Characterization of monocarboxylate transporter activity in hepatocellular carcinoma

Table 1 Immunohistochemistry protocols used to characterize the proteins expression

Code	Industry	Dilution	Epitope retrieval	Amplification system
MCT1 (sc-365501)	Santa Cruz Biotechnology, Santa Cruz, CA, United States	1:500	EDTA (1 mmol/L, pH = 8);	Vectastain® Elite ABC reagent
			98 °C; 20 min	(Burlingame, CA, United States)
MCT2 (sc-14926)	Santa Cruz Biotechnology, Santa Cruz, CA, United States	1:200	Citrate buffer (10 mmol/L, pH = 6); 98 °C; 20 min	Ultravision Detection System Anti-polyvalent, HRP, Lab Vision Corporation, Fremont, CA, United States)
MCT4 (sc-50329)	Santa Cruz Biotechnology, Santa Cruz, CA, United States	1:500	Citrate buffer (10 mmol/L, pH = 6); 98 °C; 20 min	Ultravision Detection System Anti-polyvalent, HRP, Lab Vision Corporation, Fremont, CA, United States)
CD147 (sc-71038)	Santa Cruz Biotechnology, Santa Cruz, CA, United States	1:400	EDTA (1 mmol/L, pH = 8);	Vectastain® Elite ABC reagent (Burlingame, CA, United States)
			98 °C; 20 min
GLUT1 (ab15309)	Abcam, Cambridge, United Kingdom	1:500	Citrate buffer (10 mmol/L, pH = 6); 98 °C; 20 min	Ultravision Detection System Anti-polyvalent, HRP, Lab Vision Corporation, Fremont, CA, United States)
Caspase 3	Diagnostic BioSystems (DBS) Pleasanton, CA, United States	1:160	Citrate buffer (10 mmol/L, pH = 6); 98 °C; 40 min	NovoLink (Novocastra Laboratories Ltd, Newcastle Upon Tyne, United Kingdom)
(3C SP03)
Keratin 19 (K19, b170)	Novocastra Laboratories Ltd, Newcastle Upon Tyne, United Kingdom	1:300	Citrate buffer (10 mmol/L, pH = 6); 98 °C; 40 min	NovoLink (Novocastra Laboratories Ltd, Newcastle Upon Tyne, United Kingdom)
Ki-67 (MIB-1)	(Dako, Glostrup, Denmark)	1:400	Citrate buffer (10 mmol/L, pH = 6); 98 °C; 40 min	NovoLink (Novocastra Laboratories Ltd, Newcastle Upon Tyne, United Kingdom)
EGFR human (DAK-H1-WT)	(Dako, Glostrup, Denmark)	1:200	Citrate buffer (10 mmol/L, pH = 6); 98 °C; 40 min	NovoLink (Novocastra Laboratories Ltd, Newcastle Upon Tyne, United Kingdom)

MCT: Monocarboxylate transporter; GLUT: Glucose transporter; EGFR: Epidermal growth factor receptor.

Table 2 Association of overall expression of cell metabolism markers with the neoplastic (including metastasis) and non-neoplastic status of the liver n (%)

		MCT1			MCT2			MCT4			CD147			GLUT1
	n	Positive	P value	n	Positive	P value	n	Positive	P value	n	Positive	P value	n	Positive	P value
Non-neoplastic	26	25 (96.2)		26	23 (88.5)		26	3 (11.5)		26	22 (84.6)		26	0 (0.0)
Primary tumor	73	46 (63.0)		74	52 (70.3)		76	31 (40.8)		75	36 (48.0)		76	7 (9.2)
Metastasis	16	9 (56.2)		16	6 (46.2)		17	11 (64.7)		17	10 (58.8)		17	5 (29.4)
			0.003			0.02			0.001			0.005			0.007

MCT: Monocarboxylate transporter; GLUT: Glucose transporter.

Table 3 Association of the plasma membrane expression of cell metabolism markers with the neoplastic (including metastasis) and non-neoplastic status of the liver n (%)

		MCT1			MCT2			MCT4			CD147			GLUT1
	n	Positive	P value	n	Positive	P value	n	Positive	P value	n	Positive	P value	n	Positive	P value
Non-neoplastic	26	25 (96.2)		26	0 (0.0)		26	1 (3.8)		26	20 (76.9)		26	0 (0.0)
Primary tumor	73	40 (54.8)		74	9 (12.2)		76	29 (38.2)		75	32 (42.7)		76	4 (5.3)
Metastasis	16	9 (56.2)		16	1 (7.7)		17	8 (47.1)		17	9 (52.9)		17	3 (17.6)
			0.001			0.169			0.002			0.011			0.052

MCT: Monocarboxylate transporter; GLUT: Glucose transporter.

Table 4 Association of the plasma membrane expression of monocarboxylate transporters with the plasma membrane expression of the chaperone CD147 and the glycolytic protein glucose transporter 1 n (%)

		CD147			GLUT1
	n	Positive	P value	n	Positive	P value
MCT1			< 0.001			1.000
Negative	40	4 (10.0)		40	2 (5.0)
Positive	74	56 (75.7)		74	4 (5.4)
MCT2			0.766			0.434
Negative	102	56 (54.9)		103	5 (4.9)
Positive	10	5 (50.0)		10	1 (10.0)
MCT4			0.114			0.004
Negative	80	46 (57.5)		81	1 (1.2)
Positive	36	15 (41.7)		38	6 (15.8)

MCT: Monocarboxylate transporter; GLUT: Glucose transporter.