orthologues illustrates conservation of the RRX(S/T) motif in MARK2 between species. The kinases and transcription factors that contain the RRX(S/T) motif were confirmed as phosphorylation substrates of PKA. NMDAR, NMDA receptor. C, recombinant MARK2 WT or MARK2 S409A (400 nm) was incubated with or without PKAc in the presence of 100 μm ATP and 5 μCi of [γ-32P]ATP per reaction at 30 °C for 4 h. Reaction products were analyzed by autoradiography. D, recombinant MARK2 WT were incubated with or without recombinant PKAc (400 nm) at 30 °C for 4 h in 50 mm HEPES, pH 7.5, 10 mm MgCl2, 1 mm EGTA, 0.01% Brij-35, and 100 μm ATP. Because the E. coli-derived recombinant MARK2 has no phosphorylation modification and low kinase activity, 20 nm LKB1 was added to pre-phosphorylate and activate MARK2 proteins in vitro. The analysis of the indicated protein kinase activity was performed using a 33P incorporation assay against a well known substrate of MARK2, the AMARA peptide. Radioactivity that had been incorporated in AMARA was determined by liquid scintillation counting in a Wallac MicroBeta plate counter. Neither PKAc nor LKB1 showed any activity against AMARA peptide. E, analysis of the activity of MARK2 WT (400 nm) with or without a kinase-dead mutant of PKAc (PKAc K72I, PKA-PD) (400 nm) were performed using a 33P incorporation assay against AMARA. F, analyses of the activities of MARK2 proteins (MARK2 WT with or without PKAc, MARK2 S409A with or without PKAc, MARK2 S409E) were performed using a 33P incorporation assay against AMARA. All reactions were repeated in three independent experiments. **, p < 0.005; ***, p < 0.001. G, detection of phosphorylation at Ser-409 in MARK2 by MS/MS analysis. Recombinant MARK2 WT were incubated with or without recombinant PKAc (400 nm) at 30 °C for 4 h in 50 mm HEPES, pH 7.5, 10 mm MgCl2, 1 mm EGTA, 0.01% Brij-35, and 100 μm ATP. Shown is a high resolution MS/MS spectrum of a tryptic peptide (RFpSDQAAGPAIPTSNSYSK) from MARK2 by higher energy collision dissociation (HCD) in a Q Exactive mass spectrometer. The fragmented y and b ions indicated that Ser-409 of the protein was phosphorylated. The neutral loss of 98 Da corresponding to the mass of a phosphoryl group is marked.

Article Snippet: Recombinant liver kinase B1 (LKB1) protein was bought from Carna Biosciences.

Techniques: Western Blot, Immunoprecipitation, Sequencing, Recombinant, Incubation, Autoradiography, Derivative Assay, Modification, Activity Assay, In Vitro, Radioactivity, Mutagenesis, Tandem Mass Spectroscopy, Mass Spectrometry

Journal: The Journal of nutritional biochemistry

Article Title: Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway.

doi: 10.1016/j.jnutbio.2014.06.001

Figure Lengend Snippet: Fig. 1. ALA activates SIRT1/LKB1 pathway in HepG2 cells. (A) The dose–response effect of ALA on SIRT1 deacetylase activity. HepG2 cells were treated with 0, 50, 125, 250 and 500 μM ALA, or 0, 10, 25, 50 and 100 μM resveratrol used as a positive control for 24 h. Data are presented as mean±S.E.M. (n=5). *Pb.05, **Pb.01 vs. control (0 μM ALA); #Pb.05, ##Pb.01 vs. control (0 μM resveratrol). (B) Intracellular NAD+/NADH ratio. HepG2 cells were treated with 0 (control), 250, 500 and 1000 μM ALA for 24 h. Data are presented as mean±S.E.M. (n=6). *Pb.05, **Pb.01 vs. control (0 μmol ALA). HepG2 cells were transfected with SIRT1siRNA or scramble siRNA for 24 h after incubation with ALA (250 μM, 6 h). (C) IP of acetylated liver kinase B1 (LKB1). *Pb.05 vs. control (untreated HepG2 cells); #Pb.05 vs. ALA group; ΔPb.05 vs. ALA+scramble siRNA group. (D) COIP of SIRT1 and LKB1. Nonspecific IgG was used as control.

Article Snippet: Samples were incubated with 20 μl Protein A-G (Santa Cruz Biotechnology) and 1–2 μg primary antibodies [anti-rabbit-SIRT1 antibody (Santa Cruz Biotechnology) and anti-rabbit-LKB1 antibody (Cell Signaling Technology)] for 1–2 h at 4°C under constant shaking.

Techniques: Histone Deacetylase Assay, Activity Assay, Positive Control, Control, Transfection, Incubation

Journal: The Journal of nutritional biochemistry

Article Title: Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway.

doi: 10.1016/j.jnutbio.2014.06.001

Figure Lengend Snippet: Fig. 3. ALA causes redistribution of transcription factors FoxO1 and SREBP-1 via the SIRT1/LKB1/AMPK signaling pathway. (A and B) HepG2 cells were treated with 125 μM PA and 250 μM ALA for 12 h. Starvation: HepG2 was incubated with serum-free medium. FoxO1 and SREBP-1 protein expression and distribution were determined by immunofluorescence staining (magnification, 400×). Green: FITC; blue: DAPI. (C) HepG2 cells were treated with 0 (control, C), 50, 125, 250, 500 and 1000 μM ALA for 12 h. *Pb.05, #Pb.05 vs. control (untreated cells). (D) Effect of ALA (250 μM, 6 h) on FoxO1 and SREBP-1 phosphorylation levels in the presence or absence of AMPK inhibitor (CC, 20 μM, 0.5 h), SIRT1 inhibitor (NA, 10 mM, 24 h) and AMPK activator (AICAR, 2 mM, 1 h), respectively. *Pb.05, #Pb.05 vs. ALA group.

Article Snippet: Samples were incubated with 20 μl Protein A-G (Santa Cruz Biotechnology) and 1–2 μg primary antibodies [anti-rabbit-SIRT1 antibody (Santa Cruz Biotechnology) and anti-rabbit-LKB1 antibody (Cell Signaling Technology)] for 1–2 h at 4°C under constant shaking.

Techniques: Incubation, Expressing, Immunofluorescence, Staining, Control, Phospho-proteomics

Journal: The Journal of nutritional biochemistry

Article Title: Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway.

doi: 10.1016/j.jnutbio.2014.06.001

Figure Lengend Snippet: Fig. 6. Proposed scheme illustrating the role of ALA in the regulation of hepatocyte lipid metabolism and antioxidation. ALA reverses the HFD-induced changes in AMPK expression, which is involved in SIRT1/LKB1/AMPK-mediated signaling, and transcrip- tion factors FoxO1, SREBP-1 and nuclear Nrf2 protein expression, along with changes in the lipid metabolism and antioxidative enzyme expression.

Article Snippet: Samples were incubated with 20 μl Protein A-G (Santa Cruz Biotechnology) and 1–2 μg primary antibodies [anti-rabbit-SIRT1 antibody (Santa Cruz Biotechnology) and anti-rabbit-LKB1 antibody (Cell Signaling Technology)] for 1–2 h at 4°C under constant shaking.

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

doi: 10.1128/MCB.00154-13

Figure Lengend Snippet: LKB1 controls apical junction formation in 16HBE cells. (A) At 2 days after infection with lentiviral vectors harboring control or 4 distinct shRNAs targeting LKB1 (shLKB1 to shLKB4), 16HBE cells were seeded on coverslips. At 2 days after plating, confluent cells were fixed and stained with anti-ZO-1 (top) and anti-E-cadherin (bottom). Bar, 20 μm. (B) (Top) Quantification of apical junction formation from three independent experiments. Error bar, SEMs. **, P < 0.01; ***, P < 0.001. (Bottom) At 5 days after infection, cells were lysed and analyzed by Western blotting with the indicated antibodies. Con, control. (C) (Top) Myc-tagged LKB1WT, LKB1KD (K78M), or LKB1SL26 was transiently transfected into 16HBE cells stably expressing HA-STRAD. One day later, cells were lysed and analyzed by Western blotting with the indicated antibodies. (Bottom) 16HBE cells stably expressing mLKB1WT, mLKB1KD (K78M), mLKB1SL26, or mLKB1C433A were infected with shRNA targeting LKB1 lentiviral vector, and 2 and 5 days later cells were lysed and analyzed by Western blotting with the indicated antibodies. (D) Cells described for panel C were replated, grown to confluence, and fixed and stained with anti-ZO-1 (bottom) and anti-myc (top). (E) Quantification of apical junction formation as described for panel D from three independent experiments. Error bars, SEMs. **, P < 0.01; ***, P < 0.001.

Article Snippet: Primary antibodies used were ZO-1 (clone 1A12; 339100; Invitrogen, Carlsbad, CA), ZO-1 (617300; Invitrogen, Carlsbad, CA) E-cadherin (clone ECCD-2; 131900; Invitrogen, Carlsbad, CA), E-cadherin (clone 34; 610404; BD Transduction, Lexington, KY), α-tubulin (clone YL1/2; AbD Serotec, Raleigh, NC), hemagglutinin (HA; clone 3F10; Roche, Indianapolis, IN), green fluorescent protein (GFP; rabbit polyclonal; Invitrogen, Carlsbad, CA), myc (clone 9E10; Cancer Research UK, London, United Kingdom), phospho-PRK (p-PRK; 2611; Cell Signaling, Beverly, MA), PAK4 (3242; Cell Signaling, Beverly, MA), p114RhoGEF (EB06163; Everest Biotech Ramona, CA), LKB1 (27D10; Cell Signaling, Beverly, MA), and RhoA (sc-418; Santa Cruz, Santa Cruz, CA).

Techniques: Infection, Control, Staining, Western Blot, Transfection, Stable Transfection, Expressing, shRNA, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

doi: 10.1128/MCB.00154-13

Figure Lengend Snippet: LKB1 and STRAD activate RhoA and enhance phospho-PRK recruitment to apical junctions. (A) 16HBE cells stably expressing HA-tagged STRAD were infected with pLL3.7 lentiviral vectors encoding GFP-tagged LKB1 constructs. Twenty-four hours later, cells were harvested and Rho activity (Rho.GTP) was determined using a standard pulldown assay. LKB1WT, LKB1KD (K78M), and LKB1P38A induced 6- to 8-fold increases in the levels of active RhoA.GTP relative to the level for the control, LKB1SL26, or LKB1SL26/P38A. Total phospho-PRK was determined in the input cell lysates. Error bars, SEMs. **, P < 0.01; *, P < 0.05. WB, Western blotting. (B) 16HBE cells expressing GFP-tagged hLKB1P38A and hLKB1SL26/P38A were fixed and stained with DAPI (4′,6-diamidino-2-phenylindole). GFP signals were visualized directly. Bar, 20 μm. (C) At 1 day after infection, cells were stained for anti-phospho-PRK. GFP signals were visualized directly. GFP indicated infected cells. Bar, 20 μm.

Article Snippet: Primary antibodies used were ZO-1 (clone 1A12; 339100; Invitrogen, Carlsbad, CA), ZO-1 (617300; Invitrogen, Carlsbad, CA) E-cadherin (clone ECCD-2; 131900; Invitrogen, Carlsbad, CA), E-cadherin (clone 34; 610404; BD Transduction, Lexington, KY), α-tubulin (clone YL1/2; AbD Serotec, Raleigh, NC), hemagglutinin (HA; clone 3F10; Roche, Indianapolis, IN), green fluorescent protein (GFP; rabbit polyclonal; Invitrogen, Carlsbad, CA), myc (clone 9E10; Cancer Research UK, London, United Kingdom), phospho-PRK (p-PRK; 2611; Cell Signaling, Beverly, MA), PAK4 (3242; Cell Signaling, Beverly, MA), p114RhoGEF (EB06163; Everest Biotech Ramona, CA), LKB1 (27D10; Cell Signaling, Beverly, MA), and RhoA (sc-418; Santa Cruz, Santa Cruz, CA).

Techniques: Stable Transfection, Expressing, Infection, Construct, Activity Assay, Control, Western Blot, Staining

Journal: Molecular and Cellular Biology

Article Title: LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

doi: 10.1128/MCB.00154-13

Figure Lengend Snippet: p114RhoGEF controls apical junction formation in 16HBE cells and interacts with LKB1. (A) At 2 days after infection with lentiviral vectors harboring shRNAs targeting p114RhoGEF, 16HBE cells were seeded on coverslips. Two days later, confluent cells were fixed and stained with anti-ZO-1 (top) and anti-E-cadherin (bottom). Bar, 20 μm. (B) (Top) Quantification of apical junction formation from three independent experiments. Error bars, SEMs. *, P < 0.05; **, P < 0.01. (Bottom) At 5 days after infection, cells were lysed and analyzed by Western blotting with the indicated antibodies. (C) Schematic organization of hLKB1 and mouse p114RhoGEF (numbers represent amino acids). hLKB1 possesses a farnesylation site at aa 430. SL26 represents a 9-bp, in-frame deletion in the kinase domain. p114RhoGEF has a potential PBM at its C terminus. NRD, N-terminal regulatory domain; DH, Dbl homology domain. (D to F) HEK293T cells cotransfected with the indicated combinations of constructs. Proteins were immunoprecipitated (IP) from cell lysates using anti-mouse IgG or anti-Flag antibody. Input and immunoprecipitated lysates were analyzed by Western blotting.

Article Snippet: Primary antibodies used were ZO-1 (clone 1A12; 339100; Invitrogen, Carlsbad, CA), ZO-1 (617300; Invitrogen, Carlsbad, CA) E-cadherin (clone ECCD-2; 131900; Invitrogen, Carlsbad, CA), E-cadherin (clone 34; 610404; BD Transduction, Lexington, KY), α-tubulin (clone YL1/2; AbD Serotec, Raleigh, NC), hemagglutinin (HA; clone 3F10; Roche, Indianapolis, IN), green fluorescent protein (GFP; rabbit polyclonal; Invitrogen, Carlsbad, CA), myc (clone 9E10; Cancer Research UK, London, United Kingdom), phospho-PRK (p-PRK; 2611; Cell Signaling, Beverly, MA), PAK4 (3242; Cell Signaling, Beverly, MA), p114RhoGEF (EB06163; Everest Biotech Ramona, CA), LKB1 (27D10; Cell Signaling, Beverly, MA), and RhoA (sc-418; Santa Cruz, Santa Cruz, CA).

Techniques: Infection, Staining, Western Blot, Construct, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

doi: 10.1128/MCB.00154-13

Figure Lengend Snippet: LKB1 and p114RhoGEF C-terminal domains have a dominant negative effect on apical junction formation in 16HBE cells. (A) 16HBE cells stably expressing GFP-tagged LKB1WT, LKB1KD, LKB1SL26, LKB1CRD (aa 309 to 433), or LKB1CRDΔCAAX (aa 309 to 429) were stained with anti-ZO-1 (bottom) and visualized directly with GFP signaling (top). (B) 16HBE cells stably expressing HA-tagged p114RhoGEF-FL (p114-FL), p114RhoGEF-N (p114-N), p114RhoGEF-M (p114-M), p114RhoGEF-C (p114-C), or p114RhoGEF-CΔPBM (p114-CΔPBM) were stained with anti-ZO-1 and anti-HA antibodies. Bars, 20 μm.

Article Snippet: Primary antibodies used were ZO-1 (clone 1A12; 339100; Invitrogen, Carlsbad, CA), ZO-1 (617300; Invitrogen, Carlsbad, CA) E-cadherin (clone ECCD-2; 131900; Invitrogen, Carlsbad, CA), E-cadherin (clone 34; 610404; BD Transduction, Lexington, KY), α-tubulin (clone YL1/2; AbD Serotec, Raleigh, NC), hemagglutinin (HA; clone 3F10; Roche, Indianapolis, IN), green fluorescent protein (GFP; rabbit polyclonal; Invitrogen, Carlsbad, CA), myc (clone 9E10; Cancer Research UK, London, United Kingdom), phospho-PRK (p-PRK; 2611; Cell Signaling, Beverly, MA), PAK4 (3242; Cell Signaling, Beverly, MA), p114RhoGEF (EB06163; Everest Biotech Ramona, CA), LKB1 (27D10; Cell Signaling, Beverly, MA), and RhoA (sc-418; Santa Cruz, Santa Cruz, CA).

Techniques: Dominant Negative Mutation, Stable Transfection, Expressing, Staining

Journal: Molecular and Cellular Biology

Article Title: LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

doi: 10.1128/MCB.00154-13

Figure Lengend Snippet: PLA to visualize the interaction between LKB1 with p114RhoGEF in 16HBE cells. (A) 16HBE cells stably expressing HA-tagged p114RhoGEF were fixed and stained with a mixture of mouse (Ms) anti-HA and rabbit (Rb) anti-LKB1 (A1), with anti-HA alone (A2), or with anti-LKB1 alone (A3). PLA signals were visualized by addition of a mixture of anti-mouse and anti-rabbit antibody reagents provided by the company (see Materials and Methods). Bar, 20 μm. (B) 16HBE cells stably expressing HA-tagged p114RhoGEF and either myc-LKB1WT (B1) or myc-LKB1SL26 (B2) were fixed and stained with a mixture of mouse anti-HA and rabbit anti-myc antibodies (left). PLA signals were visualized by addition of a mixture of anti-mouse and anti-rabbit antibody reagents provided by the company (see Materials and Methods). (C) Quantification of PLA signals. Data represent the number of positive dots per cell. Six random nonoverlapping images were taken, and signals were quantified using Volocity image analysis software. Error bars, SEMs. **, P < 0.01. (D) Fluorescent images of stable cells expressing HA-p114RhoGEF and myc-mLKB1WT stained with anti-HA antibody (left) and anti-myc antibody (right). Bar, 20 μm. (E) Fluorescent images of stable cells expressing HA-p114RhoGEF and myc-mLKB1SL26 stained with anti-HA antibody (left) and anti-myc antibody (right). (F) Lysates from 16HBE cells stably expressing HA-tagged p114RhoGEF and control cells were examined on Western blots with anti-p114RhoGEF or anti-LKB1 antibodies.

Article Snippet: Primary antibodies used were ZO-1 (clone 1A12; 339100; Invitrogen, Carlsbad, CA), ZO-1 (617300; Invitrogen, Carlsbad, CA) E-cadherin (clone ECCD-2; 131900; Invitrogen, Carlsbad, CA), E-cadherin (clone 34; 610404; BD Transduction, Lexington, KY), α-tubulin (clone YL1/2; AbD Serotec, Raleigh, NC), hemagglutinin (HA; clone 3F10; Roche, Indianapolis, IN), green fluorescent protein (GFP; rabbit polyclonal; Invitrogen, Carlsbad, CA), myc (clone 9E10; Cancer Research UK, London, United Kingdom), phospho-PRK (p-PRK; 2611; Cell Signaling, Beverly, MA), PAK4 (3242; Cell Signaling, Beverly, MA), p114RhoGEF (EB06163; Everest Biotech Ramona, CA), LKB1 (27D10; Cell Signaling, Beverly, MA), and RhoA (sc-418; Santa Cruz, Santa Cruz, CA).

Techniques: Stable Transfection, Expressing, Staining, Software, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

doi: 10.1128/MCB.00154-13

Figure Lengend Snippet: p114RhoGEF is required for LKB1-dependent RhoA activation. (Left) 16HBE cells stably expressing HA-tagged STRAD cells were transfected with siRNA control (siCon) or siRNA oligonucleotide targeting p114RhoGEF. Three days later, cells were infected with lentiviral vectors encoding LKB1WT, LKB1KD, and LKB1SL26. Twenty-four hours later, cells were harvested and the levels of activated Rho (Rho.GTP) were determined using a pulldown assay, followed by Western blotting. (Right) Quantification of the data from three experiments is shown. Error bars, SEMs. **, P < 0.01.

Article Snippet: Primary antibodies used were ZO-1 (clone 1A12; 339100; Invitrogen, Carlsbad, CA), ZO-1 (617300; Invitrogen, Carlsbad, CA) E-cadherin (clone ECCD-2; 131900; Invitrogen, Carlsbad, CA), E-cadherin (clone 34; 610404; BD Transduction, Lexington, KY), α-tubulin (clone YL1/2; AbD Serotec, Raleigh, NC), hemagglutinin (HA; clone 3F10; Roche, Indianapolis, IN), green fluorescent protein (GFP; rabbit polyclonal; Invitrogen, Carlsbad, CA), myc (clone 9E10; Cancer Research UK, London, United Kingdom), phospho-PRK (p-PRK; 2611; Cell Signaling, Beverly, MA), PAK4 (3242; Cell Signaling, Beverly, MA), p114RhoGEF (EB06163; Everest Biotech Ramona, CA), LKB1 (27D10; Cell Signaling, Beverly, MA), and RhoA (sc-418; Santa Cruz, Santa Cruz, CA).

Techniques: Activation Assay, Stable Transfection, Expressing, Transfection, Control, Infection, Western Blot

Journal: Molecular and Cellular Biology

Article Title: LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

doi: 10.1128/MCB.00154-13

Figure Lengend Snippet: LKB1 and p114RhoGEF regulate the maturation of primordial junctions to apical junctions. 16HBE cells infected with lentiviral vectors harboring control shRNA (A), shRNA2 targeting LKB1 (B), and shRNA3 targeting p114RhoGEF (C) were tested. At 5 days after infection, confluent monolayers were subjected to a calcium-switch assay. After calcium readdition, cells were fixed at 1 h and 6 h and stained with anti-ZO-1 (top) and anti-E-cadherin (bottom). Bar, 20 μm.

Article Snippet: Primary antibodies used were ZO-1 (clone 1A12; 339100; Invitrogen, Carlsbad, CA), ZO-1 (617300; Invitrogen, Carlsbad, CA) E-cadherin (clone ECCD-2; 131900; Invitrogen, Carlsbad, CA), E-cadherin (clone 34; 610404; BD Transduction, Lexington, KY), α-tubulin (clone YL1/2; AbD Serotec, Raleigh, NC), hemagglutinin (HA; clone 3F10; Roche, Indianapolis, IN), green fluorescent protein (GFP; rabbit polyclonal; Invitrogen, Carlsbad, CA), myc (clone 9E10; Cancer Research UK, London, United Kingdom), phospho-PRK (p-PRK; 2611; Cell Signaling, Beverly, MA), PAK4 (3242; Cell Signaling, Beverly, MA), p114RhoGEF (EB06163; Everest Biotech Ramona, CA), LKB1 (27D10; Cell Signaling, Beverly, MA), and RhoA (sc-418; Santa Cruz, Santa Cruz, CA).

Techniques: Infection, Control, shRNA, Staining

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a The UMAP plots of scRNA-seq analyses show the expression of Scgb1a1 , Foxj1 , or Stk11 in adult airway epithelial cells from adult lungs. b The pseudo-time trajectories show that the expression level of Scgb1a1 decreased during the ciliated cell differentiation process, whereas the expression levels of Foxj1 and Stk11 increased during the ciliated cell differentiation process. c The dot plot of Stk11 expression score in different cell types. The dot size represents the proportion of cells in a cluster that express the gene. The dot color corresponds to the average expression level of the gene. d E16.5 lungs were stained with antibodies against Acetylated-α-Tubulin and STK11. Scale bars: 25 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Expressing, Cell Differentiation, Staining

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a E16.5 lungs were stained with antibodies against FOXJ1 and SCGB1A1. b The proportion of FOXJ1 + cells (Left, n=3) and SCGB1A1 + cells (Right, n=4) in the intrapulmonary airways of E16.5 lungs. c Pregnant mice carrying Sox2 -CreER; Rosa26 -mTmG; Stk11 F/+ (Control) and Sox2 -CreER; Rosa26 -mTmG; Stk11 F/F ( Sox2 - Stk11 ) embryos were treated with tamoxifen at E13.5 and analyzed at E16.5. d E16.5 lungs were stained with antibodies against GFP, FOXJ1, and SCGB1A1. The yellow arrowheads indicate cells expressing both GFP and FOXJ1. e The proportion of FOXJ1 + GFP + cells to GFP + cells (Left) (Control, n=6; Sox2 - Stk11 , n=5) and SCGB1A1 + GFP + cells to GFP + cells (Right) (n=3) in the intrapulmonary airways of E16.5 lungs. f 10 weeks-old mice were treated with three doses of tamoxifen and analyzed after 30 days. g Lungs were stained with antibodies against GFP, FOXJ1, and SCGB1A1 at day 30. The yellow arrowheads indicate cells expressing both GFP and FOXJ1. h The proportion of FOXJ1 + GFP + cells to GFP + cells at day 30 (n=4). *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: a , d : 25 μm, g : 20 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Staining, Control, Expressing

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a Heat map of the expression values of genes expressed differentially between E16.5 Shh - Stk11 lungs and control lungs. b Fold change of relative mRNA expression of airway epithelial cell genes in E16.5 Shh - Stk11 lungs compared to Control lungs (n=3). c Fold change of relative mRNA expression of cell-cycle genes in E16.5 Shh - Stk11 lungs compared to Control lungs (n=3). d Pregnant females were oral gavaged daily with a CDK4/6 inhibitor (PD0332991) each day from E12.5 to E14.5. Lungs were analyzed at E16.5. e Cell proliferation was analyzed by antibody staining against Ki67 and E-cadherin (E-cad). f The proportion of Ki67 + cells in the intrapulmonary airways of E16.5 lungs (vehicle Control, n=5; vehicle Shh - Stk11 , n=3; PD0332991 Control, n=7; PD0332991 Shh - Stk11 , n=8). g Immunofluorescence staining with antibodies against FOXJ1 and SCGB1A1 in lungs after either vehicle or PD0332991 treatment. h The proportion of FOXJ1 + cells (vehicle Control, n=3; vehicle Shh - Stk11 , n=4; PD0332991 Control, n=3; PD0332991 Shh - Stk11 , n=5) and the proportion of SCGB1A1 + cells (n=3) in the intrapulmonary airways of E16.5 lungs after either vehicle or PD0332991 treatment. i 10 week-old mice were treated with three doses of tamoxifen. Mice were treated with PD0332991 or vehicle every other day via oral gavage and fed with water containing IdU for 30 days before collecting lungs for analysis. j Immunofluorescence staining with antibodies against IdU and GFP in lungs after either vehicle or PD0332991 treatment for 30 days. k The proportion of IdU + cells in the GFP labeled intrapulmonary airway epithelium (n=3). l Immunofluorescence staining with antibodies against FOXJ1, GFP, and SCGB1A1 in lungs after either vehicle or PD0332991 treatment for 30 days. The yellow arrows indicate cells expressing both GFP and FOXJ1. m The proportion of FOXJ1 + GFP + cells to GFP + cells in lungs after either vehicle or PD0332991 treatment for 30 days (n=3). *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: e , g : 11 μm; j , l : 20 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Expressing, Control, Staining, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a A schematic illustration showing the air-liquid interphase trachea culture system of this study. Adenovirus (Ad)-GFP, STK11 WT , or STK11 KD particles were added into the medium at day 0. The tracheal rudiments were cultured with different adenovirus for 5 days. b - c The proportion of Ki67 + cells in the epithelium of cultured tracheal rudiments at day 5 (c) were quantified by Ki67 immunostaining (b) (Ad-GFP Control, n=5; Ad-GFP Shh - Stk11 , n=6; Ad-STK11 WT Control, n=4; Ad-STK11 WT Shh - Stk11 , n=5; Ad-STK11 KD Control, n=4; Ad-STK11 KD Shh - Stk11 , n=6). d - f Cultured tracheal rudiments were stained with antibodies against FOXJ1 and GFP at day 5. g The proportion of FOXJ1 + cells in the epithelium of cultured tracheal rudiments at day 5 (Ad-GFP Control, n=3; Ad-GFP Shh - Stk11 , n=3; Ad-STK11 WT Control, n=3; Ad-STK11 WT Shh - Stk11 , n=3; Ad-STK11 KD Control, n=5; Ad-STK11 KD Shh - Stk11 , n=3). ns, not significant; *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: 11 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Cell Culture, Immunostaining, Control, Staining

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a Immunofluorescence staining with antibodies against p-MARK3 and Acetylated-α-Tubulin in E16.5 lungs. Yellow arrows indicate ciliated cells expressing both Acetylated-α-Tubulin and p-MARK3. b E13.5 tracheal rudiments were cultured with Ad-GFP, Ad-MARK3 WT , or Ad-MARK3 CA viruses for 5 days. c - d Cultured tracheal rudiments were stained with antibodies against FOXJ1 at day 5 (c). The proportion of FOXJ1 + ciliated cells were quantified (Ad-GFP Control, n=3; Ad-GFP Shh - Stk11 , n=3; Ad-MARK3 WT Control, n=3; Ad-MARK3 WT Shh - Stk11 , n=4; Ad-MARK3 CA Control, n=4; Ad-MARK3 CA Shh - Stk11 , n=3) (d). e E16.5 lungs were stained with antibodies against p-ERK1/2 and SOX2. f Pregnant female mice were oral gavaged with p-ERK1/2 inhibitor PD0325901 or vehicle at E13.5. Lungs were analyzed at E16.5. g - h Immunofluorescence staining with antibodies against FOXJ1 in the vehicle-treated or PD0325901 treated lungs (g). The proportion of FOXJ1 + cells (n=3) and the proportion of SCGB1A1 + cells (n=3) in the intrapulmonary airways of E16.5 lungs were quantified (h). i E13.5 tracheal rudiments were cultured with vehicle or PD0325901 for 5 days. j - k Cultured tracheal rudiments were stained with antibodies against FOXJ1 at day 5 (j). The proportion of FOXJ1 + ciliated cells were quantified (vehicle Control, n=4; vehicle Shh - Stk11 , n=3; PD0325901 Control, n=3; PD0325901 Shh - Stk11 , n=5) (k). l A schematic illustration showing that STK11-mediated signaling cascade accounts for the normal program of ciliated cell differentiation in airways. ns, not significant; *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: 11 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Immunofluorescence, Staining, Expressing, Cell Culture, Control, Cell Differentiation

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a Immunofluorescence staining with antibodies against FOXJ1 and SCGB1A1 in the E16.5 lungs. White arrowheads indicate cells that do not express SCGB1A1 and FOXJ1. b The proportion of SCGB1A1 - FOXJ1 - cells (Left), and the proportion of FOXJ1 + cells (Right) in the intrapulmonary airways of E16.5 lungs (n=3). c Immunofluorescence staining with antibodies against FOXJ1 and SCGB1A1 in the E18.5 lungs. White arrowheads indicate cells that do not express SCGB1A1 and FOXJ1. d The proportion of SCGB1A1 - FOXJ1 - cells (Left), and the proportion of FOXJ1 + cells (Right) in the intrapulmonary airways of E18.5 lungs (n=3). e Immunofluorescence staining with antibodies against Acetylated-α-tubulin and JAG1 (JAGGED1) in E16.5 lungs. Yellow arrowheads indicate the Acetylated-α-tubulin - cells that express JAG1. f The proportion of JAG1 + cells in the intrapulmonary airways of E16.5 lungs (Control, n=4; Shh - Stk11 , n=4). g Immunofluorescence staining with antibodies against Acetylated-α-tubulin and JAG1 in E18.5 lungs. Yellow arrowheads indicate the Acetylated-α-tubulin - cells that express JAG1. h The proportion of JAG1 + cells in the intrapulmonary airways of E18.5 lungs (Control, n=4; Shh - Stk11 , n=5). ns, not significant; *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: 11 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Immunofluorescence, Staining, Control

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 1 Reduced m6A modification is associated with the aggressiveness of lung cancer with KRAS mutation and LKB1 loss. A, B Representation and quantification of LKB1 expression and m6A level in lung cancer patients. Data were mean ± SD. Bar = 50 µm. C Spearman correlation analysis of m6A level with the clinical characters of KRAS mutant or wild-type lung cancer patients. D The m6A level in lung cancer patients with TTF1 positive or negative. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. **P < 0.01, ***P < 0.001 by Student’s t-test. KRAS Mut; LKB1 Loss (KL); KRAS Mut; LKB1 Wt (K); KRAS Wt; LKB1 Loss (L) and KRAS Wt; LKB1 Wt (WT).

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Mutagenesis, Expressing

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 2 LKB1 loss upregulated ALKBH5 responses to m6A reduction in KRAS mutant lung cancer. A, B Quantification and representation of LKB1 and m6A modulators in KL relative to K lung cancer patients. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. C Spearman correlation analysis of ALKBH5 expression with the KRAS mutant lung cancer pathological characters. D Venn diagram showing the differentially expressed genes (DEGs) of LKB1, m6A modulators, and readers between KL and K lung cancer. E Kaplan–Meier survival curve of patients with high and low ALKBH5 mRNA expression from the TCGA dataset. F Global m6A level regulation by LKB1 in lung cancer cell lines. G Western blot showing the LKB1 and ALKBH5 protein expression. H Co-immunofluorescence staining for LKB1 and ALKBH5 in A549 cells. Arrows show LKB1 positive and ALKBH5 negative cells. I LKB1 and m6A co-staining by LKB1 and/or ALKBH5 overexpression in A549 cells. Arrows show cells of LKB1 positive and m6A with strong (Second panel) or weak (Forth panel) intensity. Data were mean ± SD in F (n = 5). *P < 0.05 (Student’s t-test). Si-CN, SiRNA-A; OE-CN, OE-c-Flag pcDNA3; OE, overexpression. KRAS Mut; LKB1 Loss (KL); KRAS Mut; LKB1 Wt (K).

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Mutagenesis, Expressing, Western Blot, Staining, Over Expression

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 3 ALKBH5 increases the lung cancer cell proliferation and migration. A Western blot analysis shown that LKB1 and ALKBH5 were successfully knocked down in H1792 cells and overexpressed in A549 cells. B, C The measurement of global m6A by ELISA assay in H1792 (B) or A549 (C) cells, respectively. The colony formation ability (D) and cell migration (E) were reduced at both of basal level and LKB1 silenced level by ALKBH5 knockdown in H1792 cells, respectively. The reversible effect was found at both of basal level and LKB1 overexpressed level by ALKBH5 transfected in A549 cells using colony formation (F) and transwell assay (G). Data were mean ± SD (n = 5) and were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparison test for B–G. *P < 0.05. Bar = 5 mm in D and F; Bar = 50 µm in E and G.

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Transfection, Transwell Assay, Comparison

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 4 DNA hypermethylation upregulates ALKBH5 in KL lung cancer cells. A 5mC DNA methylation was detected by ELISA assay. Data as mean ± SD (n = 5), *P < 0.05 (Student’s t-test). B–D Representation and quantification of ALKBH5 expression at protein (B, C) and mRNA levels (D) in response to 5-aza treatment in A549 and H1792 cells for 72 h. Error bars, SD (n = 4 for WB; n = 5 for qRT-PCR), *P < 0.05 as compared their corresponding controls (2-way ANOVA with Bonferroni multiple comparison post hoc test). E CTCF peak located in the CpGs Island of human ALKBH5 gene promoter. Bottom panel: the Methprimer histogram of CpG islands (CpGI, red box) and CpG dinucleotides (red vertical lines) in the regulatory region of ALKBH5. F MeDIP assay showing the decrease of 5mC enrichment on the ALKBH5 promoter containing CTCF motif fragment by treated with 5-aza and LKB1 overexpression. G, H Representative bisulfite sequencing of four clones (G) and quantification of DNA methylation (H) of ALKBH5 promoter containing CTCF motif. n = 4 in F and H, mean ± SD, * P < 0.05 vs. A549 DMSO by one-way ANOVA followed by Tukey’s test in F and H. Si- CN, Si-RNA-A. OE-CN, OE-c-Flag pcDNA3. OE overexpression.

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Comparison, Methylated DNA Immunoprecipitation, Over Expression, Sequencing, Clone Assay

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 5 Suppressor-CTCF is required for ALKBH5 downregulation by LKB1. A–C Representative images and quantification of CTCF and ALKBH5 protein expression by WB and qRT-PCR in A549 cells with LKB1 overexpression and/or CTCF knockdown for 48 h. Error bars, SD (n = 4 for WB; n = 5 for qRT-PCR), *P < 0.05 vs. Si-CN or OE-CN (one-way ANOVA with Bonferroni multiple comparison post hoc test). D Luciferase reporter assay of A549 cells transfected with pGL3-basic constructs containing serial LKBH5 promoters or deletion of CTCF peak fragment. n = 8/group, mean ± SD. * P < 0.05 by one-way ANOVA followed by Tukey’s test. E Reduction of ALKBH5 Luc:-1168 bp wild-type (WT) construct activities by treatment of 5-aza or LKB1 overexpression in A549 cells, but not for ALKBH5 Luc:-1168 bp deletion (Del). Data as mean ± SD (n = 5), *P < 0.05 (Student’s t-test). F ChIP-qPCR showing that the ALKBH5-CTCF peak region was occupied by the suppressor of CTCF and activators of histone modulators in A549 cells. G–J ChIP- qPCR analyses of 5-aza treated or LKB1 overexpressed A549 cells. n = 5/group, mean ± SD. *P < 0.05 vs. IgG in F, vs. A549-DMSO in G–J by one-way ANOVA followed by Tukey’s test.

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Comparison, Luciferase, Reporter Assay, Transfection, Construct, ChIP-qPCR

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 6 ALKBH5 erases m6A modification on SOX2, SMAD7, and MYC mRNA to increase their stability and expression. A, B Representative western blotting and quantification of SOX2, SMAD7, MYC, and ALKBH5 protein. Data as mean ± SD (n = 4). C Mutation of m6A site or ALKBH5 overexpression released the SOX2, SMAD7, and MYC gene posttranscriptional repression by LKB1 in A549 cells (n = 4). D m6A-RIP analysis demonstrated that SOX2, SMAD7, and MYC were subjected to ALKBH5-mediated m6A modifications (n = 5). E YTHDF2-RIP-qPCR shown that YTHDF2 could occupy m6A sites of SOX2, SMAD7, and MYC, which was mediated by ALKBH5 (n = 4). F qRT-PCR was performed to indicate that knockdown of YTHDF2 significantly upregulated SOX2, SMAD7, and MYC gene mRNA expression at basal level, LKB1 overexpressed or ALKBH5 silenced A549 cells (n = 6). Data as mean ± SD. *P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for B–F. Si-CN, Si-RNA-A. OE-CN, OE-c-Flag pcDNA3. OE overexpression.

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Expressing, Western Blot, Mutagenesis, Over Expression, Quantitative RT-PCR, Knockdown, Comparison

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 7 Loss of LKB1 clinically associates with the increase of ALKBH5 and decrease of m6A modification on SOX2, SMAD7, and MYC genes. A Representative immunostaining images and quantification of 5mC DNA in lung cancer patients with KW and KM. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. B, C High levels of ALKBH5 DNA methylation and mRNA expression in cases of LKB1 loss compared with that of LKB1 positive expression in KM patients. D–F The correlation of ALKBH5 protein with the global m6A modification (D), m6A levels of SOX2, SMAD7, and MYC genes (E, F) in KM and KW groups. G–I Negative correlations of the m6A enrichment with mRNA expressions of SOX2, SMAD7, and MYC in KM group. J Representative immunostaining images and quantification of SOX2, SMAD7, and MYC in KM group with LKB1 loss or not. K The positive correlations of ALKBH5 with SOX2, SMAD7, and MYC protein expression in KM group. Data as mean ± SD. KRAS mutation (KM). KRAS wild-type (KW). *P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for A, B, and C. Student’s t-test for J.

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Immunostaining, DNA Methylation Assay, Expressing, Mutagenesis, Comparison

Journal: Cell death & disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Fig. 8 Proposed model for LKB1-mediated m6A modification in KRAS-mutated lung cancer progression. Loss of LKB1-induced DNA hypermethylation, which prevents CTCF binding on the ALKBH5 gene promoter, maintains ALKBH5 expression, and further represses global RNA methylation. Oncogenic SMAD7, SOX2, and MYC are crucial targets of m6A meditated by LKB1 deficiency, and are involved in KRAS mutation lung cancer progression.

Article Snippet: RNAi and protein overexpression transfection To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), YTHDF2 (sc-78661), and CTCF (sc35124) from Santa Cruz and transiently transfected these si-RNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Binding Assay, Expressing, Methylation, Mutagenesis