Journal: PLoS ONE

Article Title: Inhibition of pulmonary β-carotene 15, 15’-oxygenase expression by glucocorticoid involves PPARα

doi: 10.1371/journal.pone.0181466

Figure Lengend Snippet: A . [α- 32 P]-labeled human BCO1 cRNA probes were hybridized to human poly(A) + RNA (1 μg/lane) from the indicated human tissues. Human tissue filters were exposed to Hyperfilm MP with two intensifying screens at –80°C for 3 days. The position of a molecular size marker is shown at the left . B . Expression of BCO1 mRNA in human cell lines: intestinal TC7, embryonic kidney HEK 293, placental BeWo, and pulmonary alveolar epithelial A549. Total RNA was isolated and RT-PCR was performed as described in “Materials and Methods”. C . Expression of BCO1 protein in the indicated human cell lines: Cell lysates were subjected to western blot analysis using anti-rabbit antibody against the BCO1 protein C-terminal peptide. Equal loading was confirmed by stripping and re-probing the same blot with β-actin antibody.

Article Snippet: A BCO1 antisense complementary RNA (cRNA) probe was synthesized from a unique BCO1 coding region sequence obtained using a PCR amplified template and in vitro transcription (Promega, Madison, WI), T7 RNA polymerase and [α- 32 P]UTP (Amersham Biosciences).

Techniques: Labeling, Marker, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stripping Membranes

Journal: PLoS ONE

Article Title: Inhibition of pulmonary β-carotene 15, 15’-oxygenase expression by glucocorticoid involves PPARα

doi: 10.1371/journal.pone.0181466

Figure Lengend Snippet: A . BCO1 mRNA was determined by RT-PCR. Two x 10 6 cells were plated in 10-cm dishes. After 24 h, cells were treated with DEX (10 −7 M) or FSK (10 −5 M) for 24 h. Cells were harvested, total RNA extracted, and RT-PCR performed. GAPDH served as a loading control. B . Quantification of BCO1 was examined by real time PCR. Relative fold changes are presented as 2 -ΔΔCt . Results are presented as means ± S.D. of five independent experiments, each performed in triplicate. * P <0.05; ** P<0 . 01 . C . BCO1 protein from each treatment was analyzed by western blot using rabbit anti-BCO1 antibody with β-actin serving as a loading control ( top panel ). The bottom panel indicates the adjusted BCO1 levels (mean ± S.D., n = 3). D . A549 cells were treated with β-carotene (10 −6 M) in the absence or presence of DEX (10 −7 M) for 24 h. Carotenoids and retinoids were extracted from culture media as described in “Materials and Methods”. Retinoic acid isomers were analyzed by HPLC as described above. * P < 0.05 ; ** P < 0.01 compared to respective controls.

Article Snippet: A BCO1 antisense complementary RNA (cRNA) probe was synthesized from a unique BCO1 coding region sequence obtained using a PCR amplified template and in vitro transcription (Promega, Madison, WI), T7 RNA polymerase and [α- 32 P]UTP (Amersham Biosciences).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Inhibition of pulmonary β-carotene 15, 15’-oxygenase expression by glucocorticoid involves PPARα

doi: 10.1371/journal.pone.0181466

Figure Lengend Snippet: A . Knockdown of endogenous PPARα mRNA and protein by PPARα-specific siRNA: A549 cells were transfected with non-specific control siRNAs or a PPARα-specific siRNA pool. After 72 h transfection, total RNA was isolated and RT-PCR performed as described in Materials and Methods. Cell lysates were subjected to western blot analysis using the indicated antibodies. Equal loading was confirmed by stripping and reprobing with β-actin antibody. B . Effects of siRNA-mediated knockdown of PPARα expression on DEX-inhibited BCO1 expression: A549 cells were transfected with non-specific control siRNAs or PPARα-specific siRNAs. After 48 h transfection, cells were treated with vehicle, DEX, or forskolin for 24 h. Equal amounts of cell lysates from each treatment were subjected to western blot analysis using the indicated antibodies. Equal loading was confirmed by stripping and reprobing blots with β-actin antibody. C . Effects of siRNA knockdown of PPARα on DEX inhibition of BCO1 mRNA. A549 cells were transfected with the non-specific siRNA or PPARα siRNA. After 48 h transfection, cells were treated with vehicle, DEX, or forskolin for 24 h. BCO1 mRNA expression was quantified by qRT-PCR. Relative fold changes are presented as 2 -ΔΔCt . Results are presented as means ± S.D. of three independent experiments each performed in triplicate. * P < 0.05; ** P < 0.01 compared to vehicle control.

Article Snippet: A BCO1 antisense complementary RNA (cRNA) probe was synthesized from a unique BCO1 coding region sequence obtained using a PCR amplified template and in vitro transcription (Promega, Madison, WI), T7 RNA polymerase and [α- 32 P]UTP (Amersham Biosciences).

Techniques: Knockdown, Transfection, Control, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stripping Membranes, Expressing, Inhibition, Quantitative RT-PCR

Journal: Cell Reports Methods

Article Title: A highly optimized human in vitro translation system

doi: 10.1016/j.crmeth.2024.100755

Figure Lengend Snippet:

Article Snippet: PCR Primers for creation of linear templates for in vitro transcription from plasmids (see ) , Microsynth , N/A.

Techniques: Luciferase, Virus, Recombinant, Western Blot, In Vitro, Plasmid Preparation, Software