PMC

The MAL protein interferes with the proliferation, metastasis and invasion of GC cells. Western blotting confirmed that transfection of MGC-803 and HGC-27 cells with the siRNA significantly downregulated the expression of the MAL protein, while transfection with the MAL expression plasmid upregulated MAL expression (A). The CCK-8 results showed that MAL knockdown increased the proliferation of MGC-803 and HGC-27 cells, while MAL overexpression inhibited the proliferation of MGC-803 and HGC-27 cells (B). MAL silencing increased the metastasis of MGC-803 cells, and MAL overexpression reduced the metastasis of MGC-803 cells (C–F). MAL knockdown increased the metastasis of HGC-27 cells, and MAL overexpression inhibited the metastasis of HGC-27 cells (G–J). MAL silencing increased the invasion of MGC-803 cells, and MAL overexpression inhibited the invasion of MGC-803 cells (K–N). MAL knockdown enhanced the metastasis of HGC-27 cells, and MAL overexpression inhibited the metastasis of HGC-27 cells (O–R). The experiments were performed at least 3 times independently, and one representative result is shown. The results are presented as the means ± SD. *P < 0.05 and **P < 0.01. C–E, G–I, K–M and O–Q, Scale bars = 100 μm

a. Mal class. b. Mal association. c. Mal lifeline. d. Mitigation lifeline. e. Mal message. f. Mitigation message. g. Mal occurrence specification. h. Mitigation occurrence specification. i. Mal comment. j. Mal comment.

(A) HEK293T cells were plated into 100-mm tissue culture dishes and transiently transfected with pCDNA3-CD14 (1 µg per dish), pCDNA3-TLR4 (4 µg per dish), pEFBOS-HA-MD-2 (1 µg per dish), and pEFBOS-Flag-Mal (WT, 4 µg per dish). To study LPS-inducible Mal tyrosine phosphorylation, the 293/TLR4/MD-2 stable cell line (B) and human monocytes (C) were used. Cells were treated with medium or stimulated with 100 ng/ml LPS as shown. Flag-Mal or endogenous Mal were immunoprecipitated with α-Flag or α-Mal Abs, respectively, and subjected to immunoblotting with α-p-tyrosine Ab PY20 (Mal phosphorylation) and α-Flag (A) or α-Mal (B and C, top panels) Abs (total protein expression of overexpressed or endogenous Mal). Alternatively, total tyrosine-phosphorylated proteins were immunoprecipitated with PY20 Ab, followed by immunoblot detection of p-Mal within this fraction, using Abs against endogenous Mal (B and C, middle panels). To examine total levels of endogenous Mal proteins, Mal proteins were immunoprecipitated with Ab against endogenous Mal and subjected to immunoblotting with α-Mal Ab (B and C, bottom panels). Results of a representative experiment (n=3) are shown.

(A) Uninfected cells (top panel) or cells expressing MAL (bottom two panels) were lysed in ice-cold buffer containing 0.4 % SDS and 0.2 % Triton X-100, then lysates were loaded on top of 5–30 % linear sucrose gradients and centrifuged at 192 000 g for 18 h. Fractions were immunoblotted for MAL and MAL2 as indicated on the left. Arrows above the top panel mark the distributions of the molecular-mass standards (in kDa). The arrow above the bottom two panels indicates fraction 8. (B) MAL-expressing cells were treated in the absence (+ MAL) or presence of 5 mM mβCD (MAL + mβCD) for 30 min and lysed and centrifuged as described in (A). Fractions were immunoblotted for MAL. The fractions containing monomeric MAL are indicated. The arrow points to a higher-density fraction containing MAL. (C) The percentage of total oligomeric MAL or MAL2 in control (+ MAL) or cholesterol-depleted cells (MAL + mβCD) are plotted in the histogram on the left. Results for untreated (− mβCD) cells (black bars) are means ± S.E.M. Measurements were performed on at least three independent experiments. Results for the cholesterol-depleted cells are expressed as the average from two independent experiments. The histogram on the right shows the percentage of MAL present in the high-density fractions. Results for untreated (− mβCD) cells (black bars) are means ± S.E.M. Measurements were performed on at least three independent experiments. Results for cholesterol-depleted cells are expressed as the average from two independent experiments.