Ab187149

DLBCL tissue microarray (TMA) blocks were constructed using 166 blocks of primary DLBCL and 11 RLH FFPE tissues from cohort 1 and 51 DLBCL FFPE tissues from cohort 2. IHC of SMYD3, PKM2, and Ki-67 expression was performed on 4-μm-thick TMA sections using anti-SMYD3 (Abcam, ab187149, 1:400 dilution), anti-Ki-67 (Abcam, ab92742, 1:500 dilution) and anti-PKM2 (Cell Signaling Technology, #4053, 1:400 dilution) antibodies. The H scoring system (ranging between 0 and 300) was used to semiquantitatively score the expression levels of SMYD3 and PKM2 by two experienced pathologists (Yu and Li) based on the staining intensity and proportion of positively stained cells. We then used the cutoff value calculated by the receiver operating characteristic (ROC) curve to divide patients into groups of high vs. low expression of SMYD3 and PKM2.

CHIP assays were carried out following a standard protocol. Briefly, HepG2 cells were cross-linked with 1% formaldehyde (Sigma, Steinheim, Germany) for 15 min at room temperature with gentle shaking. Cells were harvested with a cell scraper, and the chromatin was fragmented to an average of 300 bp in length with a sonicator. Diluted whole cell sonicates were incubated with anti-SMYD3 (Abcam, ab187149) or anti-RNAPII antibodies (MMS-126R-200) (Covance, Madison, WI, USA). After the protein A beads (Millipore, 17-295) were bound, washed, and eluted, the CHIP products were purified and measured by real-time qPCR.

IHC staining was performed to examine the expression of SMYD3 and EMP1 in GC samples with the anti-SMYD3 antibody (Abcam, ab187149, 1:200) and the anti-EMP1 antibody (Abcam, ab230445, 1:100). The staining index (SI) was evaluated by the intensity and proportion of positively stained tumor cells as follows. Scores of staining intensities were: 0, negative; 1, weak; 2, moderate; 3, strong. Scores of positively stained cell proportion were: 0, no positive; 1, <10%; 2, 10%–35%; 3, 35%–75%; 4, >75%. Using this method, SI with possible scores of 0, 1, 2, 3, 4, 6, 8, 9, and 12 were obtained among the GC samples. High and low expression was then defined with the optimal cutoff value of 6.

RIPA cell lysate was used to extract cellular proteins, and the BCA Protein Assay Kit (Abcam) was used to quantify the proteins. The SDS-PAGE gel was prepared, and 6 μL of denatured protein was added to each well for electrophoresis and transferred to a PVDF membrane. The primary antibodies were as follows: rabbit anti-β-actin (4970, Cell Signaling), rabbit anti-cleaved CASP-3 (ab32042, Abcam), rabbit anti-BIRC5 (ab196495, Abcam), rabbit anti-CASP-9 (ab2324, Abcam), rabbit anti -EZH2 (49–1043, Invitrogen), rabbit anti-SMYD3 (ab187149, Abcam), rabbit anti-KDM6A (ab253183, Abcam), rabbit anti-KDM6B/JMJD3 (ab169197, Abcam), rabbit anti-ESET (2196, Cell Signaling), and rabbit anti-histone H3 (tri methyl K27) (ab192985, Abcam). Goat anti-rabbit IgG H&L (HRP) (ab6721, Abcam) was used as the secondary antibody. Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used for exposure; images were acquired using a gel imaging system, and quantitative analysis was performed by ImageJ.

Western blotting was performed on extracted protein samples using anti-SMYD3 (Abcam, ab187149, 1:1000 dilution), and anti-vinculin (Abcam, ab219649, 1:1000 dilution) antibodies as well as anti-PKM2 antibody (Cell Signaling Technology, #4053, 1:3000 dilution). Vinculin was used as the loading control. Briefly, harvested cells were washed with cold PBS and then lysed in RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein was separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, MA, USA). The membranes were incubated with antibodies overnight at 4 °C. After incubation, the membranes were washed with TBST (1*TBS containing 0.1% Tween-20), followed by incubation with HRP-conjugated secondary antibody for 1 h at room temperature. Washed again with TBST, the immunoreactive bands were visualized with Bio-rad Image analysis systems (Bio-Rad, Hercules, CA, USA).One loading control was performed for the proteins on the same membrane. Full length western blots were provided in the Supplementary materials.