Fig 2: Overexpression of Nurr1 in HC69 cells enhances HIV silencing.A, RNA-Seq confirmation of overexpression (OE) of Nurr1 in HC69 cells. Sequence read histograms for the Nurr1 locus is shown for control (vector) and Nurr1 overexpression. Annotated genes for the shown locus are indicated on the top, and the position of the locus on chromosome 2 is shown both at the top and the bottom. A read scale for each row is shown on the right, with the values for the overexpression studies drawn on a log2 scale. B, Verification of Nur77, Nurr1, and Nor1 overexpression by Western blot analysis in HC69 cell lines stably expressing 3X-FLAG-tagged Nur77, Nurr1, and Nor1 respectively by using a mouse monoclonal anti-FLAG M2 antibody (Sigma, Cat# F1804) and the nuclear receptor specific antibodies described in Fig 2B. HC69 cells stably carrying the 3X-FLAG-empty vector were used as a reference for comparison. The level of ß-tubulin was used as a loading control. Notably, the levels of endogenous Nur77 and Nor1 in HC69 cells were very limited. In contrast, Nurr1 was constitutively expressed in HC69 cells. C, Schematic depicting the TNF-a stimulation and chase studies. The four cell lines described in B were either untreated or treated with high dose (400 pg/ml) TNF-a for 24 hr. To examine HIV silencing, one set of TNF-a induced cells were used in a chase experiment by continuous culture of the cells in the absence of TNF-a for an additional 48 hr. The time points at which TNF-a is added or removed are shown by arrows on the top. D, Expression of HIV Nef protein in the different cell lines before and after TNF-a stimulation and at the end of the chase experiment was measured by Western blot analysis. The level of ß-tubulin was used as a loading control. E, Expression level of HIV mRNA (black bar graph) and Nurr1 (red rectangles and lines) in transcripts per million cellular transcripts are shown for each of the treatment steps shown in panel C in both vector-infected cells (on the left) and Nurr1 overexpressing cells (on the right half of the graph). For the 24 hr TNF-a stimulation step, both a low dose (20 pg/ml) and a high dose (400 pg/ml) are used. The values shown are the average of three replicate RNA-Seq samples with two standard deviations as error bars. The expression values for HIV and Nurr1 are shown on Y axes to the left and right, respectively.

Fig 3: KLF12 impaired hESC decidualization in vitro by repressing Nur77 expression. (a) and (b) hESCs were infected with Ad-Flag-KLF12, Ad-Flag-Nur77 or Ad-LacZ (MOI = 20). After 24 h, the cells were treated with 0.5 mM 8-Br-cAMP and 1 µM MPA as indicated for an additional 72 h. dPRL released into the medium was detected by ELFA. dPRL mRNA levels were measured by real-time PCR. *** P < 0.01 compared with the 8-Br-cAMP + MPA (-) group; # P < 0.05 and ### P < 0.001 compared with the 8-Br-cAMP + MPA (+) group; and @ P < 0.05 and @@ P < 0.01 compared with the Ad-Flag-KLF12 (MOI = 20) plus 8Br-cAMP + MPA group, ANOVA followed by a Student–Newman–Keuls test. (c) and (d) Attachment of BLSs and their expansion onto hESC monolayers. hESCs were infected with Ad-Flag-KLF12, Ad-Flag-Nur77 or Ad-LacZ (MOI = 20). After 24 h, the cells were treated with 0.5 mM 8-Br-cAMP and 1 µM MPA as indicated for an additional 72 h. Several spheroids were transferred to confluent monolayers of hESCs subjected to the different treatments. Cells were imaged after 12 h, 24 h, 36 h, and 48 h. The area of BLS expansion on the hESCs was detected at different time points and is presented relative to that of the Ad-LacZ group (area was set to 1). * P < 0.05 and ** P < 0.01 compared with Ad-LacZ group at the same time point; # P < 0.05 compared with Ad-Flag-KLF12 group at the same time point, repeated measures data ANOVA. e Analysis of blastocyst attachment and expansion. hESCs were infected with Ad-Flag-KLF12, Ad-Flag-Nur77 or Ad-LacZ (MOI = 20). After 24 h, the cells were treated with 0.5 mM 8-Br-cAMP and 1 µM MPA for an additional 72 h. Blastocysts were collected from the uterus of a pregnant mouse in the afternoon at 3.5 days post-coitus (dpc). Several blastocysts were transferred to confluent monolayers of hESCs subjected to the different treatments. Blastocysts were imaged after 24 h and 48 h

Fig 4: Enhanced KLF12 expression led to a decrease in Nur77 expression. a hESCs were infected with the indicated adenoviruses for 24 h, transfected with NBRE-Luc (300 ng/well), and then treated with 8-Br-cAMP and MPA. After 48 h, luciferase assays were performed, and the data were plotted after normalization to Renilla luciferase activity. ** P < 0.01 and *** P < 0.001 compared with the Ad-LacZ without 8-Br-cAMP and MPA group; ## P < 0.01 compared with the Ad-LacZ with 8-Br-cAMP and MPA group, ANOVA followed by a Student–Newman–Keuls test. (b) and (c) hESCs were infected with an adenovirus expressing either LacZ or KLF12 for 48 h at the indicated MOI. The KLF12 transcript copy number and protein expression were determined by real-time PCR and Western blotting. *** P < 0.001 compared with the Ad-LacZ only-treated group, ANOVA followed by a Student–Newman–Keuls test. (d) and (e) hESCs were infected with Ad-LacZ or Ad-Flag-KLF12 at the indicated MOI for 48 h and then treated with 8-Br-cAMP and MPA for another 2 or 3 days; subsequently, the Nur77 mRNA and protein levels were measured by real-time PCR and Western blotting, respectively. ** P < 0.01 compared with Ad-LacZ alone; # P < 0.05 and ## P < 0.01 compared with the Ad-LacZ plus 8-Br-cAMP and MPA group, ANOVA followed by a Student–Newman–Keuls test

Fig 5: KLF12 directly repressed Nur77 transcription. a hESCs were infected with the indicated adenoviruses for 24 h and then transfected with Nur77-Luc (-1604/+98 bp) or deleted Nur77-Luc (-710/+98 bp) (300 ng/well). After 48 h, luciferase assays were performed, and the data were plotted after normalization to Renilla luciferase activity. ** P < 0.05 compared with Ad-LacZ alone, Student’s t-test. b ChIP-PCR amplification using primers against the human Nur77 promoter region (top). PCR products were separated by agarose gel electrophoresis. Quantitative ChIP analysis was performed by real-time PCR. The results are shown as fold changes relative to LacZ (after normalization to the input DNA, bottom). Input (non-precipitated) chromatin was utilized as a positive control in these analyses. * P < 0.05 compared with Ad-LacZ alone, Student’s t-test. c ABCD assays were performed using biotinylated or non-biotinylated (competitor) double-stranded Nur77 wild-type (WT), conserved element-deleted (DEL) and conserved element-mutated (MUT) oligonucleotides with whole-cell extracts from hESCs