Journal: Scientific Reports

Article Title: TGF-β1 increases viral burden and promotes HIV-1 latency in primary differentiated human bronchial epithelial cells

doi: 10.1038/s41598-019-49056-6

Figure Lengend Snippet: TGF-β signaling alters mRNA expression of HIV-1 host restriction factors in NHBE cells. NHBE ALI cultures were treated with recombinant TGF-β1 (10 ng/ml; vehicle as control). 48 hours post-treatment, total RNA was isolated and mRNA levels of IFITM3, PSIP1 and BLIMP-1 were determined by qRT-PCR. ( a ) TGF-β1 does not alter expression of IFITM3 mRNA. ( b ) TGF-β1 suppresses PSIP1 mRNA. ( c ) TGF-β1 increases mRNA levels of BLIMP-1 compared to vehicle treated controls. n = NHBE ALI cultures from 3 different lungs; *significant (p < 0.05).

Article Snippet: To examine mRNA expression, total RNA was extracted using the Qiagen RNeasy mini kit (Cat # 74104) and complementary DNA (cDNA) was reverse transcribed using the Applied Biosystems high-capacity cDNA reverse transcription kit (Cat # 4368814). qRT-PCR was performed on the Bio-Rad CFX96 real-time system using validated TaqMan probes (Life Technologies/Applied Biosystems HIV-LTR, Cat # Pa03453409_s1; IFITM3, Cat # Hs03057129_s1; BST2, Cat # Hs01561315_m1; BLIMP-1, Cat # Hs00153357_m1; PSIP1, Cat # Hs01045711_g1; and GAPDH, Cat # Hs02786624_g1). qRT-PCR results are represented as relative quantification normalized against internal control (GAPDH).

Techniques: Expressing, Recombinant, Control, Isolation, Quantitative RT-PCR

Journal: Animal Cells and Systems

Article Title: Biochemical functions of integrase-binding domain of lens epithelium-derived growth factor

doi: 10.1080/19768354.2015.1008040

Figure Lengend Snippet: Figure 1. LEDGF-IBD stimulates HIV-1 IN strand transfer activities. (A) Schematic illustration of in vitro HIV-1 IN activities. The 20/ 20 bp oligonucleotide of which one 5′-end was labeled with radioactivity of 32P (★) was used as the substrate for the endonucleolytic (or 3′-end processing) activity whereas the 18/20 by oligonucleotide which already recessed two nucleotides from the 3′-end was used as substrate for the strand transfer activity. (B) Endonucleolytic (3′-end processing) assay in the presence of LEDGF-IBD. The 0.1 pmol labeled 20/20 bp oligonucleotide substrates were incubated with purified HIV-1 IN of 50 µM as a final concentration and LEDGF-IBD of 0–7 µM as final concentrations at 33°C for 90 min. Conversion of 20-mer oligonucletides to 18-mer oligonucleotides was observed in a 15% polyacrylamide gel. (C) Strand transfer (integration) assay in the presence of LEDGF-IBD. The 0.1 pmol labeled 18/20 bp oligonucleotide substrates were incubated with purified HIV-1 IN of 50 µM as described in B. Strand transfer (integration) reaction will produce various oligonucleotides whose sizes are larger than 18-mer (18 + n), which is visualized in a 15% polyacrylamide gel.

Article Snippet: LEDGF-IBD was amplified by pfu polymerase using a LEDGF human cDNA (Origene, sc323120) and *Corresponding author.

Techniques: In Vitro, Labeling, Radioactivity, Activity Assay, Incubation, Concentration Assay

Journal: Animal Cells and Systems

Article Title: Biochemical functions of integrase-binding domain of lens epithelium-derived growth factor

doi: 10.1080/19768354.2015.1008040

Figure Lengend Snippet: Figure 3. LEDGF-IBD increases the concerted integration activities of HIV-1 IN. (A) LEDGF-IBD increases the concerted integration reaction of the 18/20 bp donor substrate. 80 nM 18/20 bp donor substrates were incubated with purified HIV-1 IN of 50 µM and LEDGF-IBD of the concentrations indicated above. Integration products were analyzed as above. TM: labeled target DNA marker [pGEM9Zf(–), 2925 bp], the plasmid was linearized by HindIII and labeled with 32P-γ-ATP. (B) LEDGF-IBD stimulates the concerted integration reaction of the 211/211 bp donor substrate. 40 nM 211/211 bp donor substrates were incubated with purified HIV-1 IN of 50 µM and LEDGF-IBD of the concentrations indicated above. FS, full-site integration; HS, half-site integration; M, molecular marker, a 100 bp marker DNA.

Article Snippet: LEDGF-IBD was amplified by pfu polymerase using a LEDGF human cDNA (Origene, sc323120) and *Corresponding author.

Techniques: Incubation, Labeling, Marker, Plasmid Preparation