The cDNA was prepared using MMLV Reverse Transcriptase (Invitrogen, USA) followed by PCR ampli- ﬁcation for HSP40 and human b-Actin with Taq polymer- ase (Invitrogen, USA) using standard conditions and gene-speciﬁc primers listed in Supplementary Table S1. Construction of HSP40 promoter point mutant reporter vectors by site-directed mutagenesis HSP40 wild-type promoter construct was used as a template for creating mutation at different transcription factor binding sites by PCR ampliﬁcation using different mutagenic primers (see Supplementary Table S2) and Quik change site directed mutagenesis kit (Stratagene, USA) following manufacturer’s instructions. Preparation of nuclear and cytoplasmic fraction Uninfected and HIV-1-infected Jurkat cells were used to prepare nuclear and cytoplasmic extracts using ProteoJet Cytoplasmic and Nuclear Extraction Kit (Fermentas, Germany). These extracts were used to study phosphoryl- ation of HSF1 using pHSF1 (Ser230) antibody. Chemical cross-linking by ethylene glycol bis(succinimidylsuccinate) Uninfected and HIV-1-infected CEM-GFP cells (5 106) were resuspended in 100–200 ml cold lysis buffer followed by lysis on ice for 30 min. The cell lysate was spun at 12 000 rpm for 10 min at 4 C. About 100 mg of protein was used for cross-linking using ethylene glycol bis(succini- midylsuccinate) (EGS) as described in details elsewhere (32). Electrophoretic mobility shift assay Various synthetic oligonucleotide probes spanning the NF-kB-Sp1 region of HIV-1 LTR were labeled using [g32P] dATP by T4 polynucleotide kinase (NEB, USA) for 30 min at 37 C and were puriﬁed by Probequant G-50 columns (Amersham Biosciences, USA). Binding re- actions were set up with both recombinant His-HSF1 protein and nuclear extract from HIV-1-infected Jurkat cells. Binding reactions were incubated at 37 C for 15 min and loaded on 6% polyacrylamide gel. The nucleo- tide sequence of probes P1, P2, P3 and P4 are listed in Supplementary Table S3. Chromatin immunoprecipitation CEM-GFP cells (2 107) were infected with NL4-3 virus at 0.1 MOI. On Day 5 post-infection, cells were harvested and ﬁxed with 1% formaldehyde. Following ﬁxation, chromatin immunoprecipitation (ChIP) was performed by primary immunoprecipitation with Nef polyclonal antibody using ChIP assay kit (Upstate Biotechnology, USA) according to manufacturer’s instructions. In se- quential ChIP experiment, the eluate after Nef immuno- precipitation was re-incubated with either rat monoclonal HSF1 antibody or rabbit polyclonal Sp1 antibody over- night at 4 C. The occupancy on HSP40 promoter was checked using different primers as schematically shown in Figure 2C. The primers used for PCR ampliﬁcation of HSP40 promoter region is given in Supplementary Nucleic Acids Research, 2011, Vol. 39, No. 14 5881 Table S4. Similar ChIP analysis was also performed for recruitment of HSF1 on the HIV-1 LTR promoter using HSF1 antibody. The sequence of primers used in the LTR ChIP is listed in Supplementary Table S5. Immunoﬂuorescence microscopy HEK-293T cells grown on cover slips were transfected with both pcDNA-Nef and pCMV-HSF1 or pNL4-3 by Lipofectamine 2000 and harvested 24 h post-transfection for immunoﬂuorescence studies. Paraformaldehyde- ﬁxed and permeabilized cells were blocked with 10% FCS and stained with HSF1 and Nef antibody, respect- ively. The secondary antibodies used for HSF1 and Nef were Cy3-conjugated goat anti-rabbit IgG and Cy2- conjugated to goat anti-mouse IgG, respectively. After washing, cells were counterstained with DAPI present in the mounting media and the slides were analyzed with a confocal microscope (Zeiss LSM 510, Germany). Similarly, uninfected and infected Jurkat cells were ﬁxed and were spun on a glass slide to obtain a monolayer of cells. Then the cells were stained with polyclonal HSF1 antibody or pHSF1 (Ser230) antibody. The secondary antibody used was Cy3-conjugated goat anti-rabbit IgG. Cells were counterstained with DAPI and analyzed with a confocal microscope. Quantitation of HSF1 expression by RT–PCR HSF1 expression was analyzed by quantitative real-time RT–PCR in a 10 ml reaction mixture containing SYBR Green IQ supermix (Bio-Rad, USA) and 10 pmol concen- tration of each of the human GAPDH and HSF1 primer pairs (see Supplementary Table S6) using the Realplex4 Mastercycler (Eppendorf, Germany). The ampliﬁcation was performed using one cycle of 95 C for 2 min and 40 cycles of 94 C for 1 min, 60 C for 30 s and 68 C for 1 min followed by melt curve analysis. The changes in the threshold cycle (CT) values were calculated by the equation CT = CT,target CT,input. The fold difference was calculated as follows: fold difference ¼ 2ð Statistical analysis CTÞ: All experiments were repeated at least three times. The error bars represent the mean ± SEM of three independ- ent experiments. Statistical analysis of the experimental data was performed using Student’s t-test, with the levels of signiﬁcance deﬁned as *P < 0.05. RESULTS HIV-1 Nef induces HSP40 promoter activity We have reported earlier that HSP40 is induced in Nef- dependent manner in HIV-1 NL4-3 virus transfected HEK-293T cells (15). In order to conﬁrm this ﬁnding in HIV-1-infected T cells, we ﬁrst infected CEM-GFP cells with wild-type and nef-deleted NL4-3 virus and analyzed HSP40 expression on different days post- infection by both RT–PCR and immunoblotting.