Journal: EMBO Reports

Article Title: Kin17 promotes rDNA transcription, ribosomal biogenesis, and cortical lamination

doi: 10.1038/s44319-025-00524-3

Figure Lengend Snippet: ( A ) Neurospheres were subjected to ChIP assays using an anti-Kin17 antibody, followed by qPCR-based detection of rDNA (Kin17, P = 0.000053; UBF, P = 0.000017) ( n = 3). UBF was included as positive control. ( B ) The global distribution pattern of Kin17 and UBF along the rDNA locus was determined by ChIP-qPCR. Kin17- or UBF-bound rDNA was quantified relative to input rDNA ( n = 3). ( C ) rDNA promoter reporter plasmid was transfected into control or Kin17 KD 293T cells. Luciferase activity was determined by Dual-Luciferase reporter assay (Kin17 KD1, P = 0.0024; Kin17 KD2, P = 0.0035) ( n = 3 per group). ( D ) Schematic illustrating the experimental strategy for measuring CpG methylation at rDNA promoter regions. Mouse rDNA was digested with the HpaII enzyme, followed by PCR detection using the indicated primers. Digestion efficiency was assessed by PCR amplification of Pbluescript sequences using the corresponding primers. ( E ) Kin17-associated rDNA was obtained by chromatin IP using an anti-Kin17 antibody. The relative abundance of methylated rDNA was quantified relative to input. As a positive control, UBF associated rDNA was isolated via chromatin IP using an anti-UBF antibody and the corresponding methylation status was determined. The distributions of methylated (orange bars) and unmethylated (grey bars) rDNA copies were plotted accordingly (Kin17, P = 0.0004; UBF, P = 0.0006) ( n = 3). ( F ) The interaction of Kin17 and NCL in neurospheres, determined by co-IP with an anti-Kin17 antibody, followed by immunoblotting detection with an anti-NCL antibody, and reciprocally by co-IP with an anti-NCL antibody followed by immunoblotting detection with an anti-Kin17 antibody ( n = 3). ( G ) The interaction of exogenous HA-Kin17 and Flag-NCL in 293T cells co-transfected with HA-Kin17 and Flag-NCL, determined by co-IP with an anti-HA antibody and immunoblotting detection with an anti-Flag antibody ( n = 3). ( H ) Generation of Kin17 or NCL truncation fragments. ( I ) Full-length Flag-NCL and each of truncated GST-Kin17 plasmids were transfected into 293T cells for expression, followed by co-IP with an anti-Flag antibody and immunoblotting with an anti-GST antibody ( n = 3). ( J ) Full-length GST-Kin17 and each of truncated Flag-NCL plasmids were transfected into 293T cells for expression, followed by co-IP with an anti-Flag antibody and immunoblotting with an anti-GST antibody ( n = 3). ( K ) 45S pre-rRNA level in control N2a cells, N2a cells with NCL KD ( P < 0.0001), N2a cells with Kin17 KD ( P = 0.001), and N2a cells with both NCL and Kin17 KD ( P < 0.0001), by qPCR ( n = 3 per group). ( L ) Relative Polr1a enrichment at the promoter of 45S rDNA in control N2a cells, N2a cells with NCL KD ( P < 0.0001), N2a cells with Kin17 KD ( P < 0.0001), and N2a cells with both NCL and Kin17 KD ( P < 0.0001), by ChIP-qPCR ( n = 3). ( M ) Relative NCL enrichment on the promoter of 45S rDNA in control and Kin17 KD N2a cells, by ChIP-qPCR analysis ( P = 0.004262) ( n = 3). ( N ) Relative Kin17 enrichment on the promoter of 45S rDNA, in control and NCL KD N2a cells, by ChIP-qPCR analysis ( P = 0.801241) ( n = 3). For this and subsequent figures, KD represents knockdown. Statistical analysis was performed using one-way ANOVA ( C , K , L ) with Bonferroni correction or two-tailed Student’s t test ( A , E , M , N ). Error bars denote the SEM. ** P < 0.01, *** P < 0.001; **** P < 0.0001; ns not significant.

Article Snippet: Pbluescript (pBS) , Addgene , Cat #212205.

Techniques: Positive Control, ChIP-qPCR, Plasmid Preparation, Transfection, Control, Luciferase, Activity Assay, Reporter Assay, CpG Methylation Assay, Amplification, Chromatin Immunoprecipitation, Methylation, Isolation, Co-Immunoprecipitation Assay, Western Blot, Expressing, Knockdown, Two Tailed Test

Journal: EMBO Reports

Article Title: Kin17 promotes rDNA transcription, ribosomal biogenesis, and cortical lamination

doi: 10.1038/s44319-025-00524-3

Figure Lengend Snippet: ( A ) Neurospheres were subjected to ChIP assays using an anti-Kin17 antibody, followed by qPCR-based detection of rDNA (Kin17, P = 0.000053; UBF, P = 0.000017) ( n = 3). UBF was included as positive control. ( B ) The global distribution pattern of Kin17 and UBF along the rDNA locus was determined by ChIP-qPCR. Kin17- or UBF-bound rDNA was quantified relative to input rDNA ( n = 3). ( C ) rDNA promoter reporter plasmid was transfected into control or Kin17 KD 293T cells. Luciferase activity was determined by Dual-Luciferase reporter assay (Kin17 KD1, P = 0.0024; Kin17 KD2, P = 0.0035) ( n = 3 per group). ( D ) Schematic illustrating the experimental strategy for measuring CpG methylation at rDNA promoter regions. Mouse rDNA was digested with the HpaII enzyme, followed by PCR detection using the indicated primers. Digestion efficiency was assessed by PCR amplification of Pbluescript sequences using the corresponding primers. ( E ) Kin17-associated rDNA was obtained by chromatin IP using an anti-Kin17 antibody. The relative abundance of methylated rDNA was quantified relative to input. As a positive control, UBF associated rDNA was isolated via chromatin IP using an anti-UBF antibody and the corresponding methylation status was determined. The distributions of methylated (orange bars) and unmethylated (grey bars) rDNA copies were plotted accordingly (Kin17, P = 0.0004; UBF, P = 0.0006) ( n = 3). ( F ) The interaction of Kin17 and NCL in neurospheres, determined by co-IP with an anti-Kin17 antibody, followed by immunoblotting detection with an anti-NCL antibody, and reciprocally by co-IP with an anti-NCL antibody followed by immunoblotting detection with an anti-Kin17 antibody ( n = 3). ( G ) The interaction of exogenous HA-Kin17 and Flag-NCL in 293T cells co-transfected with HA-Kin17 and Flag-NCL, determined by co-IP with an anti-HA antibody and immunoblotting detection with an anti-Flag antibody ( n = 3). ( H ) Generation of Kin17 or NCL truncation fragments. ( I ) Full-length Flag-NCL and each of truncated GST-Kin17 plasmids were transfected into 293T cells for expression, followed by co-IP with an anti-Flag antibody and immunoblotting with an anti-GST antibody ( n = 3). ( J ) Full-length GST-Kin17 and each of truncated Flag-NCL plasmids were transfected into 293T cells for expression, followed by co-IP with an anti-Flag antibody and immunoblotting with an anti-GST antibody ( n = 3). ( K ) 45S pre-rRNA level in control N2a cells, N2a cells with NCL KD ( P < 0.0001), N2a cells with Kin17 KD ( P = 0.001), and N2a cells with both NCL and Kin17 KD ( P < 0.0001), by qPCR ( n = 3 per group). ( L ) Relative Polr1a enrichment at the promoter of 45S rDNA in control N2a cells, N2a cells with NCL KD ( P < 0.0001), N2a cells with Kin17 KD ( P < 0.0001), and N2a cells with both NCL and Kin17 KD ( P < 0.0001), by ChIP-qPCR ( n = 3). ( M ) Relative NCL enrichment on the promoter of 45S rDNA in control and Kin17 KD N2a cells, by ChIP-qPCR analysis ( P = 0.004262) ( n = 3). ( N ) Relative Kin17 enrichment on the promoter of 45S rDNA, in control and NCL KD N2a cells, by ChIP-qPCR analysis ( P = 0.801241) ( n = 3). For this and subsequent figures, KD represents knockdown. Statistical analysis was performed using one-way ANOVA ( C , K , L ) with Bonferroni correction or two-tailed Student’s t test ( A , E , M , N ). Error bars denote the SEM. ** P < 0.01, *** P < 0.001; **** P < 0.0001; ns not significant.

Article Snippet: ChIP and input samples (20 μL each) were digested by adding 2 μL of HpaII, 3 μL 10× NEBuffer 1, 2 μL of 1 ng/μL Pbluescript (pBS) plasmid, and 3 μL of water, followed by incubation at 37 °C for 2 h. After heat inactivation for 10 min, 70 μL of water was added to each sample, and DNA was purified using the SanPrep Column PCR Product Purification Kit.

Techniques: Positive Control, ChIP-qPCR, Plasmid Preparation, Transfection, Control, Luciferase, Activity Assay, Reporter Assay, CpG Methylation Assay, Amplification, Chromatin Immunoprecipitation, Methylation, Isolation, Co-Immunoprecipitation Assay, Western Blot, Expressing, Knockdown, Two Tailed Test