IHCwasperformed Proteintech). Sequencing-grade trypsin (Promega, Madison, WI, USA)

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 formationassay,600cellswereseededin10cmdishes(or200cellsina Cruz, Xena browser (UCSC Xena, https://xenabrowser.net/). Pearson\'s 6-well plate) and stained with 0.05% crystal violet after 2–3 weeks of correlation was computed among features using log-transformed ex- cell culture. pression values. 2.8. Cell migration and cell invasion assays 2.13. Statistical analysis 4 For the cell migration assay, 3 × 10 were seeded into the Statistical differences between two groups were tested using upper chamber of a collagen-precoated transwell filter (8 μm pores; Student\'s t-test. Comparisons among three or more groups were con- Costar, Acton, MA, USA) with RPMI-1640 medium without FBS. RPMI- ducted using one-way ANOVA. The chi-squared test was used to eval- 1640 medium containing 15% FBS was added to the lower chamber. uate the significance of correlation. Survival analysis was performed The plates were maintained in the cell culture incubator overnight. using the Kaplan–Meier method. P-values<0.05 were considered sta- After being fixed by methanol and stained with 0.05% crystal violet, tisticallysignificant(*,P < 0.05;**,P < 0.01;***,P < 0.001;****, fourdifferentareas(up,down,left,andright)oftheplateswereimaged P < 0.0001). Correlation between MICAL2 and myosin-9 in LUAD using an optical microscope (Leica DMi1). For the cell invasion assay, tissue arrays was analyzed using nonparametric Spearman correlation. 4 5×10 cells per well were seeded into the upper chamber of a Error bars represent standard error of the mean. All calculations were Matrigel-precoated transwell filter (8 μm pores; Costar) or a transwell performed with SPSS Version 20.0 (IBM Corp., Armonk, NY, USA) or filter (8 μm pores; BD, Franklin Lakes, NJ, USA) coated with 100 μlof GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA). 300 μl/ml Matrigel (BD). 3. Results 2.9. Immunoprecipitation mass spectrometry (IP-MS) 3.1. MICAL2 was overexpressed and cytoplasm-enriched in LUADs PC-9 cell lysis solution was assessed to investigate the binding compared to normal lung tissues protein of endogenic MICAL2. The antibody used for im- munoprecipitation was rabbit polyclonal anti-MICAL2 (1:100; ToassessMICAL2expressionpatternsinLUADs,IHCwasperformed Proteintech). Sequencing-grade trypsin (Promega, Madison, WI, USA) onhumanLUADtissuearrayscontaining126individualLUADsandthe was used for tryptic digestion. One-half of the volume of each sample paired normal lung tissues. MICAL2 expression was detected solely in was separated and analyzed with an EASY-nLC™ 1200 nano-UPLC the nucleus of approximately 62% of normal lung tissues, while it was coupledtoaQExactive™massspectrometer(ThermoFinnigan/Thermo expressed in the nucleus and/or cytoplasm in approximately 84% of FisherScientific,SanJose,CA,USA).Separationwasperformedusinga LUADs (Fig. 1A). We then compared thetotal expression ofMICAL2 by reversed-phase column (100 μm, ID × 15 cm, Reprosil-Pur 120 C18- comparing the mean integrated optical density (MI) of MICAL2 using AQ, 1.9μm; Thermo Fisher Scientific). Data-dependent acquisition was Image-Pro Plus 6.0 software. The average MI for MICAL2 expression in performed in profile and positive modes on an Orbitrap analyzer LUADs was 0.106 ± 0.049, which was much higher than that in (Thermo Fisher Scientific). Potential MICAL2-binding proteins were n###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-3.png####ormallungtissues(0.037 ± 0.027; P < 0.0001;Fig.1A).Compared selected by intensity ratios>10 (MICAL2-Ab: IgG > 10). The false with the matched normal lung tissues, the expression levels of nuclear discoveryrate(FDR)wasanalyzedbyperformingaconcatenateddecoy MICAL2 in LUADs were dramatically decreased in 74 of 126 (58.7%) database search and the identified proteins were reported at caseswhilecytoplasmicMICAL2 increased in124of126(98.4%) cases FDR < 1%. (P < 0.0001; Fig. 1A). Together, MICAL2 was overexpressed and nu- clear exported in LUAD tissues.

 2.10. Immunoprecipitation parallel reaction monitoring (IP-PRM) 3.2. High cytoplasmic MICAL2 or total MICAL2 expression positively PC-9 cell lysis solution was used to test the binding interaction be- correlated with lymphatic metastasis and poor prognosis in LUADs tweenendogenousmyosin-9andMICAL2.Rabbitpolyclonalanti-MYH9 antibody (1:1000; Proteintech) was used for IP. After tryptic digestion, The different expression patterns of MICAL2 in LUADs and paired 2μgpeptidewasseparatedonanEASY-nLC™1200nano-UPLCcoupled normal lung tissues led us to further analyze the relationship between to the Q Exactive™ mass spectrometer system mentioned above clinicopathological characteristics and MICAL2 expression levels and (Thermo Finnigan) cck-8 ic50. Data-dependent acquisition was performed in subcellular location. As shown in Table 1 and Fig. 1B, both the overall profile and positive modes on the Orbitrap analyzer (Thermo Fisher expressionandthecytoplasmictranslocation ofMICAL2intheprimary Scientific). The collected PRM data were imported into Skyline for LUADs were positively associated with lymph node metastasis peptide matching [27]. (P < 0.05 and P < 0.0001, respectively) and advanced TNM stage (P < 0.05 and P < 0.01, respectively). In contrast, MICAL2 expres- 2.11. Tumor xenograft model sion levels were not associated with gender, age, tumor size, or differ- entiation (Table 1). Accordingly, high levels of total and cytoplasmic 6 For the in vivo tumor metastasis assay, 1 × 10 A549 stably MICAL2 expression were correlated with poor overall survival (OS) in −ΔC expressing MICAL2-Flag, MICAL2 -Flag or vector-CTRL were in- LUAD patients (Fig. 1C; P < 0.05). jectedintothetailveinsoffemaleBABL/cathymicnude mice(age4–5 weeks). 

After 5 weeks, all animals were sacrificed. The lung tissues 3.3. MICAL2 promoted LUAD cell proliferation, migration, invasion, and were excised and fixed in formalin. Hematoxylin and eosin (H&E) epithelial to mesenchymal transition staining was performed to detect metastatic focus and IHC to detect −ΔC MICAL2-Flag or MICAL2 -Flag expression levels. All animal experi- GiventhatMICAL2 wasoverexpressedintheLUAD tissues 3x FLAG stability, wenext ments were approved by the Central South University Animal Care investigated MICAL2 function in LUAD cell lines. Western blotting re- Commission. vealed that MICAL2 was highly expressed in PC-9, BEAS-2B, and HTB- 182cells,butnotinA549,H446,andLTEP-α-2cells(Fig.2A).Basedon 2.12. Analysis of LUAD data sets these results, knockdown of MICAL2 was performed in the MICAL2- high PC-9 cell line using three independent shRNAs. We confirmed RNAsequencingdatafromTheCancerGenomeAtlas(TCGA)LUAD significant knockdown in two—namely BCI-121, the PC-9_sh1 and PC-9_sh3 (706 samples) were obtained from the University of California, Santa cells (Fig. 2B and C).