Journal: Nature cancer

Article Title: Targeting PAK4 to reprogram the vascular microenvironment and improve CAR-T immunotherapy for glioblastoma

doi: 10.1038/s43018-020-00147-8

Figure Lengend Snippet: a, ECs isolated from human GBM tumor (patient #5377) were transduced to express CRISPR targeting PAK4 or a random sequence, followed by transfection with plasmids expressing WT PAK4 or kinase-dead K350M mutant PAK4 or empty vector (EV). Cell lysates were analyzed by immunoblot. b, Purified MEF2D and PAK4 proteins were incubated in kinase buffer, followed by immunoblot analysis. These experiments were repeated independently twice with similar results.

Article Snippet: Recombinant human PAK4 (Abcam; ab96405) and MEF2D (Novus; H00004209-P01) proteins were incubated with 200 μM ATP (Cell Signaling Technology; 9804) in 40 μl 10× kinase buffer (Cell Signaling Technology; 9802) for 2 h at 30°C.

Techniques: Phospho-proteomics, Isolation, CRISPR, Sequencing, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Western Blot, Purification, Incubation

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 1 | Global redistribution of OCT4 binding in PGCs compared with ESCs. a, Cross-section of a human fetal testis (22 weeks) immunostained for OCT4. The panel on the right is an enlargement of the region enclosed within the white broken lines of the left panel. Scale bars, 50 µm; inset, 100 μm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. b, Left panel: heatmap visualization of OCT4 ChIP–seq data, depicting all binding events centred on the peak region within a 5 kb window around the peak. Right panel: distribution and peak heights of OCT4 peaks around the transcription start site (TSS). Peak heights are reported in reads per million. c, Scatterplot comparing OCT4 binding in PGCs and ESCs. Selected genes known to be associated with pluripotency are highlighted in blue, and those highly expressed in the germline are highlighted in red. d, Genome browser representation of ChIP–seq tracks for OCT4 in ESCs (red) and PGCs (yellow) at the OCT4 and PIWIL1 loci. Regions that were bound exclusively by OCT4 in ESCs or PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR (signals per million reads). ChIP–seq were independently repeated twice and similar results were obtained. e, Venn diagram of unique and shared genes bound by OCT4 in ESCs and PGCs. GO analysis results are shown to the right and bottom of the venn diagram. Y-axes represent each category of molecular functions and biological processes. The analysis was performed twice and similar results were obtained based on two independent ChIP–seq data.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: Binding Assay, Immunostaining, ChIP-sequencing

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 2 | Co-occurrence of OCT4 with PAX5 and PRDM1 in PGCs. a, The position weight matrix of an enriched motif found in OCT4 ChIP–seq data from PGCs. The motif resembles the binding motifs for PRDM1 and PAX5. b, Cross-section of a human fetal testis (22 weeks). Upper panel: immunostained for PAX5 (red) and OCT4 (green), and DAPI stained for nuclei (blue). Lower panel: immunostained for PRDM1 (red) and OCT4 (green), and DAPI stained for nuclei (blue). Enlarged panels on the right represent the region enclosed within the white broken lines of the far left panel. White arrows indicate co-localization of PAX5 and OCT4 or PRDM1 and OCT4. Scale bars (orignal images), 100 µm; (expanded images) 50 µm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. c, Venn diagram of unique and shared genes bound by OCT4, PRDM1 and PAX5 in PGCs. The number of genes bound exclusively by each transcription factor or co-bound by multiple transcription factors are labelled. d, Genome browser representation of ChIP–seq tracks for OCT4 (yellow), PAX5 (blue) and PRDM1 (green) at the TBX3 and PIWIL1 loci. Regions that are bound collectively by OCT4, PAX5 and PRDM1 in PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR (signals per million reads). ChIP–seq were independently repeated twice and similar results were obtained. e, GO analysis of co-bound genes. The analysis was performed twice and similar results were obtained. f, GST pull-down assay performed using OCT4 and PAX5 recombinant proteins. Pull-down was repeated three times and similar results were obtained. Unprocessed scans of western blots are shown in Supplementary Fig. 8.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Binding Assay, Staining, Immunostaining, Pull Down Assay, Recombinant, Western Blot

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 5 | PAX5 acts upstream of OCT4. a, Genome browser representation of ChIP–seq tracks at the OCT4 locus. Enhancer regions are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR. ChIP–seq was independently repeated twice and similar results were obtained. b, OCT4 expression in hESCs, control cells and cells overexpressing PAX5 during in vitro differentiation. Data represent the mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. c, Mouse testis xenografts immunostained for GFP and c-KIT. Scale bar, 50 µm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. DAPI stained for nuclei. d, Flow cytometry analysis for GFP and c-KIT of mouse testis xenografts. e, RT-qPCR analysis of OCT4 expression in hPGCs formed in mouse seminiferous tubules by cells overexpressing PAX5, PAX5 knockout cells or control hESCs. Data represent the mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. f, Reporter construct used for measuring OCT4 enhancer activity is shown. The genomic fragment bound by PAX5 and OCT4 (red) was inserted upstream of a luciferase gene driven by a minimal promoter. The y axis represents the fold enrichment of luciferase activity. Data represent the mean ± s.d. of n = 3 independent replicates. Source data for b, e and f are provided in Supplementary Table 2.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Expressing, Control, In Vitro, Two Tailed Test, Immunostaining, Staining, Flow Cytometry, Quantitative RT-PCR, Knock-Out, Construct, Activity Assay, Luciferase

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 6 | PAX5 and OCT4 act upstream of PRDM1. a, Genome browser representation of ChIP–seq tracks at the PRDM1 locus. Enhancer regions bound by OCT4 and PAX5 in PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR. ChIP–seq was independently repeated twice and similar results were obtained. b, PRDM1 expression in control cells, cells overexpressing PAX5 and PAX5 knockout cells during in vitro differentiation. Data are represented as mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. c, RT-qPCR analysis of PRDM1 expression in hPGCs formed in the mouse seminiferous tubules by PAX5 overexpressing, PAX5 knockout and control hESCs. Data are represented as mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. d, Reporter construct used for measuring PRDM1 enhancer activity is shown. The genomic fragment bound by PAX5 and OCT4 (red) was inserted upstream of a luciferase gene driven by a minimal promoter. The y axis represents the fold enrichment of luciferase activity. Data represent the mean ± s.d. of n = 3 independent replicates. e, RT-qPCR analysis of the expression of genes associated with germline programming. Data represent the mean ± S.D. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. f, Model for gene regulation in pluripotency and germline programmes. In pluripotent stem cells, OCT4, together with other transcription factors (TFs) and cofactors, binds to its own enhancer to activate and maintain its high expression. While differentiating towards germline cells, PAX5 replaces OCT4 and binds to the enhancer of OCT4 to maintain a moderate expression of OCT4. Meanwhile, PAX5 and OCT4 bind to the enhancer of PRDM1 and activate its expression to initiate the germline programme. Source data for b, c, d and e are provided in Supplementary Table 2.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Expressing, Control, Knock-Out, In Vitro, Two Tailed Test, Quantitative RT-PCR, Construct, Activity Assay, Luciferase

Journal: bioRxiv

Article Title: Inhibition of Mul1-mediated ubiquitination promotes mitochondria-associated translation

doi: 10.1101/2021.07.28.454107

Figure Lengend Snippet: A, Detection of K63 ubiquitination of PABPC1 purified by specific K63-ubiquitin binding domain Vx3K0 by IP/WB using both whole cell extracts and fractionated mitochondrial extracts in GPS2 WT and KO MEFs. B, Detection of K63 ubiquitination by IP/WB of PABPC1 in mitochondria extracts in GPS2 WT, KO and KO treatment with UBC13 inhibitor MEFs. C, FCCP-induced ubiquitination of PABPC1 is mediated by mitochondria specific E3 ligase MUL1. D, PABPC1 is polyubiquitinated by the E3 ubiquitin ligase MUL1 by in vitro ubiquitination assay. E, Polyubiquitination on PABPC1 mediated by E3 ubiquitina ligase MUL1 is significantly inhibited by GPS2 in in vitro ubiquitination assay. F, WB analysis of binding of PABPC1 with EIF4G, PAIP1 and PAIP2 on mitochondrial extracts from GPS2 WT and KO MEFs. G, WB analysis of binding of PABPC1 with PAIP1 and PAIP2 on mitochondrial extracts from GPS2 WT, KO and KO knockdown MUL1 (siMul1) MEFs. H, Puromycin labeling of de novo protein synthesis on mitochondrial extracts from GPS2 WT, KO and KO knockdown MUL1 (siMul1) MEFs. Anti-GAPDH blot was used as loading control.

Article Snippet: Ubiquitination assays were carried out at 30°C for 2h in 50mM Tris-HCl, pH7.6, containing 50mM NaCl, 5mM MgCl2, 5mM ATP, 1x Ubiquitin Aldheide, 50nM E1, 5μg ubiquitin, 200 nM UbcH13– Uev1a E2 complexes (Boston Biochem), 0.1ug recombinant human MUL1 Protein (H00079594-P01, Novus Biologicals).

Techniques: Ubiquitin Proteomics, Purification, Binding Assay, In Vitro, Knockdown, Labeling, Control