Hif 1α antibody

HPAs were seeded in a 100-mm petri dish at the density of 2 × 10⁶ cells and incubated overnight at 37°C in a humidified, 5% CO₂ incubator. Cells were serum starved at approximately 70%–80% confluency, followed by exposure to HIV-1 Tat. Control and HIV-1 Tat–treated HPAs were harvested and RIP assay was performed with EZ-Magna RIP kit (Millipore Sigma, St. Louis, MO, 17–701) as per the manufacturer’s protocol. HIF-1α antibody (Millipore Sigma, St. Louis, MO, MAB5382) was used to perform the immunoprecipitation of RNA-binding protein/RNA complexes. Isolated RNAs from the input and enrichment fractions were used to either prepare cDNA using the verso cDNA synthesis kit (Thermo Fischer Scientific, Waltham, MA, AB 1453A) or for RNA sequencing. The reverse transcribed RNA was analyzed with the 7500 Fast Real-Time PCR System (Applied Biosystems, Grand Island, NY) with the TaqMan Universal PCR Master Mix (Applied Biosystems, Grand Island, NY 4304437, Thermo Fisher Scientific, Waltham, MA,), and BACE1-AS primers were used for qRT-PCR.

Similar to the RIP assay, HPAs were seeded in a 100-mm petri dish at the density of approximately 2 × 10⁶ cells and incubated overnight at 37°C in a humidified, 5% CO₂ incubator. Cells were serum starved at approximately 70%–80% confluency, followed by HIV-1 Tat exposure. Control and HIV-1 Tat–treated HPAs were harvested, and ChIP assay was performed using the EZ-Magna ChIP A/G ChIP kit (Millipore Sigma, St. Louis, MO, 17–10086) as per the manufacturer’s protocol. HIF-1α antibody (Millipore Sigma, St. Louis, MO, MAB5382) was used to perform the immunoprecipitation of DNA-binding protein/DNA complexes. Isolated DNAs were analyzed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Grand Island, NY) with the RT² SYBR Green Fluor qPCR Mastermix (Qiagen, Hilden, Germany, 330510) or TaqMan Universal PCR Master Mix (Applied Biosystems, 4304437, Thermo Fisher Scientific, Waltham, MA,) for the BACE1 promoter. PCR products were subjected to agarose gel electrophoresis to confirm the binding of HIF-1α to the promoter of BACE1.

HUVECs and MCF-7 cells were grown on coverslips and treated with AECHL-1 in combination with VEGF, TNF-α or Deferoxamine. Cells grown till 70 % confluence were fixed with 3.7 % paraformaldehyde for 5 min, followed by permeabilization and blocking in 0.2 % Triton X100 and 5 % milk, respectively. Cells were washed three times with 1x PBS and incubated with primary antibody (HIF-1α, WAVE-2, IQGAP-1) and their respective Alexafluor-conjugated secondary antibodies. Nuclei were labeled using DAPI. The conditions for incubations, concentration of primary and the secondary labeled antibodies were standardized. F-actin was visualized using phalloidin (Molecular Probes) conjugated to either Alexafluor 488 or 647. The cells were viewed under CFLSM. Tumor vasculature and pericyte coverage were visualized in 8- and 30-μm-thick cryosections using CD-31 (1:50 BD Bioscience) and α-SMA (1:500, Sigma) antibodies, respectively, according to established protocols [29 (link), 30 (link)]. For studies involving immunohistochemistry, formalin-fixed paraffin-embedded (FFPE) sections of tumors were incubated with HIF-1α antibody (1:50, Sigma) and processed according to previously described protocol [31 (link)].