Journal: Cancer Medicine

Article Title: Nicotinamide N‐methyltransferase is related to MELF pattern invasion in endometrioid carcinoma

doi: 10.1002/cam4.4359

Figure Lengend Snippet: RNA sequencing analyses of FFPE samples using laser microdissection. (A) Representative hematoxylin and eosin (H&E) staining of EC with MELF pattern invasion (left). Representative images of laser microdissection for isolation of the invasive front area (right). Scale bar: 200 μm. (B) RNA sequencing results plotted as a scatterplot matrix. NNMT was expressed higher in the invasive front area than in the surface area in both cases

Article Snippet: Seventy percent confluent AN3CA cells were co‐transfected with equal amounts of NNMT CRISPR/Cas9 knockout plasmid and NNMT HDR plasmid (sc‐403192 and sc‐403192‐HDR, respectively; Santa Cruz Biotechnology) using Lipofectamine 3000 reagent (Thermo Fisher Scientific) and incubated for 72 h. Transfected cells were suspended in PBS supplemented with FBS (2%).

Techniques: RNA Sequencing, Laser Capture Microdissection, Staining, Isolation

Journal: Cancer Medicine

Article Title: Nicotinamide N‐methyltransferase is related to MELF pattern invasion in endometrioid carcinoma

doi: 10.1002/cam4.4359

Figure Lengend Snippet: Immunohistochemistry of NNMT in EC with the MELF pattern. (A) Representative images of various staining intensity: 1+ (weak), 2+ (moderate), and 3+ (strong). (B) Representative images of immunohistochemical analyses of EC with the MELF pattern in the invasive front area (upper pictures). Comparison of H‐scores of the invasive front area and the surface area in G1 with MELF pattern invasion, G1 without MELF pattern invasion (non‐MELF), G2, and G3 ( n = 30, respectively) (lower 4 graphs). Each line shows the H‐score of the invasive front area and the surface area in each case. In G1 with MELF pattern invasion, the data show an increase in the invasive front area in 21 of 30 cases (red lines). Scale bars: 50 μm in (A), 500 μm in the left upper picture of (B), and 100 μm in the right upper picture of (B). We used the Wilcoxon signed‐rank test to calculate p ‐values

Article Snippet: Seventy percent confluent AN3CA cells were co‐transfected with equal amounts of NNMT CRISPR/Cas9 knockout plasmid and NNMT HDR plasmid (sc‐403192 and sc‐403192‐HDR, respectively; Santa Cruz Biotechnology) using Lipofectamine 3000 reagent (Thermo Fisher Scientific) and incubated for 72 h. Transfected cells were suspended in PBS supplemented with FBS (2%).

Techniques: Immunohistochemistry, Staining, Immunohistochemical staining, Comparison

Journal: Cancer Medicine

Article Title: Nicotinamide N‐methyltransferase is related to MELF pattern invasion in endometrioid carcinoma

doi: 10.1002/cam4.4359

Figure Lengend Snippet: Immunoblotting of NNMT in EC cells and generation of NNMT knockout AN3CA and NNMT‐expressing HEC1B and HEC108. (A) NNMT protein levels in AN3CA, HEC1A, HEC1B, HEC108, HEC116, and SNG‐M. (B) Confirmation of depletion of NNMT in NNMT knockout AN3CA cells (KO1 and KO2) and enrichment of NNMT expression in NNMT‐expressing HEC1B and HEC108 cells by immunoblotting. EV, empty vector control cells

Article Snippet: Seventy percent confluent AN3CA cells were co‐transfected with equal amounts of NNMT CRISPR/Cas9 knockout plasmid and NNMT HDR plasmid (sc‐403192 and sc‐403192‐HDR, respectively; Santa Cruz Biotechnology) using Lipofectamine 3000 reagent (Thermo Fisher Scientific) and incubated for 72 h. Transfected cells were suspended in PBS supplemented with FBS (2%).

Techniques: Western Blot, Knock-Out, Expressing, Plasmid Preparation, Control

Journal: Cancer Medicine

Article Title: Nicotinamide N‐methyltransferase is related to MELF pattern invasion in endometrioid carcinoma

doi: 10.1002/cam4.4359

Figure Lengend Snippet: Functional analyses of NNMT. (A) Migration assay. The migration distance was obtained by dividing the width of the wound at 40 h (AN3CA) or 50 h (HEC1B and HEC108) by that at 0 h. The migration distance of control cells (EV or Empty pVec) is expressed as 1. The relative migration distance means a ratio to that of control cells. (B) Matrigel invasion assay. Invading cells are shown in images. We counted invasive cells in five random fields per well. (C) Proliferation assay. (D) Colony formation assay. Colonies were counted per well. (E) Cell morphology. Data are representative of three (AN3CA and HEC1B) or two (HEC108) independent experiments and are shown as means ± SEs. Asterisks mean significant differences, which are determined by the Dunnett's test (AN3CA) and the Student's t‐ test (HEC1B and HEC108) (* p < 0.05, ** p < 0.01). Scale bars: 500 µm in (A), 50 μm in (B), 200 µm in (D), and 100 µm in (E). N.S., not significant

Article Snippet: Seventy percent confluent AN3CA cells were co‐transfected with equal amounts of NNMT CRISPR/Cas9 knockout plasmid and NNMT HDR plasmid (sc‐403192 and sc‐403192‐HDR, respectively; Santa Cruz Biotechnology) using Lipofectamine 3000 reagent (Thermo Fisher Scientific) and incubated for 72 h. Transfected cells were suspended in PBS supplemented with FBS (2%).

Techniques: Functional Assay, Migration, Control, Invasion Assay, Proliferation Assay, Colony Assay

Journal: Cancer Medicine

Article Title: Nicotinamide N‐methyltransferase is related to MELF pattern invasion in endometrioid carcinoma

doi: 10.1002/cam4.4359

Figure Lengend Snippet: NNMT regulates ERK phosphorylation, MMP2 secretion, N‐cadherin expression, chemoresistance, and H3K9me2 methylation. (A) Immunoblotting of ERK1/2 and phospho‐ERK1/2 protein expression in KO1, KO2, and EV. (B) The expression of pro‐MMP2 in the supernatants obtained KO1, KO2, and EV cells. It was evaluated by gelatin zymography. Equal total protein loading was confirmed using a BCA Protein Assay Kit. The results were quantified using ImageJ. (C) Immunoblotting of N‐cadherin expression in KO1, KO2, and EV cells. (D) Cell viability was compared in the presence of cisplatin or carboplatin. The value of 0 μM is expressed as 1. The relative values of various concentrations of cisplatin (1, 2, 4, 8, 16, 32, or 64 μM) and carboplatin (15.6, 31.3, 62.5, 125, 250, 500, or 1000 μM) are presented as ratios relative to 0 μM. (E) Cellular ROS levels in KO1, KO2, and EV cells. We calculated the change in median fluorescence intensity (MFI) by adding cisplatin and compared the results between KO1, KO2, and EV cells. The change in MFI in EV cells is expressed as 1. (F) Representative images of immunohistochemically staining for cleaved caspase‐3 using cell blocks. Positive cells were counted in five random fields per slide. (G) Immunoblotting to determine H3K4me3, H3K9me2, and H3K27me3 protein levels in KO1, KO2, and EV cells. Equal protein loading was confirmed by quantifying histone H3. Data are representative of three independent experiments and are shown as means ± SEs. Asterisks indicate significant differences as determined by the Dunnett's test (* p < 0.05, ** p < 0.01). Scale bars: 100 µm in (F)

Article Snippet: Seventy percent confluent AN3CA cells were co‐transfected with equal amounts of NNMT CRISPR/Cas9 knockout plasmid and NNMT HDR plasmid (sc‐403192 and sc‐403192‐HDR, respectively; Santa Cruz Biotechnology) using Lipofectamine 3000 reagent (Thermo Fisher Scientific) and incubated for 72 h. Transfected cells were suspended in PBS supplemented with FBS (2%).

Techniques: Phospho-proteomics, Expressing, Methylation, Western Blot, Zymography, Bicinchoninic Acid Protein Assay, Fluorescence, Staining