Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis

Table 1 Antibody dilution, unmasking, incubation time, temperature, and immunodetection used for immunofluorescence

Antibody (source)	Unmasking of antigens	Dilution	Incubation	Detection system
NF-κB p65 (Neomarkers, United States)	Citrate buffer pH 6.0, 95 °C, 10 min, water bath	1:1000	18 h at 4 °C	IF with Alexa Fluor 546 (Invitrogen, Germany) as secondary antibody

NF-κB: Nuclear factor kappa B; IF: Immunofluorescence.

Table 2 Primer sequences for real-time reverse transcription-polymerase chain reaction

mRNA	Accession No.	Sense primer, 5'→3’	Antisense primer, 5'→3’
18S	NR_003278.1	GTAACCCGTTGAACCCCATT	CCATCCAATCGGTAGTAGCG
PPARa	NM_001113418.1	CCTTCCCTGTGAACTGACG	CCACAGAGCGCTAAGCTGT
IL-1B	NM_008361.3	GAGAGCCTGTGTTTTCCTCC	GAGTGCTGCCTAATGTCCC
TNF	NM_013693.2	CCATTCCTGAGTTCTGCAAAGG	AGGTAGGAAGGCCTGAGATCTTATC
HMGCR	NM_008255.2	ATCCAGGAGCGAACCAAGAGAG	CAGAAGCCCCAAGCACAAAC
SCD1	NM_009127.4	AGATCTCCAGTTCTTACACGACCAC	CTTTCATTTCAGGACGGATGTCT
CPT1a	NM_013495.2	CTCAGTGGGAGCGACTCTTCA	GGCCTCTGTGGTACACGACAA
NOS2	NM_010927.3	CTCACTGGGACAGCACAGAA	GATGTGGCCTTGTGGTGAA
PTGS/COX2	XM_192868	TGACCCCCAAGGCTCAAATAT	TGAACCCAGGTCCTCGCTTA
FASN	NM_007988.3	GGCTGCTACAAACAGACCAT	CACGGTAGAAAAGGCTCAGT
SREBF2	NM_033218.1	ACCTAGACCTCGCCAAAGGT	CGGATCACATTCCAGGAGA

PPARa: Peroxisome proliferator-activated receptor a; IL-1B: Interleukin 1B; TNF: Tumor necrosis factor; HMGCR: 3-hydroxy-3-methyl-glutaryl-CoA reductase; SCD1: Stearoyl-CoA desaturase 1; CPT1a: Carnitine palmitoyl transferase 1a; NOS2: Nitric oxide synthase 2; PTGS/COX2: Prostaglandin-endoperoxide synthase 2; FASN: Fatty acid synthase; SREBF2: Sterol regulatory element binding transcription factor 2.

Table 3 Scoring system for hepatocellular iron and nuclear factor kappa B-p65 nuclear translocation

Scoring system		Assessed by
Hepatocellular iron
Score 0	No granules	Prussian blue
Score 1	Zone 1, granules seen at × 40
Score 2	Granules seen at × 20
Score 3	Granules seen at × 10
Score 4	Granules seen at × 10 in zone 1 and 2

NF-κB: Nuclear factor kappa B; IF: Immunofluorescence.

Table 4 Liver weight and serum parameters of p62 transgenic and wild-type mice fed a methionine-choline deficient diet or control diet for two and four weeks

	2 wk				4 wk
	ctrl		MCD		ctrl		MCD
	wt	tg	wt	tg	wt	tg	wt	tg
Number of animals	10	10	12	12	10	10	12	12
Relative liver weight (% of body weight)	4.8 ± 0.1	4.6 ± 0.1	3.4 ± 0.1c	4.0 ± 0.2ace	4.2 ± 01	4.1 ± 0.1	3.4 ± 0.1c	3.5 ± 0.2ce
Serum ALT (U/L)	289 ± 83	233 ± 20	418 ± 36c	469 ± 73ce	189 ± 38	222 ± 25	235 ± 40	209 ± 46
Serum AST (U/L)	1568 ± 224	1535 ± 162	2404 ± 125c	2544 ± 207ce	1845 ± 204	1712 ± 253	2813 ± 286c	3057 ± 346c
Serum triglycerides (mg/dL)	244 ± 19	219 ± 17	107 ± 5c	128 ± 11ce	211 ± 18	203 ± 21	95 ± 7c	106 ± 5ce
Serum HDL (mg/dL)	94 ± 6	98 ± 4	24 ± 2c	21 ± 4ce	112 ± 8	119 ± 8	15 ± 2c	18 ± 2ce
Serum glucose (mg/dL)	234 ± 27	179 ± 15	67 ± 10c	59 ± 7ce	193 ± 13	253 ± 36	59 ± 7c	40 ± 5ace

^aP < 0.05 vs wild-type;

^cP < 0.05 vs control diet;

^eP < 0.05 vs wild-type on control diet; Values are expressed as the mean ± SEM; tg: Transgenic; wt: Wild-type; ctrl: Control; MCD: Methionine-choline deficient.

Table 5 Gas-chromatography mass-spectrometry fatty acid analyses of mice fed the methionine-choline deficient or control diet for two weeks

Fatty acid	ctrl wt	ctrl tg	P value1	MCD wt	MCD tg	P value2	P value3
14:0	0.25 ± 0.04	0.25 ± 0.05	0.970	0.24 ± 0.04	0.43 ± 0.04	0.003	0.005
15:0	0.01 ± 0.003	0.01 ± 0.003	0.113	0.02 ± 0.01	0.05 ± 0.01	0.035	0.002
16:0	17.23 ± 0.79	16.08 ± 1.43	0.623	17.16 ± 1.63	23.85 ± 1.65	0.006	0.0033
16:1	1.69 ± 0.29	1.51 ± 0.30	0.570	0.57 ± 0.10	1.13 ± 0.12	0.005	0.138
17:0	0.11 ± 0.01	0.06 ± 0.01	0.021	0.23 ± 0.03	0.34 ± 0.03	0.010	0.0001
17:1	0.02 ± 0.01	0.01 ± 0.01	0.046	0.003 ± 0.003	0.01 ± 0.01	0.514	0.117
18:0	12.64 ± 0.57	9.41 ± 0.81	0.006	13.08 ± 0.97	18.15 ± 1.00	0.0014	0.0009
18:1	20.13 ± 2.51	17.98 ± 2.07	0.678	24.11 ± 1.28	39.66 ± 3.27	0.001	0.0009
18:2	17.88 ± 0.87	16.42 ± 1.60	0.241	23.90 ± 2.15	36.28 ± 6.34	0.040	0.004
18:3	0.23 ± 0.05	0.27 ± 0.10	0.571	1.50 ± 0.28	2.99 ± 0.46	0.003	0.00009
20:0	0.22 ± 0.05	0.10 ± 0.03	0.045	0.14 ± 0.03	0.33 ± 0.05	0.0007	0.138
20:1	0.40 ± 0.03	0.30 ± 0.04	0.045	0.81 ± 0.14	1.99 ± 0.27	0.002	0.00009
20:2	0.45 ± 0.04	0.53 ± 0.06	0.385	1.67 ± 0.17	2.94 ± 0.25	0.0006	0.00009
20:3	1.09 ± 0.09	0.89 ± 0.14	0.186	2.87 ± 0.37	5.15 ± 0.44	0.0006	0.00009
20:4	13.69 ± 0.58	10.37 ± 0.80	0.011	16.49 ± 1.15	23.20 ± 1.81	0.006	0.00015
22:0	0.59 ± 0.11	0.29 ± 0.07	0.031	0.31 ± 0.03	0.50 ± 0.03	0.0007	0.921
22:1	0.01 ± 0.003	0.003 ± 0.003	0.387	0.01 ± 0.01	0.04 ± 0.01	0.091	0.129
22:4	0.57 ± 0.05	0.51 ± 0.08	0.273	4.24 ± 0.48	6.25 ± 0.56	0.0036	0.00009
22:6	4.72 ± 0.28	3.82 ± 0.29	0.054	12.08 ± 0.90	14.06 ± 1.05	0.214	0.00009
23:0	0.08 ± 0.01	0.04 ± 0.004	0.0003	0.08 ± 0.01	0.10 ± 0.01	0.194	0.223
24:0	0.42 ± 0.02	0.31 ± 0.02	0.003	0.45 ± 0.02	0.63 ± 0.05	0.0013	0.0009

¹P value of the comparison between ctrl wt and ctrl tg;

²P value of the comparison between MCD wt and MCD tg;

³P value of the comparison between ctrl wt and MCD tg; Values (μg/mg dry liver tissue) are expressed as the mean ± SEM. Liver tissues were lyophilized and analyzed by gas-chromatography mass-spectrometry. tg: Transgenic; wt: Wild-type; ctrl: Control; MCD: Methionine-choline deficient.