Osteopontin is an important mediator of alcoholic liver disease via hepatic stellate cell activation

Table 1 Characteristics of participants for Osteopontin enzyme-linked immunosorbent assay

	Cases2	Control3	P-value
Age (yr)	57.24	51.95	0.08171
ALT (IU/L)	36.59	31.41	0.21631
AST (IU/L)	51.45	33.41	0.0180b
GGT (IU/L)	220.76	93.32	0.0121b
INR	1.46	1.04	0.0017d
Albumin (g/L)	35.41	43.55	0.0004f
Bilirubin (μmol/L)	48.38	11.36	0.0065d
EtOH lifetime (kg)	1685	1555	0.99161

Patient characteristics identify cases (drinkers with liver cirrhosis) from controls (drinkers without liver disease) by significant differences in the liver function tests and other biochemical measures of liver injury.

¹Not significant;

²[n = 25 (M); n = 4 (F)]; [n = 17 (M); n = 4 (F)].

^bP < 0.01 vs control;

^dP < 0.001 vs control;

^fP < 0.0001 vs control. INR: International normalised ratio for prothrombin time to predict survival; M: Male; F: Female; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: γ-glutamyl transferase.

Table 2 Antibodies for Western blots, immunofluorescence and enzyme-linked immunosorbent assay

Specificity	Host	Isotype	Use	Dilution	Samples	Source (Catalog No.)
Primary antibodies
OPNK20 (SPP1)	Goat	Polyclonal IgG	WB	1:1000	Human, LX2	Santa Cruz, Santa Cruz, CA, United States (sc-10591)
			Blocking	200 ìg/mL
OPN (AF808)	Goat	Polyclonal IgG	WB	1:1000	Mice	RD (AF808)
Capture anti-OPN	Mouse	Monoclonal	ELISA	3 ìg/mL	Human, LX2	RD (MAB14331)
Detection biotynlated anti-OPN	Goat	Polyclonal IgG	ELISA	200 ng/mL	Human, LX2	RD (BAF1433)
OPN-R3 aptamer blocks SPP1 binding to receptors				100 nmol/L	LX2	Duke University, NC, United States
αvβ3-integrin (CD51/61) (ITGAV)	Mouse	Monoclonal	Blocking	10 ìg/mL	LX2	RD, Minneapolis, MN, United States (MAB3050)
CD44v6	Mouse	Monoclonal IgG1	Blcoking	1:1000	LX2	RD (BBA13)
Vimentin	Rat	Monoclonal	IF	1:200	LX2	RD (MAB2105)
Phospho-Akt Ser473 (P-Akt)	Rabbit	Polyclonal	WB	1:1000	LX2, Mice	Cell Signalling Technology (No. 9271)
Total-Akt (pan)	Rabbit	Polyclonal IgG	WB	1:1000	LX2, Mice	Cell Signalling Technology (No. 4691)
Phospho-Erk p44/42 (P-Erk)	Mouse	Monoclonal IgG	WB	1:2000	LX2, Mice	Cell Signalling Technology, Danvers, MA, United States (No. 9106)
Total-Erk (T-Erk)	Rabbit	Polyclonal	WB	1:1000	LX2, Mice	Cell Signalling Technology (No. 9102)
GAPDH	Mouse	Monoclonal	WB	1:200000	Mice	Ambion (AM4300)
Secondary antibodies
Anti-mouse HRP (P0161)				1:10000	Dako Australia Pty Ltd, Campbellfield, VIC, Australia
Anti-rabbit HRP (P0449)				1:7500
Anti-goat HRP (P0448)				1:75000
AlexaFluor 488; AlexaFluor 594				1:400	Molecular probes

WB: Western blots; ELISA: Enzyme-linked immunosorbent assay; IF: Immunofluorescence; OPN: Osteopontin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HRP: Horseradish peroxidase.

Table 3 Primer sequences for human (h-) and mouse (m-) genes

Target gene	Forward (5’→3’)	Reverse (5’→3’)	Expected size (bps)	Ref.
OPN and OPN pathway
Total h-OPN	AATGGTGCATACAAGGCCATC	TGTCCTTCCCACGGCTGT	90	NM_000582[1]
h-OPN-C	CTGAGGAAAAGCAGAATG	AATGGAGTCCTGGCTGT	149	[40]
h-CD44v6	GCTTTCAATAGCACCTTGCC	GTTGCCAAACCACTGTTCCT	867	[41]
h-αvβ3-integrin	TTGTTGCTACTGGCTGTTTTG	TCCCTTTCTTGTTCTTCTTGAG	89	NM_002210
m-OPN	CAGCCTGCAAGATCCTA	GCGCAAGGAGATTCTGCT TCT	71	[7]
m-αvβ3-integrin	GTAATCGAGATGCCCCAGAG	CTTCCATCCAGGGCAATATG	142	[42]
m-CD44	TCTGCCATCTAGCACTAAGAGC	GTCTGGGTATTGAAAGGTGTAGC	106	[43]
Fibrogenesis and ECM
h-TGFβ	CCACAACGAAATCTATGAC	CGGTGACATCAAAAGATAAC	260	NM_000660
m-TGF-β	CCTTCCTGCTCCTCATGGCCA	GTCCTTCCTAAAGTCAATGTA	149	[44]
h-Collagen 1α1	GATTCCCTGGACCTAAAGGTGC	AGCCTCTCCATCTTTGCCAGCA	106	NM_000088.3
m-Collagen 1 (Col1)	GAGCGGAGAGTACTGGATCG	GCTTCTTTTCCTTGGGGTTC	158	[45]
h-Collagen 4α4	CAGGCTCAACTGGTCTAAGAGG	AGGTGGACCAAAGTGACTGGCA	157	NM_000092.4
h-MMP2	ATGACAGCTGCACCACTGAG	ATTTGTTGCCCAGGAAAGTG	164	NM_004530.2
h-MMP3	GCAGTTTGCTCAGCCTATCC	AGTGTCGGAGTCCAGCTTC	194	NM_002422.3
h-MMP9	ACGCCGCTCACCTTCACT	AGGAGGAAAGGCGTGTGC	111	NM_004994
m-MMP7	GCGGAGATGCTCACTTTGACA	ATTCATGGGTGGCAGCAAAC	87	[46]
m-MMP9	CCCACATTTGACGTCCAGAGAAGAA	GTTTTTGATGCTATTGCTGAGATCCA	205	[46]
m-α-sma	AACAGGAATACGACGAAG	CAGGAATGATTTGGAAAGGA	134	[47]
Plasmin-Plasminogen system
h-μPA	CACACACTGCTTCATTGATTAC	TTGGTGGTGACTTCAGAGCCG	409	NM_002658
h-PLG (Plasminogen)	GCAGTGGAGAAAACTATGACGGC	GAAGATGGTGGAGGTGTTGTGC	244	NM_000301[23]
h-PAI-1	CTTTGGTGAAGGGTCTGCTGTG	TGGGTTTCTCCTCCTGTTGTCAG	183	M16006[23]
m-PLG	GCCCAACCTACCAATGTCTGAAAG	GGTGGAACTGAGGAATCTGACTGG	292	[23]
m-μPA	AGGGTGAGCGCCAATAGCAT	GATACATTCACGTGGAGCAT	530	[48]
m-PAI-1	ATGCCATCTTTGTCCAGCGG	TTGGTATGCCTTTCCACCCAG	151	[23]
Normalising
h-β-actin	TGCCATCCTAAAAGCCAC	TCAACTGGTCTCAAGTCAGTG	289	NM_001101
m-β-actin	AAATCGTGCGTGACATCAAA	AAGGAAGGCTGGAAAAGAGC	178	[49]

Table 4 Composition of buffers used in protein extraction and expression studies

Buffer	Composition
RIPA	0.5% (w/v) sodium deoxycholate, 150 mmol/L NaCl, 50 mmol/L Tris pH 8.0, 20 mmol/L NaF, 10% (v/v) glycerol, 5.0 mmol/L EDTA pH = 8.0, 0.1% (w/v) SDS, 1.0% (v/v) Igepal CA-630 Electrophoresis reagent, 1 × phosSTOP, 1 × complete protease inhibitor
TBS	50 mmol/L Tris, 150 mmol/L NaCl, pH = 7.5
TBST	0.1% (v/v) Tween 20 in TBS
Horseradish peroxidase inactivation	0.05% (v/v) of sodium azide, 3% (w/v) skim milk and 0.1% (v/v) Tween 20 in TBS