Three independent IP-MS assays were performed in the PC-9 cell line using anti-MICAL2 antibody for detecting potential endogenously MICAL2-binding protein.

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 Fig. 3. MICAL2 shuttled between the cytoplasm and the nucleus: A. MICAL2 accumulated in the nucleus in PC-9 after treatment with LMB. The bottom panelshowsnuclearandcytoplasmicpositiveratesofendogenousMICAL2:thenuclearpositiveratedramaticallyincreasedafterLMBtreatment.B.Bothnuclearand cytoplasmiclocalizationofexogenousMICAL2inA549-MICAL2cellswithoutLMBtreatment Thymoquinone,comparedtoastrongnuclearlocalizationwhentreatedwithLMB.The bottom panel shows that the MICAL2 cytoplasmic positive rate was significantly decreased after LMB treatment. C. MICAL2 locations in HeLa cells transfected with −ΔC WT and three truncated mutants of MICAL2. The right panel reveals a significant decrease in MICAL2 cytoplasmic positive rate in both MICAL2 and −ΔCΔN −ΔN MICAL2 transfected cells but not MICAL2 transfected cells compared with MICAL2-WT transfected cells. revealedinthecellexperiment,theirexpressionlevelsweredetermined LUAD metastasis. in LUAD tissues. TCGA data showed that the mRNA levels of MICAL2 and MYH9 were in accordance (n = 706, r = 0.5104, P < 0.0001) 4. Discussion (Fig. S2A). Then IHC of LUAD tissue arrays was performed for further exploration (Fig. S2B). The protein levels of total and cytoplasmic Lung cancer is the leading cause of cancer-related mortality MICAL2 were positively corelated with myosin-9. In addition, the worldwide [35]. Metastasis is the most critical parameter determining subcellular location of MICAL2 and myosin-9 were also in accordance, survivalinlungcancercases[36].Therefore,theidentificationofnovel whichformsthebasisoftheirbindingcapacityinLUADs.Furthermore, prognostic biomarkers and treatment targets of metastatic lung cancer only double-positive staining of MICAL2 and myosin-9 was positively isurgentforpatients.Inthecurrentstudy,weidentifiedanovelroleof associated with lymph node metastasis (Table S2). Overall, our results subcellular MICAL2 in the prognosis of LUAD as well as its nucleocy- suggest that MICAL2 and myosin-9 might synergistically contribute to toplasmic shuttling and tumor-promoting action in LUAD cell lines. 

81
W. Zhou, et al. Cancer Letters 483 (2020) 75–86 Fig.4. Myosin-9boundtoMICAL2anditsinhibitionorknockdownnegativelyaffectedMICAL2nuclearexport:A.LC-MS/MSspectrumofthetrypticpeptide LQQELDDLLVDLDHQR for myosin-9. Three independent IP-MS assays were performed in the PC-9 cell line using anti-MICAL2 antibody for detecting potential endogenously MICAL2-binding protein. B. IP-PRM (Parallel reaction monitoring) data for the sequence LPSPDPAASSSPSTVDSASPAR for MICAL2. The chromato- graphic traces show the overlapping transitions and retention time of the endogenous peptide of MICAL2. Three independent IP-PRM assays were performed in the PC-9 cell line using anti-myosin-9 antibody for exclusively detecting MICAL2-binding 3xFLAG for sale. C. HEK293T cells were transfected with myosin-9-Flag and MICAL2-HA plasmid (left) or myosin-9-HA and MICAL2-Flag plasmid with control siRNA or si-myosin-9 (right) Iberiotoxin. Immunoprecipitations were performed using an Anti-Flag M2 AffinityGel.Theimmunoprecipitationproducts(IP)andtotallysis(TL)weredetectedviawesternblotting(WB),whichshowedthatMICAL2andmyosin-9canbind to each other and the use of si-myosin-9 can inhibit the expression of myosin-9 as well as its binding capacity to MICAL2. D. Subcellular localizations of exogenous MICAL2 in A549-MICAL2 cells treated with or without ML7 or si-myosin-9. The positive rate of cytoplasmic MICAL2 decreased significantly after treatment. MICAL2 levels were overexpressed in cancers such as prostate promoter via various signal pathways [9,17]. 

However, MICAL2 had cancer [37], gastric carcinoma [18], brea###http://www.glpbio.com/simage/GA11366-H-D-Leu-Thr-Arg-pNA-acetate-salt-1.png####st cancer [14], and colorectal yet to be explored in LUAD cell lines. In our study, MICAL2 was found cancer [15]. However, the prognostic and predictive value of MICAL2 to be necessary for cell proliferation, migration, invasion, and EMT expression levels for LUADs remains unclear. In the current study, changes in LUAD cell lines via the AKT and myosin-9 pathways. Given MICAL2 was overexpressed and nuclear exported in LUADs compared that MICAL2 activates SRF signaling through redox-dependent depo- to the adjacent normal lung tissues (Fig. 1). Furthermore, our results lymerization of nuclear actin [16] and SRF binds to the CArG box lo- indicated that both overall levels and cytoplasmic levels of MICAL2 cated at the MYH9 promoter [38], we propose that MICAL2 promotes contributed to LUAD lymphatic metastasis and shorter OS (Table 1). 

MYH9 expression. Surprisingly, MYH9 expression levels are corelated −ΔC These significant differences in MICAL2 between LUAD tissues and withMICAL2expressionlevelsaswellasMICAL2 inLUADcelllines normal lung tissues along with its predictive value in LUAD patients (Figs. 2G and 5A), which is confirmed in TCGA data and tissue arrays make MICAL2 an ideal candidate as a cancer biomarker. (Fig. S2). Moreover, myosin-9 was identified as a tumor promoter in To assess the oncogenic activity of MICAL2, the role of MICAL2 in NSLCs (non-small cell lung cancer); it was overexpressed and sig- LUAD cell lines was explored. MICAL2 has been described as a tumor nificantly corelated with lymphatic invasion and poor prognosis [39].

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