Journal: bioRxiv

Article Title: NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

doi: 10.1101/2020.05.26.116897

Figure Lengend Snippet: A Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm. B HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90. C Empty vector (EV), FLAG-NF45 plasmid (NF45) or FLAG-NF90 plasmid (NF90) were transfected into HEK293 cells for 24 h, and then rDNA-Luc was co-transfected into the cells. Luciferase activity was measured after a further 24 h (n=3). Western blots show the protein levels of FLAG-NF45 and FLAG-NF90. D The line indicates the position of qPCR primer on the 45S pre-rRNA. E-F The qPCR analysis of 45S pre-rRNA from HEK293 cells treated with indicated shRNAs (E) and plasmids (F) for 24 h. Western blots show the expression levels of NF45, NF90, FLAG-NF45 and FLAG-NF90. (n = 3) Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (C, F). One-way ANOVA analysis followed by Tukey’s test (B, E). (* p < 0.05, *** p < 0.001). Exact p values are reported in Appendix Table S3.

Article Snippet: Primary antibodies listed as follows: p-UBF (ser 388): SC-21637, RPA194 (N-17): SC-25931, nucleolin (D-6): sc-17826, UBF (H-300): SC-9131, NF45(G-3): SC-365068, NF90 (N-18): SC-22530, NFATc1 (7A60): SC-7294, UB (P4D1): SC-8017, and Fibrillarin (G-4): SC-66021 were purchased from Santa Cruz with 1:1000 dilutions.

Techniques: Staining, Infection, Negative Control, shRNA, Transfection, Luciferase, Activity Assay, Western Blot, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

doi: 10.1101/2020.05.26.116897

Figure Lengend Snippet: A Schematic illustration of a single human rDNA gene repeat and positions of the primers used for ChIP. B In Jurkat cells, a ChIP assay was performed with control IgG, NF45 and NF90 antibodies, and then the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold-enrichment was normalized to control IgG treatment. (n=3) C The human rDNA promoter region contains one ARRE2-like sequence. The red letters in gray shaded area indicate the probe sequences used in panel D. Gray shaded area indicates the upstream control element. Blue color indicates the core promoter region. D EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section. E The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA-Luc wild type (WT) or mutated rDNA-Luc reporter plasmid (Mutant) for 24 h. (n=3) Western blot shows FLAG-NF90 protein level. Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (E). (*** p < 0.001). Exact p values are reported in Appendix Table S3.

Article Snippet: Primary antibodies listed as follows: p-UBF (ser 388): SC-21637, RPA194 (N-17): SC-25931, nucleolin (D-6): sc-17826, UBF (H-300): SC-9131, NF45(G-3): SC-365068, NF90 (N-18): SC-22530, NFATc1 (7A60): SC-7294, UB (P4D1): SC-8017, and Fibrillarin (G-4): SC-66021 were purchased from Santa Cruz with 1:1000 dilutions.

Techniques: Control, Sequencing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

doi: 10.1101/2020.05.26.116897

Figure Lengend Snippet: A The regulatory mechanism of the rDNA transcription. Firstly, UBF1 binds to the upstream control element (UCE) of rDNA promoter to recruit SL1, then SL1 recruits Pol I for rDNA transcription. B Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, GFP-C23. * represents the target protein. C Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells. D HEK293 cells were infected by lentivirus containing NF45-specfic shRNA or NF90-specfic shRNA, and then stimulated in serum for 0, 3 and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot. E ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3) F ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3) G Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm. Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (F). One-way ANOVA analysis followed by Tukey’s test (E). (** p < 0.01, *** P < 0.001). Exact p values are reported in Appendix Table S3.

Article Snippet: Primary antibodies listed as follows: p-UBF (ser 388): SC-21637, RPA194 (N-17): SC-25931, nucleolin (D-6): sc-17826, UBF (H-300): SC-9131, NF45(G-3): SC-365068, NF90 (N-18): SC-22530, NFATc1 (7A60): SC-7294, UB (P4D1): SC-8017, and Fibrillarin (G-4): SC-66021 were purchased from Santa Cruz with 1:1000 dilutions.

Techniques: Control, Co-Immunoprecipitation Assay, Transfection, Infection, shRNA, Expressing, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

doi: 10.1101/2020.05.26.116897

Figure Lengend Snippet: A ChIP analysis of NF45 and NF90 binding to the H42.9 loci in CD3 + T cells stimulated with P/I for 6 h. (n=3) B qPCR analysis of pre-rRNA in CD3 + T cells treated with P/I for 6 h. (n=3) C CD3 + T cells were first silenced with shNC, shNF45 or shNF90, and then treated with P/I to extract total RNA for qPCR analysis of pre-rRNA. (n=3) D The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3) E Confocal images of Jurkat cells to show NF45 (left panel) or NF90 (right panel) (green fluorescence) colocalized with NFATc2 (red fluorescence) in nucleoli. Scale bar, 10 μm. F Co-IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated withP/I. G ChIP analysis of NFATc2 binding to the rDNA loci in CD3 + T cells stimulated with P/I for 6 h. (n=3) H Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3) I ChIP analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3 + T cells stimulated with P/I for 6 h. (n=3) Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (A, B, C, D, G, I). One-way ANOVA analysis followed by Tukey’s test (H). (* p < 0.05, *** p <0.001). Exact p values are reported in Appendix Table S3.

Article Snippet: Primary antibodies listed as follows: p-UBF (ser 388): SC-21637, RPA194 (N-17): SC-25931, nucleolin (D-6): sc-17826, UBF (H-300): SC-9131, NF45(G-3): SC-365068, NF90 (N-18): SC-22530, NFATc1 (7A60): SC-7294, UB (P4D1): SC-8017, and Fibrillarin (G-4): SC-66021 were purchased from Santa Cruz with 1:1000 dilutions.

Techniques: Binding Assay, Activity Assay, Fluorescence, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Plasmid Preparation

Journal: bioRxiv

Article Title: NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

doi: 10.1101/2020.05.26.116897

Figure Lengend Snippet: Besides of the well-known NAFT-IL-2 T cell activation pathway, NFAT translocates to nucleolus, cooperatively with NF45/NF90 promote rDNA transcription upon T cell activation. Inhibition of rDNA transcription by NF45/NF90 knockdown or a specific polymerase I inhibitor CX5461 suppressed T cell activation and skin and heart allograft rejection.

Article Snippet: Primary antibodies listed as follows: p-UBF (ser 388): SC-21637, RPA194 (N-17): SC-25931, nucleolin (D-6): sc-17826, UBF (H-300): SC-9131, NF45(G-3): SC-365068, NF90 (N-18): SC-22530, NFATc1 (7A60): SC-7294, UB (P4D1): SC-8017, and Fibrillarin (G-4): SC-66021 were purchased from Santa Cruz with 1:1000 dilutions.

Techniques: Activation Assay, Inhibition, Knockdown