Journal: Cell stem cell

Article Title: Highly cooperative chimeric super-SOX induces naive pluripotency across species.

doi: 10.1016/j.stem.2023.11.010

Figure Lengend Snippet: Figure 1. Reprogramming screen of Sox2-Sox17 chimeric TF library (A) Bright-field and Oct4-GFP merged overview images showing retroviral reprogramming of Oct4-GFP (OG2) MEFs on 21 days post infection (scale bars, 1 mm). (B) Schematic representation of Sox2 and Sox17 structures and chimeric TFs. Sequence from Sox2 in blue and Sox17 in red. Superscripts represent residues swapped from Sox17- to Sox2-HMG, e.g., Sox242–46 has residues 43–47 from Sox17, and Sox2c17 represents a complete replacement of Sox2-CTD with Sox17-CTD. (C) Protein sequence alignment of DNA binding domains of mouse and human Sox2, Sox17, and the most crucial chimeric Soxes in this study. (D–G) Reprogramming of OG2 MEFs by retroviral vectors carrying Klf4, Sox2/Sox17 chimeric TFs, and Oct4 (D), Oct4L80A (E and F), or Brn4 (G).

Article Snippet: The mouse and human protein sequences of Sox2AV and Sox2-17 were (HMG-box domains are uppercase, Sox17 parts are in bold): >Mouse Sox2A61V mynmmetelkppgpqqasgggggggnataaatggnqknspDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRP FIDEAKRLRVLHMKEHPDYKYRPRRKTKTLMKKDKytlpggllapggnsmasgvgvgaglgagvnqrmdsyahmngwsngsysmmqeqlgypqhpglnah gaaqmqpmhrydvsalqynsmtssqtymngsptysmsysqqgtpgmalgsmgsvvkseasssppvvtssshsrapcqagdlrdmismylpgaevpepaapsrlhmaqh yqsgpvpgtaingtlplshm e3 Cell Stem Cell 31, 1–21.e1–e9, January 4, 2024 >Human SOX2A61V mynmmetelkppgpqqtsgggggnstaaaaggnqknspDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFI DEAKRLRVLHMKEHPDYKYRPRRKTKTLMKKDKytlpggllapggnsmasgvgvgaglgagvnqrmdsyahmngwsngsysmmqdqlgypqhpglnahg aaqmqpmhrydvsalqynsmtssqtymngsptysmsysqqgtpgmalgsmgsvvkseasssppvvtssshsrapcqagdlrdmismylpgaevpepaapsrlhmsqhy qsgpvpgtaingtlplshm >Mouse Sox2-17 mynmmetelkppgpqqasgggggggnataaatggnqknspDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKALTLAEKR PFIDEAKRLRVLHMQDHPNYKYRPRRRKQVKRMKRVeggflhalvepqagalgpeggrvamdglglpfpepgypagpplmsphmgphyrdcqglg apaldgyplptpdtspldgveqdpaffaaplpgdcpaagtytyapvsdyavsveppagpmrvgpdpsgpampgilappsalhlyygamgspaasagrgfhaqpqq plqpqapppppqqqhpahgpgqpspppealpcrdgtesnqptellgevdrtefeqylpfvykpemglpyqghdcgvnlsdshgaissvvsdassavyycnypdi >Human SOX2-17 mynmmetelkppgpqqtsgggggnstaaaaggnqknspDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKALTLAEKRPF IDEAKRLRVLHMQDHPNYKYRPRRRKQVKRLKRVeggflhglaepqaaalgpeggrvamdglglqfpeqgfpagppllpphmgghyrdcqslgappld gyplptpdtspldgvdpdpaffaapmpgdcpaagtysyaqvsdyagppeppagpmhprlgpepagpsipgllappsalhvyygamgspgagggrgfqmqpqh qhqhqhqhhppgpgqpspppealpcrdgtdpsqpaellgevdrtefeqylhfvckpemglpyqghdsgvnlpdshgaissvvsdassavyycnypdv iPSC generation and characterization Mouse reprogramming experiments were done as described before.13,36 Briefly, for retrovirus production monocistronic pMX-Oct4, Sox (Addgene #193350-193354), and Klf4 vectors were co-transfected with pCL-Eco (Addgene #12371)172 in HEK293 cells with FuGENE6 (Promega) using low volume transfection protocol (Steffen et al., 2017).

Techniques: Retroviral, Infection, Sequencing, Binding Assay

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Morphology of the prostatic ductal system of the dorsal prostate showing proximal (Prox) and distal regions. U, urethra. Scale bar, 0.5 mm. (B and C) The relative expression levels of Klf4 (B) mRNA and (C) protein in the proximal stem cell niche and the distal ductal region. Experiments were repeated 2 times. Data represent means ± SDs. p = 0.0005. (D) Western blot indicating Klf4 expression in proximal and distal ducts. (E) Duct-forming ability of proximal cells (3 × 10 4 ) in collagen gels infected with shCon or shKlf4 lentivirus. Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.03. (F) Proximal cells were infected with shCon or shKlf4 lentivirus and inoculated (5 × 10 4 ) sub-RC with UGM (2 × 10 5 ). Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.03. (G) Prostatic tissue generated by shCon or shKlf4 cells was examined for CK5, CK8, and Ki67 expression. Scale bars, 35 μm. (H) Proximal cells were infected with Akt lentiviruses combined with control (shCon + Akt) or shKlf4 (shKlf4 + Akt) lentiviruses and seeded (3 × 10 4 ) in collagen gels. Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.02. (I) Proximal cells were infected with shCon + Akt or shKlf4 + Akt lentiviruses and inoculated sub-RC (5 × 10 4 ) with UGM (2 × 10 5 ). Experiments were repeated 3 times. Data represent means ± SDs. p < 0.02. (J) PIN and carcinoma-containing tubules in shCon + Akt or shKlf4 + Akt grafts were determined in all fields in 3 sections from each graft (3 grafts per condition). p < 0.00001. (K) Grafts from shCon + Akt and shKlf4 + Akt cells were examined by H&E and for expression of CK5, CK8, pAkt, Ki67, and Klf4. Scale bars, 30 μm. See also Figures and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Expressing, Western Blot, Infection, Generated

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Expression of Sca-1 and CD49f by the Akt cell line (right); cells with control antibodies (left). (B) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control (shCon) or shKlf4 lentivirus. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p = 0.001. (C) Spheres from shKlf4 and shCon Akt cell line were dissociated, and equal numbers of cells (2,000) were passaged 6 times in triplicate in Matrigel. The sphere number diminished with passage as cells became more migratory and infiltrated the Matrigel (bottom). Scale bars, 30 μm. (D) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control or Klf4-expressing constructs. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p < 0.006.

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Expressing, Infection, Construct

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Cells from the Akt cell line were infected with non-silencing control (shCon) orshKlf4 (shKlf4) lentiviruses and implanted (2 × 10 6 ) sub-RC. Experiments were repeated at least 3 times. Data represent means ± SDs. p = 0.0006. (B) Gross tumors after implantation of shCon or shKlf4-infected Akt cell line after 4 weeks. T, tumor; G, glomerulus. Scale bars, 35 μm. (C) Sub-RC grafts were examined for expression of E-cadherin (E-cad), vimentin (vim), Slug, and Ki67. Scale bars, 25 μm. (D) Expression of E-cadherin (E-cad), ZO-1, CK5, CK8, and vimentin (vim) in the Akt-transformed cell line after infection with control or shKlf4-expressing lentiviruses. Scale bars, 50 μm. (E) Western blot of Klf4, E-cadherin (E-cad), Slug, Zeb1, and Snail after infection of the Akt cell line with control or shKlf4-expressing lentiviruses. See also .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Infection, Expressing, Transformation Assay, Western Blot

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Western blot of KLF4, Slug, vimentin (Vim), Zeb1, and Snail in control cells (shKlf4 + EV) and in cells rescued by expressing KLF4 (shKlf4 + KLF4). (B) Sub-RC grafts of control (shKlf4 + EV) or KLF4-expressing cells (shKlf4 + KLF4) (1 × 10 5 cells). Experiments were repeated 3 times. Data represent means ± SDs. p = 0.002. (C) Sub-RC grafts were examined by H&E (arrowhead indicates duct-like morphology) and for expression of CK8, KLF4, and Ki67. Scale bars, 45 μm. See also .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Western Blot, Expressing

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Venn diagram of differentially expressed genes (FDR <0.01, FC >2) in the Akt-transformed cell line after Klf4 knockdown (shKlf4) versus KLF4 re-expression (shKlf4 + KLF4). (B) Heatmap of EMT genes regulated by decreased (shKlf4 + empty vector [EV]) or increased KLF expression (shKlf4 + KLF4). (C) Heatmap of stem cell genes regulated by Klf4 knockdown (shKlf4) and KLF4 re-expression. (D) Ingenuity pathway analysis (canonical pathway) reveals pathways regulated by decreased (shKlf4) or increased (shKlf4 + KLF4) KLF4. Left: −log(p value), right: Z score. Pathways highlighted in red represent those that are inversely regulated by knockdown and re-expression of Klf4 (inverse Z score). (E) GSEA reveals positive enrichment of genes in a tumor metastasis gene signature after decreased (shKlf4) Klf4 expression. (F) GSEA reveals positive enrichment of genes in a tumor suppressor gene signature after increased (shKlf4 + KLF4) KLF4 expression. (G) Overlap of genes after Klf4 knockdown in the Akt-transformed cell line with those expressed in a cohort of human metastatic prostate cancer. (H) Overlap of genes after Klf4 knockdown in the Akt-transformed cell line with those expressed in a cohort of aggressive human prostate cancer. See also and Tables , , , and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Transformation Assay, Expressing, Plasmid Preparation

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Primary prostate cancer patients whose tumors express increased levels of KLF4 (21 of 131 cases [red line] have no biochemical recurrence for 10 years. (B) Increased levels of KLF4 predict prolonged survival in primary prostate cancer patients whose tumors have low Gleason scores (3 + 3 and 3 + 4). (C) Expression of KLF4 in 455 patients with primary prostate cancer from TCGA database as a function of Gleason scores. (D) Heatmap indicating the chromatin immunoprecipitation sequencing (ChIP-seq) read density within ±3 kb of the TSS region. (E) Pie chart illustrating the relative distribution of KLF4-bound sequences across the genome. (F) Motif analyses discovered Klf4 consensus motifs at ChIP-seq peak centers (p = 1e–175). (G) Heatmap representing 193 direct Klf4 target genes identified by combinatorial ChIP-seq and RNA-seq analyses of shControl, shKlf4, shKlf4 + EV, and shKlf4 + KLF4. (H) Bar graph illustrating ranked KEGG pathway analysis of 1,528 genes bound by Klf4 and regulated by Klf4 knockdown. See also and Tables and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Expressing, ChIP-sequencing, RNA Sequencing Assay

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) ChIP-seq (red) and RNA-seq (blue and purple) tracks indicate Klf4 enrichment on gene regulatory elements and transcript changes of good prognosis genes (ATF3, JUND, LIF, TEAD4) after Klf4 knockdown and rescue, respectively. shCon and shKlf4 depicted at scale 0–2.63. shKlf4 + EV and shKlf4 + KLF4 depicted at scale 0–0.46. Asterisks depict Klf4 motif locations. (B) Increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 predict prolonged survival (red line) (p = 0.003) in prostate cancer patients whose tumors have low Gleason scores. (C) Prostate cancer patients whose tumors express increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 (red line) have a good prognosis (p = 0.0002). (D) Increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 predict prolonged survival (red line) (p = 0.008) in prostate cancer patients whose tumors have low Gleason scores. (E) Prostate cancer patients whose tumors express increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 (red line) (p = 0.018) have a good prognosis. (F) Expression of ATF3, JUND, KLF4, LIF, and TEAD4 in 455 patients with prostate cancer from TCGA database as a function of Gleason scores. (G) Cox proportional hazard model on Gleason 6 and 7(3 + 4) comparing the performance of ATF3, JUND, KLF4, LIF, and TEAD4 expression with Gleason score alone or with the D’Amico classification. DF, degree of freedom. See also Figures , , and , and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: ChIP-sequencing, RNA Sequencing Assay, Expressing

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Plasmid Preparation, Recombinant, SYBR Green Assay, Transformation Assay, shRNA, Software