Journal: Genome Biology

Article Title: Alternative splicing links histone modifications to stem cell fate decision

doi: 10.1186/s13059-018-1512-3

Figure Lengend Snippet: Isoform switch from PBX1a and PBX1b during hESC differentiation. a Genome browser view shows the AS event and H3K36me3 signals of PBX1 upon hESC differentiation. The green horizontal bars below the ChIP-seq tracks indicate the narrow peaks called by MACS2. b The inclusion level for exon 7 of PBX1 is significantly correlated to the H3K36me3 signals over this exon across cell lineages. c The sequence difference of three protein isoforms of PBX1 and the main functional domains. d The relative expressions of PBX1a and PBX1b in 56 cells/tissues, representing the differential expressions of two isoforms in three groups based on their developmental states. e The expression levels of NANOG and OCT4 genes are negatively correlated with the expression of PBX1b. f The expression levels of PSIP1 and SRSF1 show significant positive correlations with the expression level of PBX1a. Also see Additional file : Figures S9, S10

Article Snippet: The SRSF1-bound PSIP1 protein (also known as LEDGF) level was determined with the PSIP1 antibody (Human LEDGF Antibody, R&D Systems) using western blot as described above.

Techniques: ChIP-sequencing, Sequencing, Functional Assay, Expressing

Journal: Genome Biology

Article Title: Alternative splicing links histone modifications to stem cell fate decision

doi: 10.1186/s13059-018-1512-3

Figure Lengend Snippet: Isoform switch of PBX1 links H3K36me3 to hESC fate decision. a qRT-PCR and western blot show the expression levels of Yamanaka factors in H1, MSC, and IMR90 cells. Whiskers denote the standard deviations of three replicates. b RT-PCR and western blot show the isoform switches between PBX1a and PBX1b from H1 cells to differentiated cells. c i. ChIP-PCR shows the differential binding of PBX1b to NANOG promoter in H1 cells and differentiated cells; ii. ChIP-PCR shows the reduced H3K36me3 signal in differentiated cells; iii. ChIP-PCR shows the differential recruitment of PSIP1 to exon 7 of PBX1. d RIP-PCR show the differential recruitment of SRSF1 around exon 7 of PBX1. e Co-IP shows the overall physical interaction between PSIP1 and SRSF1 in all studied cell types. f The mechanism by which H3K36me3 is linked to cell fate decision by regulating the isoform switch of PBX1, which functions upstream of the pluripotency regulatory network. Also see Additional file : Figures S9, S10

Article Snippet: The SRSF1-bound PSIP1 protein (also known as LEDGF) level was determined with the PSIP1 antibody (Human LEDGF Antibody, R&D Systems) using western blot as described above.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Co-Immunoprecipitation Assay

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 1. Guide RNA adjacent to the coding sequence D366 shows efficient disruption of the PSIP1 gene. (a) Schematic representation of LEDGF/p75 protein with indication of the epitope sites of respective antibodies used in Western analysis. Below the human PSIP1 locus on chromosome 9 is depicted showing the different exons as light grey boxes. IBD is underlined in green. (b) Schematic of representing the location of the different gRNA that were used (red lines), gRNA1 close to D366 and two additional supporting gRNAs (gRNA_A, gRNA_B). D366 is shown in yellow. The expected PCR fragment sizes are indicated as well as the predicted deletions for the different gRNA combinations. Below the targeted gDNA sequence is shown. D366 is boxed in green, the PAM site is shown in red and the landing site of gRNA1 is shown in blue. (c) Agarose gel analysis showing truncated amplicons generated by DNA cleavage guided by a pair of gRNAs. Genomic DNA was extracted from polyclonal cell populations and PCR amplified using Fwd and Rv primers indicated in panel (b). The WT amplicon is indicated by the large arrow head. The lower migrating bands (small arrow head) indicate segmental deletion. (d) Western blot analysis showing LEDGF protein in a polyclonal HEK293T population transfected with the indicated gRNA pairs. Wild-type 293T cells (WT) are shown as control. (e) Immunocytochemical staining of endogenous LEDGF showing nuclear localization in WT and CRISPRed polyclonal HEK239T cells. Phalloidin-stained F-actin in white is shown as a counterstain. The respective antibodies used are indicated above. Scale Bar: 10 μm.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Sequencing, Disruption, Western Blot, Agarose Gel Electrophoresis, Generated, Amplification, Transfection, Control, Staining

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 3. HDR template design and screening. (a) Schematic representation of exon12 sequence of PSIP1 gene harboring D366 residue. Below the designed HDR template as a ssDNA oligo is shown with red characters indicating the sites of nucleotide substitutions, generating a VspI restriction site at D366N and deleting the PAM site. (b) Sanger sequencing results for the HEK293T_LEDGF D366N clone that was identified, showing consensus between HDR template and allele 1 (substitution indicated in red). The sequence for allele 2 shows a 8-nucleotide deletion (“−” indicates deletion) and the translation (capital letters below) resulting in a premature stop codon (underlined). (c) Western blot analysis showing the LEDGF/p75 levels in D366N clone compared to WT levels.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Sequencing, Residue, Western Blot

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 2. Characterization of HEK293T_LEDGF KO cell lines using a single gRNA1. (a) Western blot analysis of LEDGF/p75 protein in 3 LEDGF KO clones. (b) Immunocytochemical analysis of wild-type HEK293T (WT) cells and monoclonal LEDGF KO cells showing nuclear localization of LEDGF/p75 as dense, fine speckles in WT cells (shown in green) and the lack thereof in the KO cell line. Phalloidin (white) was used to counterstain cytoplasmic F-actin. LEDGF KO (clone1) image is shown and is representative of all 3 isolated KO cell lines. (c) Sanger sequencing of the indel profile generated in the region targeted by gRNA1 (“−” indicates deletion, substitutions indicated in red) and the predicted changes in the amino acid sequence of exon 12 on the right. (d) PSIP1 mRNA expression levels with standard deviation relative to β-actin in the respective knock-out clones as determined by qPCR. Error bars represent SD. Student’s t test was performed using GraphPad Prism 7.0 software. Sample means were considered significantly different from the WT control at p < 0.05 (*). Scale Bar: 10 µm.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Western Blot, Clone Assay, Isolation, Sequencing, Generated, Expressing, Standard Deviation, Knock-Out, Software, Control

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 4. LEDGF D366N mutant is uncoupled from HIV-IN but interacts with other binding partners. (a–c) In vivo co-localization of the LEDGF D366N and cellular binding partners. Wild-type HEK293T, LEDGF KO and LEDGF D366N cells were transfected with flag-tagged IWS1, JPO2 and HIV-IN (panels a, b and c, respectively) and fixed 30 hrs later. Fluorescence microscopy to assess localization of proteins: LEDGF/p75 and LEDGF D366N were detected using αLEDGF480–530 (shown in green), whereas the transfected flag-tagged proteins were detected with αFlag Ab (shown in red). (a,b) LEDGF WT (top panel) and LEDGF D366N (lower panel) co-localizes with IWS1 and JPO2. (c) LEDGF D366N mutant did not co-localize with HIV-IN (lower panel), while in HEK293T WT cells HIV-IN is retained in the nucleus by binding to LEDGF/p75 (top panel). Scale Bar: 10 µm. (d) Co-immunoprecipitation of LEDGF/p75 and LEDGF D366N by cellular binding partners. HEK293T_LEDGF KO cells were transfected with either WT or D366N mutant LEDGF plasmid along with either one of the plasmids encoding Flag-tagged binding partners: IWS1, JPO2 or HIV IN. Cells were lysed 24 h later and lysates were incubated with anti-FLAG® M2-agarose affinity resin to capture the binding partner protein, which was then visualized by Western blot using αFlag Ab and αLEDGF480–530.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Mutagenesis, Binding Assay, In Vivo, Transfection, Fluorescence, Microscopy, Immunoprecipitation, Plasmid Preparation, Incubation, Western Blot

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 5. HIV replication is severely affected in SupT1_LEDGF KO cells. (a) Schematic representation of a detailed zoom of exon12 sequence of the PSIP1 gene harboring D366. Sanger sequencing of the indel profile generated in the region targeted by gRNA1 (“−” indicates deletion, substitution indicated in red) and the predicted changes in the amino acid sequence of exon 12 shown on the right. (b) Western analysis of LEDGF/ p75 protein in the 3 isolated LEDGF KO cell lines. (c) PSIP1 mRNA expression relative to β-actin in the respective SupT1_LEDGF KO clones as determined by qPCR. Error bars represent SD. Student’s t test was performed using GraphPad Prism 7.0 software. Sample means were considered significantly different from the WT control at p < 0.05 (*). (d) HIV-1 replication assay. The respective cell lines were challenged with the laboratory HIVNL4.3 strain at a final concentration of 5.0*102 pg p24. Viral replication was monitored by daily sampling of p24 in the cell culture supernatant. The graph shows a representative infection experiment out of three independent trials.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Sequencing, Generated, Western Blot, Isolation, Expressing, Clone Assay, Software, Control, Concentration Assay, Sampling, Cell Culture, Infection

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 6. LEDGF depletion results in an increase of the silent HIV reservoir. (a) Schematic representation of the dual-colored VSV-G pseudotyped HIVOGH reporter virus carrying an eGFP cDNA driven by the viral LTR promoter in the nef position and an entire constitutive transcriptional unit (EF1a-mKO2) inserted downstream. (b) HIV provirus integration is greatly diminished in the absence of LEDGF/p75 when compared to SupT1_WT cells, as evidenced by the lower %mKO2 positive cells for a given virus concentration. Error bars represent SD. Student’s t test was performed using GraphPad Prism 7.0 software. Sample means were considered significantly different from the WT control at p < 0.05 (*). (c) The latent fraction for the respective cell lines for different virus concentrations. LEDGF KO increases the fraction of silently infected cells ((% eGFP−, mKO2+ cells)/% mKO2+ cells) ∗ 100. eGFP, Enhanced Green Fluorescent Protein; mKO2, Mutant Kusubira Orange 2. The experiment was performed three times. The plots are representatives of one of three independent infection experiments.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Virus, Concentration Assay, Software, Control, Infection, Mutagenesis

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 8. HIV replication is hampered in SupT1_LEDGF D366N cell line as compared to the SupT1 WT cell line. (a) HIV-1 replication assay. The cell lines were challenged with the laboratory HIVNL4.3 strain at a final concentration of 5.0*102 pg p24. Replication was monitored by daily sampling of the p24 levels in the cell culture supernatant. (b) Introduction of the D366N mutation in LEDGF/p75 renders SupT1 cells refractory to transduction as shown by a 4-fold decrease in %mKO2 expressing cells for a given VSV-G pseudotyped HIVOGH virus concentration. Student’s t test was performed using GraphPad Prism 7.0 software. Sample means were considered significantly different from the WT control at p < 0.05 (*). (c) In addition to the lower transduction efficiency observed for SupT1_LEDGF D366N, the latent fraction is greater when compared to SupT1_WT cells, even at higher concentrations of HIVOGH virus. eGFP, Enhanced Green Fluorescent Protein; mKO2, mutant Kusubira Orange 2. The plots represent one of three independent infection experiments.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Concentration Assay, Sampling, Cell Culture, Mutagenesis, Transduction, Expressing, Virus, Software, Control, Infection

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 7. Characterization of SupT1_LEDGF D366N. (a) Sanger sequencing results showing consensus between HDR template and allele 1. The HDR template is shown with red characters indicating the sites of nucleotide substitutions, generating a VspI restriction site at D366N and deleting the PAM site. The sequence for allele 2 shows a 22-nucleotide deletion gRNA1 (“−” indicates deletion), resulting in KO of the PSIP1 allele. (b) Western blot analysis for the SupT1_LEDGF D366N clone using C-terminal specific LEDGF antibody, αLEDGF480–530. SupT1_WT and KO cells are included as controls. (c) Immunocytochemical analysis of LEDGF D366N protein (green) in SupT1_LEDGF D366N cells (bottom panel) recapitulates that of LEDGF/p75 in SupT1_WT cells (top panel). Phalloidin (white) was used to stain F-actin. Scale Bar: 10 µm.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Sequencing, Western Blot, Staining

Journal: Scientific reports

Article Title: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.

doi: 10.1038/s41598-019-38718-0

Figure Lengend Snippet: Figure 9. HIV replication remains hampered in SupT1 LEDGF D366N cell line at high MOI as compared to the SupT1 WT and SupT1 LEDGF KO cell line. (a) HIV-1 replication assay at low MOI. The cell lines were challenged with the laboratory HIVNL4.3 strain at a concentration of 1.5*106 pg p24 for 2 h. The cells were then washed to remove the virus to result in 5.0*102 pg/ml p24 final concentration and monitored for replication. (b) HIV-1 replication assay at high MOI. The cell lines were challenged with the laboratory HIVNL4.3 strain at a final concentration of 1.5*106 pg p24. Replication was monitored by daily sampling of the p24 levels in the cell culture supernatant. Each plot represents one of three independent infection experiments.

Article Snippet: LEDGF/p75 was detected using rabbit anti-human LEDGF polyclonal antibody at 1:500 ON at 4 °C (A300–848A and A300–847A; Bethyl Laboratories Inc., Montgomery, TX).

Techniques: Concentration Assay, Virus, Sampling, Cell Culture, Infection

Journal: Cell

Article Title: L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity

doi: 10.1016/j.cell.2016.09.031

Figure Lengend Snippet: BAZ1B, PSIP1, and TSN Mediate the L-Arginine-Dependent Reprogramming of T Cells toward Increased Survival Capacity (A) Scheme of the limited proteolysis workflow. (B) Proteins that experience a structural change in response to 1 mM L-arginine but not to 1 mM D-arginine or L-ornithine. Transcriptional regulators are in orange, proteins are grouped according to their functions. Known interactions are indicated based on http://string-db.org/ and http://www.genemania.org/ . (C) Survival experiment with human CD4 + T cell clones devoid of the indicated proteins. Control (Ctrl), n = 39; Cas9-transduced control (Cas9 Ctrl), n = 45; BAZ1B-KO, PSIP1-KO, and PTPN6-KO, n = 46, n = 9, and n = 29, respectively. Each T cell clone was analyzed in triplicate. Bars represent the mean ± SEM. (D) Same as in (C). Cas9 Ctrl, n = 20; TSN-KO and B2M-KO, n = 23 and n = 3, respectively. (E) Percentage of living cells after IL-2 withdrawal of T cells cultured in Ctrl medium. Ctrl, n = 39; Cas9 Ctrl, n = 45; BAZ1B-KO, PSIP1-KO, and TSN-KO, n = 46, n = 9, and n = 29, respectively. (F–I) Western blots or FACS analysis of T cell clones showing deletion of target proteins. C refers to Cas9 Ctrl clones. Unspecific bands are marked with asterisk. An antibody to tubulin (Tub) was used as a loading control. B2M-KO was verified by staining cells with an antibody against MHC-I. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (Student’s t test). (C–E) Error bars represent SEM throughout. See also Figure S6 and .

Article Snippet: Human PSIP1 (LEDGF/p75) polyclonal , Bethyl laboratories , Cat#A300-848A.

Techniques: Clone Assay, Control, Cell Culture, Western Blot, Staining

Journal: Cell

Article Title: L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity

doi: 10.1016/j.cell.2016.09.031

Figure Lengend Snippet:

Article Snippet: Human PSIP1 (LEDGF/p75) polyclonal , Bethyl laboratories , Cat#A300-848A.

Techniques: Purification, Recombinant, Transfection, Sequencing, Software