Volume 115, Issue 4, Pages 967-977 (October 1998) Retinoic acid receptor γ1 expression determines retinoid sensitivity in pancreatic carcinoma cells  Astrid Kaiser, Maja Wolf–Breitinger, Andreas Albers, Tomislav Dorbic, Burghardt Wittig, Ernst–Otto Riecken, Stefan Rosewicz  Gastroenterology  Volume 115, Issue 4, Pages 967-977 (October 1998) DOI: 10.1016/S0016-5085(98)70269-0 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Differential effects of ATRA on anchorage-independent growth in pancreatic carcinoma cell lines. Pancreatic tumor cell lines Capan-2 (2) and AR42J (▨) were incubated with the indicated concentrations of ATRA and then subjected to a human tumor clonogenic assay as described in Materials and Methods. Number of colonies are expressed as percentage of control cells treated with vehicle alone. The mean ± SEM of four independent experiments performed for each cell line is shown. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 RAR/RXR subtype expression in AR42J and Capan-2 cells. RT-PCR analysis was performed for Capan-2 and AR42J cells using receptor-specific oligonucleotide primers for RAR and RXR subtypes. As a positive control, receptor subtype–specific cDNAs were used as a template for the RT-PCR. Size of amplification products was determined by a 100-bp DNA ladder (M). Aliquots from each PCR product were electrophoresed on a 1.5% agarose gel. A representative of three experiments is shown. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Southern blot analysis of RARγ1-transfected AR42J cell clones. Thirty micrograms of EcoRI/SstI–digested genomic DNA isolated from wild-type and mock-transfected (mock) cells and cells transfected with RARγ (clone 3, clone 4, clone 5) were separated on a 1% agarose/TBE gel, transferred to Hybond N+ membranes, and hybridized with the radioactively labeled RARγ1 cDNA probe. For size determinations, a 1.0-kb DNA ladder (GIBCO) was electrophoresed in parallel. Arrow indicates the anticipated size of the integrated RARγ gene. The equivalent high-molecular hybridization signals correspond to the endogenous RARγ. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Northern blot analysis of RARγ1-transfected AR42J cell clones. Ten micrograms of polyA+-enriched RNA isolated from wild-type (WT) and RARγ1 transfectants (clones 3, 4, and 5) was separated on a 1% agarose-formaldehyde gel, transferred to Hybond N+ membrane, and hybridized with the radioactively labeled RARγ1 cDNA. In parallel, the 0.24-9.0-kb RNA ladder (GIBCO) was electrophoresed. The transcripts detected in all transfectants at approximately 2.2 kb represent the transfected RARγ1 gene. Equal RNA loading is shown in the lower part of the figure by the ethidium bromide stain of the Northern gel. Shown is a representative of two independent experiments, yielding identical results. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Electrophoretic mobility shift assay of RARγ1-transfected AR42J cells. Nuclear extracts (1, 2.5, and 5 μg) isolated from clone 3 and mock-transfected AR42J cells were incubated with radioactively labeled β RARE oligonucleotide and electrophoresed on a 6% PAA/TBE gel (see Materials and Methods). For competition experiments, 5 μg of nuclear extracts of mock-transfected AR42J cells were incubated with a 40-fold excess of unlabeled oligonucleotide before addition of labeled oligonucleotide. Arrow indicates position of β RARE-protein complex. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of ATRA on anchorage-independent growth in RARγ1-expressing AR42J cell clones. RARγ1-transfected cell clones 3, 4, and 5 and mock transfectants were examined in the human tumor clonogenic assay with the indicated final concentrations of ATRA. The number of colonies was evaluated and expressed as the percentage of untreated controls. The mean ± SEM of three of four experiments are shown. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Northern blot analysis of Capan-2 and RARγ1-transfected DSL-6A/C1 cells and effects of ATRA on anchorage-independent growth. (A) Forty micrograms of total RNA isolated from Capan-2 cells, DSL-6A/C1 wild-type (WT), mock-transfected (mock), and DSL-6A/C1 RARγ1-transfectants γ21, γ27, and γ50 was separated on a 1% agarose-formaldehyde gel, transferred to Hybond N+ membrane, and hybridized with the radioactively labeled RARγ1 cDNA. For size determination, a 0.24-9.0-kb RNA ladder (GIBCO) was electrophoresed in parallel. The 3.4-kb transcript detected in Capan-2 cells corresponds to the endogenous RARγ. The transcripts detected in all transfectants at approximately 2.2 kb represent the transfected RARγ1 gene. Equal RNA loading of all lanes is shown by the ethidium bromide stain of the Northern gel. (B) DSL-6A/C1 wild-type, mock-transfected, and RARγ1-transfected DSL-6A/C1 cell clones γ21, γ27, and γ50 were examined in the human tumor clonogenic assay with the indicated final concentrations of ATRA. The number of colonies was evaluated and expressed as percentage of untreated controls. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Northern blot analysis of Capan-2 and RARγ1-transfected DSL-6A/C1 cells and effects of ATRA on anchorage-independent growth. (A) Forty micrograms of total RNA isolated from Capan-2 cells, DSL-6A/C1 wild-type (WT), mock-transfected (mock), and DSL-6A/C1 RARγ1-transfectants γ21, γ27, and γ50 was separated on a 1% agarose-formaldehyde gel, transferred to Hybond N+ membrane, and hybridized with the radioactively labeled RARγ1 cDNA. For size determination, a 0.24-9.0-kb RNA ladder (GIBCO) was electrophoresed in parallel. The 3.4-kb transcript detected in Capan-2 cells corresponds to the endogenous RARγ. The transcripts detected in all transfectants at approximately 2.2 kb represent the transfected RARγ1 gene. Equal RNA loading of all lanes is shown by the ethidium bromide stain of the Northern gel. (B) DSL-6A/C1 wild-type, mock-transfected, and RARγ1-transfected DSL-6A/C1 cell clones γ21, γ27, and γ50 were examined in the human tumor clonogenic assay with the indicated final concentrations of ATRA. The number of colonies was evaluated and expressed as percentage of untreated controls. Gastroenterology 1998 115, 967-977DOI: (10.1016/S0016-5085(98)70269-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions