Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A functional screen identifies transcriptional networks that regulate HIV-1 and HIV-2.

doi: 10.1073/pnas.2012835118

Figure Lengend Snippet: Fig. 3. KLF2 and KLF3 regulate HIV-1 transcription in CD4+ T cells. (A) Schematic of HIV LTR highlighting where ChIP primers bind and approximate location of previously described transcription factor binding sites for NF-κB, Sp1, and TATA as well as the transcription start site (arrow +1) and TAR el- ement. (B) Unstimulated resting CD4+ T cells that were depleted of CD25+, CD69+, and HLA_DR+ cells were infected with HIV (40). After 72 h, cells were either activated with anti-CD3/CD28 (gray bars) or left unstimulated (black bars) for 24 h. ChIPs using anti-KLF2 or anti-KLF3 and mouse IgG were per- formed. Associated HIV-1 LTR DNA was measured by RT-qPCR and normal- ized against input chromatin. n = 6 independent ChIPs for resting cells; n = 5 independent ChIPs for activated cells. Data are presented as mean ± SEM. (C) Unstimulated resting CD4+ T cells were infected with HIV and siRNA tar- geting KLF2, KLF3, or nontargeting control were nucleofected into cells. Cells were harvested for HIV transcriptional analysis 48 h later. KLF2 and KLF3 mRNAs were measured by RT-qPCR in HIV-infected CD4+ T cells tar- geted by the indicated siRNAs. n = 9 independent knockdowns for siCtrl, n = 8 independent knockdowns for siKLF2 and siKLF3, and n = 4 independent knockdowns for siKLF2+siKLF3. Data are presented as mean ± SEM (Mann–Whitney U test). (D) KLF2 and KLF3 protein expression were mea- sured by Western blot in unstimulated resting CD4+ T cells nucleofected with siKLF2 or siKLF3 (n = 1; representative of three experiments). β-actin was the loading control. (E) HIV-1 RNA expression in cells treated with control, KLF2, or KLF3 siRNAs was measured by RT-qPCR. n = 9 experiments for siCtrl, n = 8 experiments for siKLF2 and siKLF3, and n = 4 experiments for siKLF2 + siKLF3. Data are shown as box and whisker. Bars are the range of values, and the horizontal line is the median. Significance was determined by Mann– Whitney U test. **P < 0.005, ***P < 0.00005.

Article Snippet: Transcription factor expression constructs were KLF2 (Origene, SC127849), KLF3 (Addgene, 49102), PLAGL1 (Origene, SC115928), or empty pcDNA3 vector.

Techniques: Binding Assay, Infection, Quantitative RT-PCR, Control, MANN-WHITNEY, Expressing, Western Blot, RNA Expression, Whisker Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A functional screen identifies transcriptional networks that regulate HIV-1 and HIV-2.

doi: 10.1073/pnas.2012835118

Figure Lengend Snippet: Fig. 4. KLF2 and KLF3 bind the HIV-1 LTR and correlate with H3 acetylation. HEK293T cells were infected with HIV-1 for 24 h. Expression constructs were transfected into cells and harvested 24 h later. ChIPs with antibodies specific to (A) KLF2, (B) KLF3, and (C) acetylated H3 and control mouse or rat IgG were performed. Associated LTR DNA was measured by RT-qPCR and normalized to input chromatin. Representative data from two experiments are shown. Data are presented as mean ± SD. *P < 0.05. (D) Western blot for H3 in cytoplasmic and nuclear lysates prepared from transfected cells. β-actin was used as loading control for cytoplasmic extracts, and HDAC1 was used as a control for nuclear extracts (n = 1; representative of three independent transfections).

Article Snippet: Transcription factor expression constructs were KLF2 (Origene, SC127849), KLF3 (Addgene, 49102), PLAGL1 (Origene, SC115928), or empty pcDNA3 vector.

Techniques: Infection, Expressing, Construct, Transfection, Control, Quantitative RT-PCR, Western Blot