Table S1. Results of qualitative RT-PCR to determine the mRNA expression of ALPL, SPP1, SPARC and BGLAP. ALPL. SPP1. SPARC. BGLAP. Amplicon MC.
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Supplementary Materials: Establishment and Characterization of New Canine and Feline Osteosarcoma Primary Cell Lines Florian R. L. Meyer and Ingrid Walter 1. PCR By means of PCR, the presence of mRNA of tissue unspecific alkaline phosphatase (ALPL) and osteonectin (SPARC) was detected positively in all tested feline and canine tissue and cell culture samples, except for the cell culture samples of COS_1220 which lost ALPL expression, mirroring the results obtained by immunohistochemistry. However, we failed to amplify osteocalcin (BGLAP) mRNA in feline and canine samples. Amplification of the genes in the individual tumour samples and the results of the corresponding melting curve analysis are depicted in Table S1. Table S1. Results of qualitative RT-PCR to determine the mRNA expression of ALPL, SPP1, SPARC and BGLAP.
ALPL Amplicon
MC
SPP1 Amplicon
MC
SPARC Amplicon
MC
BGLAP Amplicon
MC
sample 1033 1033
Tumour CC
+ +
+ +
+ ~
+ +
+ +
+ +
-
-
1077 1077
Tumour CC
+ ~
+ +
~ +
+ +
+ +
+ +
-
-
1140 1140
Tumour CC
+ ~
+ +
+ +
+ +
+ +
+ +
-
-
1186 1186h 1186w
Tumour CC CC
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
-
-
1189
Tumour
+
+
+
+
+
+
-
-
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Table S1. Cont.
1189 1220 1220 MC + ~ -
ALPL Amplicon + +
MC CC + Tumour CC + melting curve positive result very late amplification signal negative result +
SPP1 Amplicon + + +
MC + + +
SPARC Amplicon + + +
MC + + +
BGLAP Amplicon -
MC -
Positive result; ~ very late amplification signal; − negative result.
2. DNA Fingerprint Assay Microsatellite analysis of the established primary cell lines confirmed their origin from the corresponding tumor. In feline samples 16 markers were tested. All markers were identical between tumor tissue and cells in culture. In dogs, 18 markers were tested. COS_1033 and COS_1186h cells and tumor were completely identical in their marker setup, although one marker failed to amplify in the latter, both in tumor and in the cell line. COS_1220 showed one allele changed in a single marker, COS_1189 had alterations in two alleles. Three markers differed between COS_1186w and the tumor, although one marker failed to amplify in the tumor sample, but not in the cell line. A complete list of the obtained microsatellite data as first published in [1] is supplied in Table S2 and S3. These list can be helpful in future experiments to exclude cross contaminations. Table S2. Length of microsateites in dogs used to confirm the cell cultues origins.
Microsatelite a AHT121: AHT137: AHTH171: AHTH260: AHTK211: AHTK253:
1033 Tumour 102/102 131/135 233/233 238/244 87/95 288/292
COS_1033 Cell Culture 102/102 131/135 233/233 238/244 87/95 288/292
1186 Tumour -/135/137 221/229 238/244 91/91 288/292
COS_1186h Cell Culture -/135/137 221/229 238/244 91/91 288/292
COS_1186w Cell Culture 98/102 135/137 221/229 238/244 91/91 288/292
1189 Tumour 106/112 131/139 229/229 246/250 91/99 290/292
COS_1189 Cell Culture 106/116 131/139 229/229 246/250 91/99 286/290
1220 Tumour 102/102 131/131 219/219 244/244 95/95 288/288
COS_1220 Cell Culture 102/102 131/131 219/219 244/244 95/95 288/288
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Table S2. Cont.
Microsatelite a CXX279: FH2054: FH2848: INRA21: INU005: INU030: INU055: REN162C04: REN169D01: REN169O18: REN247M23: REN54P11:
1033 Tumour 120/124 152/156 236/242 99/105 124/124 144/156 210/222 200/200 216/216 164/168 272/272 226/226
COS_1033 Cell Culture 120/124 152/156 236/242 99/105 124/124 144/156 210/222 200/200 216/216 164/168 272/272 226/226 a
1186 Tumour 124/124 152/156 242/246 105/105 122/126 144/150 210/218 206/206 212/216 162/164 268/268 226/232
COS_1186h Cell Culture 124/124 152/156 242/246 105/105 122/126 144/150 210/218 206/206 212/216 162/164 268/268 226/232
COS_1186w Cell Culture 124/126 152/156 242/246 99/105 122/126 144/150 210/218 206/206 212/216 162/164 268/268 226/232
1189 Tumour 118/126 152/156 232/238 95/95 110/124 150/150 210/210 202/206 210/212 162/172 268/272 224/226
COS_1189 Cell Culture 118/126 152/156 232/238 95/95 110/124 150/150 210/210 202/206 210/212 162/172 268/272 224/226
Microsatelite names are based on the standards of the ISAG Comparison Test 2006.
Table S3. Length of microsateites in cats used to confirm the cell cultures origins.
Microsatelite a FCA 069 FCA 075 FCA 105 FCA 149 FCA 220 FCA 229 FCA 310 FCA 441 FCA 678 FCA 005
1140 Tumour 103/107 132/134 197/205 124/124 214/214 168/168 138/138 155/155 190/192 148/148
FOS_1140 Cell Culture 103/107 132/134 197/205 124/124 214/214 168/168 138/138 155/155 190/192 148/148
1077 Tumour 105/109 126/134 201/203 124/130 212/214 168/168 128/130 155/159 190/192 144/148
FOS_1077 Cell Culture 105/109 126/134 201/203 124/130 212/214 168/168 128/130 155/159 190/192 144/148
1220 Tumour 120/124 168/172 230/236 101/101 124/132 144/144 210/222 202/202 214/216 164/170 268/272 226/236
COS_1220 Cell Culture 120/124 168/172 230/236 101/101 124/132 144/144 210/222 202/202 216/216 164/170 268/272 226/236
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Table S3. Cont.
Microsatelite a FCA 026 FCA 201 FCA 224 FCA 293 FCA 453 FCA 649 a
1140 Tumour 146/146 145/155 160/160 187/187 188/200 138/138
FOS_1140 Cell Culture 146/146 145/155 160/160 187/187 188/200 138/138
1077 Tumour 156/158 143/161 160/160 187/187 188/196 126/140
FOS_1077 Cell Culture 156/158 143/161 160/160 187/187 188/196 126/140
Microsatelite names are based on the standards of the ISAG Comparison Test 2006.
References 1.
Meyer, F.R.L.; Steinborn, R.; Grausgruber, H.; Wolfesberger, B.; Walter, I. Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma. Vet. J. 2015, 206, 67–74. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).