the expression and distribution of MICAL2 negative

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 In the present study, the expression and distribution of MICAL2 negative; 1, weak; 2, moderate; and 3, strong. PP was defined as fol- wereanalyzedinLUADtissuearrays,aswellasitsrelationshipwiththe lows: 0, negative; 1, 1–10% positive cells; 2, 11–50% positive cells; 3, clinicopathological characteristics of LUAD patients. Cell experiments 51–80% positive cells; and 4, more than 80% positive cells. Slices revealed tumor-promoting effects (growth, invasion, and EMT) invol- scoringnolessthan4pointswereclassifiedasimmunoreactive[24,25]. ving MICAL2 pathways in LUAD cell lines. Furthermore, our findings identifiedMICAL2asanucleocytoplasmicshuttlingprotein.Finally,the 2.4. Plasmids and constructs regulation and cytological function of MICAL2 nuclear export were investigated both in vitro and in vivo. The shMICAL2s were synthesized and purified by Hanyin Biotech Co. (Shanghai, China) and integrated into pHY-316 vector (Hanyin 2. Materials and methods Biotech), targeting MICAL2 with the following primers: 5′-CCTCCAGG CCTTCAACATT-3′,5′-CCAAAGCCCTGTGGTACAA-3′,5′-GCGCACTGC 2.1. Tissue specimens CATTGAACTT-3’. Constructs containing human MICAL2-Flag fusions were integrated into pHY-023 (Hanyin Biotech) and GV141 vectors Paired human tumor/normal tissue samples were collected from (Genechem, Shanghai, China). Coding sequences of truncated mutants −ΔC June 2004 through September 2015 (first collection, n = 126), with of MICAL2 were obtained using specific primers: MICAL2 :5′-ACG follow-up for 4–5 years after surgery. The second round (n = 74) of GGCCCTCATGGGGGAAAACGAGGATGAGAAGCAG-3′ and 5′-CCAAGC −ΔN LUAD tissue samples were collected from April 2014 to January 2018. TTGGCTGCTTCATGAGTGAGGGGTT-3’; MICAL2 :5′-ACGGGCCCT Pathologicaldiagnosiswasrenderedbyexperiencedpathologists.Upon CATGAACAAACGGAGACGGAAGGGCTTCA-3′ and 5′-TTAAGCTTGGG −ΔNΔC the approval of the relevant ethics committee of Xiangya Hospital of CCAAGAAGTGGGTGTAGCACTGGAAG-3’; MICAL2 :5′-ACGGGC Central South University and written consent from patients, samples CCTCATGCTGGGAGTTGAAATCCATGTG-3′ and 5′-CCAAGCTTGGCTG were collected in the operating room, formalinfixed, and embedded in CTTCATGAGTGAGGGGTT-3’. The pENTER-Myosin9-Flag/His plasmid paraffin. The paired normal tissue wascollected more than 10cmfrom was purchased from Vigene Bioscience (Shandong, China). Both the tumor edge. Two hundred pairs of LUAD samples were ultimately MICAL2-HAandmyosin9-HAweresubclonedfromFlag-taggedplasmid appropriated for the study, from patients with an average age of 57 into GV366 plasmid (Genechem) using XbaI (TaKaRa, Shiga, Japan) (ranging from 20 to 84 years): 87 were from female patients and 113 and NotI (TaKaRa). All gene cloning was verified by sequencing 3x FLAG. The werefrommalepatients;81containedlymphnodemetastases;86cases MYH9 siRNAs were synthesized by Guangzhou RiboBio Co. were in stage I, 46 were in stage II, 68 were no less than stage III. We (Guangzhou, China) targeting MYH9 with the following primers: excluded sub-standard cases such as patients who had undergone pre- 5′-GCATCAACTTTGATGTCAA-3′,5′-GCAACACGGAGCTGATCAA-3’. operative radiotherapy or chemotherapy, as well as those with lung squamous cell carcinoma or benign tumors. 2.5. Western blotting and immunoprecipitation 2.2. Cell lines and culture Western blotting (WB) was performed according to standard pro- tocols using 10% Bis-Tris gels. Membranes were incubated in primary PC9, A549, BEAS-2B, LTEP-α-1, HeLa, and HEK293T cells were [rabbit anti-MICAL2, 1:1000 (Abnova); rabbit anti-ZO1, authenticate###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-2.png####dviashorttandemrepeat(STR)analysis.Allcelllineswere 1:1000 (Abcam, Cambridge, UK); rabbit anti-E cadherin, 1:200 passagedlessthan10timesaftertheirinitialrevivalfromfrozenstocks. (Abcam); rabbit anti-β catenin, 1:3000 (Abcam); rabbit anti-vimentin, The cells were cultured in RPMI-1640 or DMEM (for HEK293T cells 1:3000 (Abcam); rabbit anti-MYH9, 1:2000 (Proteintech); rabbit anti- only) growth medium (Gibco/Thermo Fisher Scientific, Waltham, MA, AKT, 1:3000 (Cell Signaling Technology, CST, Danvers, MA, USA); USA) with 10% fetal bovine serum (FBS; Gibco).

 

 The cell culture in- rabbit anti-AKT(Ser473), 1:3000 (CST); rabbit anti-GSK-3β, 1:1000 cubator was set at 37 °C with 5% CO . (CST);rabbitanti-GSK-3β(Ser9),1:1000(CST);mouseanti-Flag,1:4000 2 (MilliporeSigma, Burlington, MA, USA); mouse anti-HA, 1:500 (Santa 2.3. Immunohistochemistry (IHC) Cruz Biotechnology, Dallas, TX, USA)] overnight at 4 °C. Immunoprecipitationwasperformedasdescribedpreviously[26]using Sample slides with 3-μm thick tissue sections were deparaffined in EZview™ Red ANTI-FLAG® M2 Affinity Gel (MilliporeSigma) overnight xylene, rehydrated in descending concentrations of ethanol and PBS, at 4 °C. and subjected to epitope retrieval by citrate buffer (pH 6.0). Endogenous peroxidases were blocked by 3% hydrogen peroxide in 2.6. Immunofluorescence methanol. Slides were then incubated overnight in the indicated anti- bodies[rabbitanti-MICAL2(1:50;Abnova,Taipei,Taiwan);rabbitanti- Primary  for immunofluorescence (IF) were sourced as Flag (1:200; Proteintech, Rosemont, IL, USA); rabbit anti-myosin-9 follows: anti-MICAL2 from Abnova; monoclonal anti-Flag M2 from (1:400;Proteintech)]at4°Cfollowedby15minat20°C,theninbiotin- Sigma-Aldrich (St. Louis, MO, USA). Treatment: PC-9 cells or A549- conjugated goat anti-rabbit antibody (1:200; Vector® Laboratories, MICAL2 cells were treated with or without 10 nM LMB (leptomycin B; Burlingame, CA, USA) for 1 h at 20 °C, in ABC HRP reagent (Vector®) CST) or 10 μM ML7 (myosin-9 inhibitor; Selleck Chemicals, Houston, for 1 h at 20 °C and DAB Peroxidase Substrate Kit (Vector®) for ap- TX, USA) overnight, then harvested for detection of MICAL2 localiza- proximately 3 min in the dark. Positive (hepatic tissue) and negative tion by indirect immunofluorescence using anti-MICAL2 antibody or controls were assayed at the same time. Results were obtained on an anti-Flag antibody followed by Alexa Fluor®-conjugated secondary an- Aperio digital pathology slide scanner (Leica Biosystems, Nußloch, tibody (green; Abcam). They were then scanned with a confocal ima- Germany) andaLeica DM6B microscope (LeicaMicrosystems, Wetzlar, ging system (Leica SP8) orfluorescence microscope (Leica DM LB2). Germany). 

 

Staining was evaluated based on both mean integrated op- tical density (MI = IOD/AREA) [21 Concanamycin A,22] using the Image-Pro Plus 6.0 2.7. Cell proliferation assays application (Media Cybernetics, Inc., Rockville, MD, USA) for overall stainingandabinarysystemfornuclearorcytoplasmicstainingtermed CellproliferationwasdetectedusingtheCCK-8(CellCountingKit-8; the immunoreactive score (IRS = SI < staining intensity> × PP < Dojindo,Kumamoto, Japan)methodandaplatecloneformation assay Cell Counting Kit-8 chemicals. percentage of positive cells>). Kaplan-Meier analysis was conducted For the CCK-8 assay, 2000 cells per well were seeded in 96-well plates using the average MI value cut-off which divided slices into high ex- and absorbance was read at 450 nm at 6, 24, 48, and 72–96 h, re- pressionandlowexpressiongroups[23].SIwasclassifiedasfollows:0, spectively,afterbeingincubatedwithCCK-8for2h.Fortheplateclone

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