Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

Table 1 PCR primers

Gene	GenBank accession size	Orientation	Primer sequence	Amplicon size
KCR	D88577	Forward	AAATGACCTCAGCTCCCAGA	103
		Reverse	TTCACCAGCCCTTTCCATAC
CSF-1 receptor (CD115)	X06368	Forward	TGGCCTTCCTTGCTTCTAAA	114
		Reverse	ATGTCCCTAGCCAGTCCAA
HMGCS1	NM_145942	Forward	GCGGCTAGAAGTTGGAACAG	196
		Reverse	AGCATATCGTCCATCCCAAG
Lipocalin 2	NM_008491	Forward	CCAGTTCGCCATGGTATTTT	169
		Reverse	GGTGGGGACAGAGAAGATGA
Hck	BC010478	Forward	GCCTCAAAAACAGAGCCAAG	150
		Reverse	GTACAGTGCGACCACAATGG
18S rRNA	XR_000144	Forward	AATGGTGCTACCGGTCATTCC	192
		Reverse	ACCTCTCTTACCCGCTCTC

Table 2 Significantly up- or down-regulated genes ontology

	Number	Percentage (%)
Cytoplasm	105	16.7
Cell growth and/or maintenance	87	13.8
Integral to membrane	85	13.5
Nucleus	57	9.0
Nucleoside and nucleic acid metabolism	54	8.6
Protein metabolism	54	8.6
Biosynthesis	34	5.4
Purine nucleotide binding	33	5.2
DNA binding	30	4.8
Signal transduction	26	4.1
Response to external stimulus	24	3.8
Transferase activity, transferring phosphorus containing groups	24	3.8
Lipid metabolism	23	3.7
Plasma membrane	19	3.0
Catabolism	18	2.9
Immune response	18	2.9
Phosphorus metabolism	18	2.9
Peptidase activity	16	2.5

Table 3 Standardized Pubmed search with terms relevant to Kupffer cells abrogating liver injury during cholestasis: Each gene up- or down-regulated > 5-fold

Gene	Fold change	Search terms									Articles of interest
		*Gene*	Kupffer cells	Cholestasis	Apoptosis	Cell death	Survival	Inflammation	Liver	Liver repair
Acetyl-coenzyme A synthetase 2	9.61	179	-	-	-	1	2	-	19	-	2
Hydroxysteroid (17-β) dehydrogenase 2	8.26	668	-	-	4	2	10	3	70	-	1
RIKEN cDNA 1110025G12	7.61	-	-	-	-	-	-	-	-	-	-
HM GCS1	5.81	181	-	-	6	6	2	-	67	-	3
RBP 4	5.76	1044	2	2	10	10	25	42	195	2	3
RIKEN cDNA 1200011D03 gene	5.75	-	-	-	-	-	-	-	-	-	-
Inter-alpha trypsin inhibitor, heavy chain 2	5.46	113	2	-	1	-	3	19	63	-	5
Lipocalin 2	5.35	375	-	1	24	25	15	48	17	1	15
Hck	-5.67	4	-	-	-	-	-	-	-	-	3
Hypothetical protein 5930431H10	-6.15	-	-	-	-	-	-	-	-	-	-
Leucine-rich repeat-containing 5	-6.26	22	-	-	1	1	-	-	1	-	2
SAM domain and HD domain, 1 (SAMHD1)	-6.81	5	-	-	-	-	-	-	-	-	-
Myristoylated alanine rich protein kinase C substrate	-7.14	458	-	-	5	7	10	5	11	-	3
Heterogeneous nuclear ribonucleoprotein A/B	-7.94	50	-	-	2	2	2	-	5	-	3
(SCAN-KRAB-) zinc finger gene 1	-9.05	6	-	-	1	-	1	-	-	-	1
Inactive X specific transcripts	-12.21	3	-	-	-	-	-	-	-	-	-

Table 4 Overview of function, source and involvement of the 10 genes identified by Pubmed-search: Degree of up- or down-regulation

Gene	Fold change	Biological function	Primary cell source	Involvement	Reference
Acetyl-Coenzyme A synthetase 2	9.61	Mitochondrial matrix enzyme involved in CoA ligation	Mitochondrial matrix	Activation during fasting	[5051]
HM GCS1	5.81	Cholesterol biosynthesis		Inhibition induces apoptosis, up-regulation involved in liver regeneration	[2526]
RBP4	5.76	An a2-globulin that transports vitamin A from the liver	Parenchymal liver cells, little in Kupffer cells	Known marker for hepatocellular necrosis	[2223]
Inter-alpha trypsin inhibitor, heavy chain 2	5.46	Plasma protease inhibitor	Kupffer cells	Endothelial growth factor	[27–29]
Lipocalin 2	5.35	Iron-siderophore-binding protein	Macrophages, neutrophils	Suppression of inflammation, tissue involution, apoptosis, differentiation of myeloid cells	[31–40]
Hck	-5.67	Protein-tyrosine kinase	Granulocytes, monocytes	Mitogenesis, differentiation, survival, migration	[41–44]
Leucine-rich repeat-containing 5	-6.26	Interaction with cellular G-proteins		Enzyme inhibition, cell adhesion, cellular trafficking, proliferation and activation of lymphocytes and monocytes	[4552]
Myristoylated alanine rich protein kinase C substrate	-7.14	Substrate of protein kinase C		Intracellular signaling, brain development, cellular migration and adhesion, phagocyte activation, phagocytosis	[464753–55]
Heterogeneous nuclear ribonucleoprotein A/B	-7.94	Chromatin-associated RNA-binding protein	Ubiquitious	RNA handling, proliferation arrest	[5657]
(SCAN-KRAB-) zinc finger gene 1	-9.05	Protein interaction domain	Ubiquitious	Cell survival, differentiation	[4849]