Immunohisto chemical analysis of MICAL2 expression in LUADs and paired normal lung tissues

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 Fig. 1. Immunohistochemical analysis of MICAL2 expression in LUADs and paired normal lung tissues and its relationship to clinicopathological char- acteristics: A. MICAL2 expression levels are relatively lower in normal lung tissues and solely located in the nucleus, while it is highly expressed in LUADs and is mainlylocatedinthecytoplasm.TheMIofMICAL2inLUADsishighercomparedtopairednormallungtissues.Incontrasttopairednormallungtissues,mostLUADs have lower nuclear IRSs yet higher cytoplasmic IRSs. B. MICAL2 expression is higher in LUADs with lymphatic metastasis than in LUADs without lymphatic metastasis. Compared withnon-lymphatic-metastasis LUADs, lymphatic-metastasis LUADs have higher total and cytoplasmic MICAL2 expression levels, while there is no significant difference in nuclear MICAL2 levels. C. Total and cytoplasmic MICAL2 expression levels, but not nuclear MICAL2 expression levels, in LUAD are related to poor prognosis. proliferation, migration, and invasion of PC-9 (Fig. 2C–E). transition (MET) (Figs. 2F, 2C, S1). 

Consistent with this, knockdown of Knockdown of MICAL2 also induced significant morphological changes MICAL2 led to reduced expression of the mesenchymal markers vi- inthePC-9cells 3xFLAG PEPTIDE structure, leadingtotheformation ofcobblestone-likecellsand mentinandβ-cateninandincreasedexpressionoftheepithelialmarkers a reduction in protuberances indicating mesenchymal to epithelial ZO-1 and E-cadherin (Fig Pimozide. 2G). Cell signaling pathways responsible for Table 1 Association between MICAL2 protein expression and the clinical pathological characteristics of patients with LUADs. Pathological category n MI(IOD/AREA) P IRS of Cytoplasmic MICAL2 P IRS of Nuclear MICAL2 P Mean ± SD Negative Positive Negtive Positve Total 126 0.106 ± 0.049 24 102 86 40 Sex Male 74 0.109 ± 0.052 0.4415 14 60 1.0000 50 24 1.0000 Female 52 0.101 ± 0.045 10 42 36 16 Age <60 68 0.112±0.047 0.1097 11 57 0.4954 46 22 1.0000 ≥60 58 0.098 ± 0.051 13 45 40 18 TNM Ⅰ 55 0.093 ± 0.039 0.0163* 18 37 0.0026* 35 20 0.2603 Ⅱ 30 0.128 ± 0.061 3 27 19 11 Ⅲ~Ⅳ 41 0.107 ± 0.048 3 38 32 9 T 1 39 0.099 ± 0.036 0.5335 7 32 0.4529 26 13 0.6036 2 61 0.110 ± 0.058 14 47 44 17 3–4 26 0.104 ± 0.046 3 23 16 10 N Negtive 74 0.095 ± 0.042 0.0044* 24 50 <0.0001* 46 28 0.0851 Positive 52 0.120 ± 0.053 0 52 40 12 Differenciation Well 19 0.081 ± 0.043 0.0584 3 16 0.9256 14 5 0.8119 Moderate 66 0.111 ± 0.046 13 53 43 23 Poor 41 0.109 ± 0.055 8 33 29 12 * indicates P < 0.05. 78
W. Zhou, et al. 

Cancer Letters 483 (2020) 75–86 Fig.2. MICAL2promotedcellgrowthandmetastasisinLUADcelllines:A.MICAL2ishighlyexpressedinthePC-9celllinewhilelessexpressedintheA549cell line. B. RT-PCR or WB confirmed the knockdown or overexpression of MICAL2 expression levels in PC-9 or A549 cell lines. C. Representative images (gross and microscopic views) illustrate PC-9_nc, PC-9_sh1, and PC-9_sh3 as well as A549-CTRL and A549-MICAL2 cells inplate colony formation. Both PC-9_sh1 and PC- 9_sh3 exhibit lower colony numbers and size compared to PC-9_nc (n = 3, *** = P < 0.001), while A549-MICAL2 exhibits colony numbers and size higher than A549-CTRL(n=3,**=P < 0.01,***=P < 0.001).MICAL2-highcellsweremoreepithelial-likecomparedtoMICAL2-lowcellsbasedonthemicroscopicview. D.ACCK8assaywas usedandabsorbance at450 nm(OD 450nm) wasmeasured (n=4,**= P < 0.01,***= P < 0.001).E.Transwell assayswithorwithout Matrigelshowedt###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-4.png####hatMICAL2promotesmigrationandinvasioninLUADcells.Fourfields(up,down,left,andright)wereimagedandthreeindependentassayswere performed (n = 3, *** = P < 0.001, **** = P < 0.0001). F. Light microscopy images show the cellular morphological changes: the cells are epithelial-like (revealing the formation of cobble stone-like cells and a reduction in protuberances) after MICAL2 was knocked down in PC-9 cells Cell Counting Kit-8 glpbio, while MICAL2-overexpressed A549 cells presented longer and more numerous protuberances compared with A549-CTRL cells. G. Knockdown of MICAL2 resulted in an increase in the epithelial markersZO-1andE-cadherinbutdecreaseinthemesenchymalmarkersvimentinandβ-cateninaswellasmyosin-9andphosphorylationlevelsofAKTandPGSK-3β, while the overexpression of MICAL2 in A549 cell lines exhibited the opposite change in those markers. Right is the quantitative analysis of the bands (n=3, *= P < 0.05, ** = P < 0.01, *** = P < 0.001).

Comments

Popular posts from this blog

The cells were counterstained with DAPI for Apoptosis rates were determined by Annexin V-/PI double staining 10 min

Control cells were incubated without mon- MYB inhibition is feasible and has therapeutic potential

Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo