Background
Methods
Cell line and cell culture
Transfection of pcDNA6/TR(c) via electroporation
Immunocytochemistry for TetR
Design of short hairpin RNAs
No. | sequence shRNA (5'3') (loop sequence) | database sequence NM_002997 |
---|---|---|
1
| top cac cag gac ttc acc ttt gaa acc cga a gg ttt caa agg tga agt cc bottom aaa agg act tca cct ttg aaa cct tcg ggt ttc aaa ggt gaa gtc ct | 874agg act tca cct ttg aaa cc 893 |
2
| top cac cag gag gaa ttc tat gcc tga cga a tc agg cat aga att cct cc bottom aaa agg agg aat tct atg cct gat tcg tca ggc ata gaa ttc ctc ct | 1162agg agg aat tct atg cct ga 1181 |
3
| top cac cgg taa gtt aag taa gtt ga c gaa tca act tac tta act tac c bottom aaa agg taa gtt aag taa gtt gat tcg tca act tac tta act tac c | 1749ggt aag tta agt aag ttg a 1767 |
Cloning of shRNAs of interest in pENTR(tm)/H1/TO and sequencing
Transfection of pENTR™/H1/TO cloned fragments in pcDNA6/TR© transfected St-T1, selection
Primers for Polymerase Chain Reaction (PCR)
sequences 5' 3' | size of the amplified fragment [bp] | |
---|---|---|
β-actin
| for - cagggtgtgatggtgggaatgg rev - caggatggcgtgagggagagca | 409 |
IGFBP-1
| for - agtttagccaaggcacagga rev - tatctggcagttggggtctc | 204 |
PRL
| for - gcttctgtatcatctggtcacg rev - tgcgtaggcagtggagcag | 247 |
GAPDH
| for - tgcaccaactgcttagc rev - acagtcttctgggtggcagtg | 131 |
RNA isolation, reverse transcription, PCR and real-time PCR
Tet induction of Sdc-1 knock-down in double transfected St-T1
Subcellular protein fractionation and dot blot analysis for Sdc-1 expression
Decidualization of St-T1 and Tet-induced KdS1
Investigation of chemokine and angiogenic factor profiles via dot blot analysis with cell culture supernatant of decidualized St-T1 and KdS1 with or without IL-1β incubation
Statistical analysis
Results
Immunocytochemistry
Tet-induction of Sdc-1 knock-down
Expression of Sdc-1 on cell membrane of KdS1
Decidualization
Dot Blots after decidualization
cytokines | dSt-T1 | dKdS1 | dSt-T1+IL-1β | dKdS1+IL-1β |
---|---|---|---|---|
CCL2
|
+
|
+
| ||
CCL5
|
+
|
+
| ||
CXCL1
|
+
|
+
|
+
|
+
|
CXCL8
|
+
|
+
|
+
|
+
|
CXCL10
|
+
|
+
| ||
CXCL11
|
+
|
+
| ||
G-CSF
|
+
|
+
| ||
GM-CSF
|
+
|
+
| ||
IL-6
|
+
|
+
|
+
|
+
|
IL-13
|
+
|
+
|
+
| |
MIF
|
+
|
+
|
+
|
+
|
sICAM-1
|
+
|
+
|
angiogenic factors | dSt-T1 | dKdS1 | dSt-T1+IL-1β | dKdS1+IL-1β |
---|---|---|---|---|
Ang-1
|
+
|
+
|
+
| |
Angiogenin
|
+
|
+
|
+
| |
CXCL16
|
+
|
+
| ||
HGF
|
+
|
+
|
+
|
+
|
IGFBP-2
|
+
|
+
|
+
|
+
|
IGFBP-3
|
+
|
+
|
+
|
+
|
MMP-9
|
+
|
+
|
+
|
+
|
PTX3
|
+
|
+
|
+
|
+
|
TIMP-1
|
+
|
+
|
+
|
+
|
TSP-1
|
+
|
+
|
+
|
+
|
uPA
|
+
|
+
|
+
|
+
|