Journal: JCI Insight

Article Title: Ciliary gene RPGRIP1L is required for hypothalamic arcuate neuron development

doi: 10.1172/jci.insight.123337

Figure Lengend Snippet: (A) Decreased neuro-confluency of human ESc–derived isogenic ARH neurons (day 30) homozygous for the FTO risk (R/R) allele at rs1421085 or rs8050136 compared with neurons homozygous for the protective (P/P) allele at rs1421085 or rs8050136. Scale bar: 20 μm. (B) Decreased FTO and RPGRIP1L expression in ARH neurons derived from the human H9 ESc line CRISPRed to homozygosity for the risk allele at rs1421085 or rs8050136. P < 0.05 was statistically significant, by 1-way ANOVA. (C) Upregulation of RPGRIP1L expression in response to increased P110 protein levels secondary to P200 overexpression (Supplemental Figure 4B), or in response to virus-induced RPGRIP1L overexpression, correlated with increased POMC neuron number. Red letters signify CRISPRed alleles. Each column represents the average of 6 isogenic cell lines. Data in A, B, and C are represented as box-and-whisker plots; boxes are the interquartile range, lines are the median value, and whiskers are minimum and maximum values. *P < 0.05, **P < 0.04, ***P < 0.01, by 2-tailed Student’s t test.

Article Snippet: Ready-to-transduce lentiviral particles carrying human CUX1 P200 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_181552","term_id":"1519242681","term_text":"NM_181552"}} NM_181552 ) and RPGRIP1L ( {"type":"entrez-nucleotide","attrs":{"text":"NM_015272","term_id":"1519246482","term_text":"NM_015272"}} NM_015272 ) were purchased by ORIGENE.

Techniques: Derivative Assay, Expressing, Over Expression, Whisker Assay

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , EMSA using NE isolated from human ECs, showing allele-imbalanced gel shifting on 22 of the 24 candidate fSNPs identified by Reel-seq screening of the CDKN2A/B locus using NE isolated from human PBMCs. Data for EMSA represent n = 3 biologically independent experiments. SNPs in red indicate no allele-imbalanced gel shifting. b , Genomic view of the 200-kb CDKN2A/B region showing the following tracks, ordered from top to bottom based on the ENCODE database. (1) SNP track showing locations of the 24 candidate fSNPs; (2–4) three epigenetic tracks for H3K27ac, H3K4me1 and H3K4me3, known as transcriptional factor-binding sites; (5) DNase I hypersensitivity sites (DNase I HS) in human astrocytes; (6) predicted regulatory elements including promoters (red) and enhancers (gray); (7) annotated genes including p14 ARF , p16 INK4a , p15 INK4b and ANRIL. c , Zoomed-in view of the 4-kb genomic region around fSNP rs1537371, showing the same tracks as above plus the negative result from CUX1 ChIP–seq assay in three human cell lines, GM12878, K562 and MCF-7. d , e , Demonstration of fSNP rs1537371 by EMSA ( d ) and luciferase reporter assay ( e ). A, risk allele; C, nonrisk allele; T, very rare allele; RLA, relative luciferase activity. Data for EMSA represent n = 3 biologically independent experiments; data for luciferase reporter assays represent n = 6 biologically independent samples. f , Sequences showing mutations around rs1537371 in three independent CRISPR–cas9 clones (nos. 2, 19 and 56), together with wild-type sequence. CON, wild-type control. g , qPCR showing decreased expression of p16 INK4a , one of the potential risk genes in the three mutants. Data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. h , Dot plot of fSNP rs1537371 and p16 INK4a mRNA levels showing significantly higher expression of p16 INK4a in healthy PBMCs carrying homozygous risk allele A/A versus nonrisk allele C/C ( P = 0.047, n = 26). P values were calculated using two-tailed Student’s t -test, and all data are presented as mean ± standard error (s.e.). h , Non-normally distributed data related to quantification of p16 INK4a expression are presented as median ± interquartile range, and P values were calculated with the nonparametric Mann–Whitney test for pairwise comparisons.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Isolation, Binding Assay, ChIP-sequencing, Luciferase, Reporter Assay, Activity Assay, CRISPR, Clone Assay, Sequencing, Control, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , ChIP assay demonstrating reduced binding of CUX1 to a DNA fragment containing rs1537371 in CUX1 shRNA knockdown ECs (left), and no specific binding of CUX1 to two randomly selected DNA fragments as controls (con; right). CUX1-Ab, anti-CUX1 antibody; IgG-Ab, anti-IgG antibody as an isotype control; NS, not significant. Data for ChIP assay represent n = 3 biologically independent experiments. b , Sequencing analysis showing significant enrichment of the A allele versus the C allele in ChIP DNA compared to input DNA ( n = 3), with P = 0.010. c , AIDP–Wb demonstrating specific binding of CUX1 to rs1537371, with risk allele A binding more CUX1 than nonrisk allele C. T is a very rare allele. Data for AIDP–Wb represent n = 3 biologically independent experiments. d , CUX1-dependent luciferase reporter assay in 293T cells showing luciferase activity in CUX1 shRNA knockdown (left) and CUX1-overexpressed ECs (right). pLVX-CUX1, CUX1 expression vector; rs1537371-A, luciferase reporter construct pGL3 (basic promoter vector, Promega); con, negative control. Data for this assay represent n = 6 biologically independent samples. e , qPCR (left) and immunoblot (right) showing downregulation of CUX1 in human ECs by shRNA knockdown. α-Tubulin was used as a loading control. Data for qPCR analysis represent n = 4 biologically independent samples, each performed in triplicate. Data for immunoblot analysis represent n = 3 biologically independent experiments. f , qPCR showing downregulation of p14 ARF , p15 INK4b , p16 INK4a and ANRIL expression in CUX1 shRNA knockdown human ECs. Data for qPCR analysis represent n = 4 biologically independent samples, each performed in triplicate. g , Immunoblot analysis showing downregulation of p14 ARF , p15 INK4b and p16 INK4a expression in CUX1 shRNA knockdown human ECs. Data for immunoblot analysis represent n = 3 biologically independent experiments. P values were calculated using two-tailed Student’s t -test, and all data are presented as mean ± s.e.).

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Binding Assay, shRNA, Knockdown, Control, Sequencing, Luciferase, Reporter Assay, Activity Assay, Expressing, Plasmid Preparation, Construct, Negative Control, Western Blot, Two Tailed Test

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: A . qPCR analysis showing that a knockdown of CUX1 by siRNA results in a downregulation of p14 arf , p15 INK4b , p16 INK4a and ANRIL expression in human primary ECs. Data for qPCR analysis represents four biologically independent samples (n=4), each performed in triplicate. B . A downregulation of CUX1 expression by siRNA in ECs represses replicative senescence ( right panel ) as is shown by both SA-β-gal ( upper ) and γ-H2AX ( lower ) staining. Data for SA-β-gal and γ-H2AX staining represents three biologically independent experiments (n=3). Quantitative plots for both β-gal positive cells (%) in SA-β-gal staining ( upper ) and γ-H2AX foci/cells (%) in γ-H2AX staining ( lower ) are shown on the right side of the panel.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Knockdown, Expressing, Staining

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , SA-β-gal (top) and γ-H2AX staining (bottom) showing an increase in replicative senescence from p5 ECs (left) to p10 ECs (middle), and a reduction in replicative senescence in CUX1 shRNA knockdown p10 ECs (right) compared to scrambled p10 ECs (middle). Right, quantitative plots are shown for both β-gal + cells (%) in SA-β-gal staining (top) and γ-H2AX foci/cells (%) with γ-H2AX staining (bottom). Data for SA-β-gal and γ-H2AX staining represent n = 3 biologically independent experiments. b , Immunoblots and qPCR showing increased expression of CUX1 and p16 INK4a in p10 ECs compared to p5 ECs. Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. c , PCR-based analysis showing significant decrease in telomeric length from p5 to p10 ECs. Data for PCR analysis represent n = 4 biologically independent samples. d , Immunoblots and qPCR showing that shRNA knockdown of CUX1 resulted in decreased expression of p16 INK4a in p10 human ECs. Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. e , qPCR showing significant downregulation of SASP genes, IL-6, IL-1β and ICAM1 in CUX1 shRNA knockdown p10 ECs. Data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. f , g , Decrease in both BrdU incorporation ( f ) and percentage of S/G2/M cell numbers ( g ) in p10 ECs (middle) versus p5 ECs (left) indicated an increase in replicative senescence. Knockdown of CUX1 in p10 ECs (right) resulted in recovery from both decreased BrdU incorporation and reduced percentage of S/G2/M cell numbers, indicating a blockage in cellular senescence in CUX1 shRNA knockdown p10 ECs. Data for BrdU incorporation represent n = 12 biologically independent samples; data for cell cycle analysis represent n = 3 biologically independent samples. P values were calculated using two-tailed Student’s t -test, and all data are presented as mean ± s.e.).

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Staining, shRNA, Knockdown, Western Blot, Expressing, BrdU Incorporation Assay, Cell Cycle Assay, Two Tailed Test

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: A . qPCR analysis showing CUX1 knockdown by shRNA results in a downregulation of p16 INK4a , p14 ARF , p15 INK4b and ANRIL expression in human primary VSMCs. Data for qPCR represents three biologically independent samples (n=3), each performed in duplicate. B . SA-β-gal ( left ) and γ-H2AX ( right ) staining showing that a knockdown of CUX1 in human primary VSMCs inhibits replicative senescence ( right panel )(n=3). Quantitative plots for both β-gal positive cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) in γ-H2AX staining are shown on the right side of the panel. C . qPCR analysis showing a significant decrease in the expression of IL-6 and ICAM1 , but not IL1β in the CUX1 shRNA knockdown VSMCs ( right lane ). Data for qPCR analysis represents three biologically independent samples (n=3), each performed in duplicate.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Knockdown, shRNA, Expressing, Staining

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , Immunoblot and qPCR analysis demonstrating that overexpression of CUX1 results in increased expression of p16 INK4a (middle) in ECs. Increased p16 INK4a expression was repressed by p16 INK4a shRNA knockdown (right). Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. b , SA-β-gal (top) and γ-H2AX (bottom) staining showing that overexpression of CUX1-induced cellular senescence in human ECs (middle versus left). Downregulation of p16 INK4a by shRNA in CUX1-overexpressed human ECs rescued senescent phenotypes (right; n = 3). Quantitative plots for both β-gal + cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) in γ-H2AX staining are shown. c , qPCR showing increased expression of SASP genes IL-6 , IL-1β and ICAM1 in CUX1-overexpressed ECs (middle versus left). Increased expression of SASP genes IL-6, IL-1β and ICAM1 remained unchanged in CUX1-overexpressed and p16 INK4a shRNA downregulated human ECs (right). Data for SA-β-gal and γ-H2AX staining represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. d , e , Decrease in BrdU incorporation ( d ) and percentage of S/G2/M cell numbers ( e ) in CUX1-overexpressed human ECs (middle) demonstrated an increase in cellular senescence. Knockdown of p16 INK4a by shRNA in CUX1-overexpressed human ECs (right) resulted in recovery from decreased BrdU incorporation and reduced percentage of S/G2/M cell numbers, indicating blockage of cellular senescence in CUX1-overexpressed and p16 INK4a -downregulated human ECs. Data for BrdU incorporation represent n = 12 biologically independent samples; data for cell cycle analysis represent n = 3 biologically independent samples. P values were calculated using two-tailed Student’s t -test, and all data are presented as mean ± s.e.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Western Blot, Over Expression, Expressing, shRNA, Knockdown, Staining, BrdU Incorporation Assay, Cell Cycle Assay, Two Tailed Test

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: A . Western blot analysis showing an over-expression of p16 INK4a using an ectopic vector p156RRL in the CUX1 shRNA knockdown ECs ( right lane ). A knockdown of CUX1 resulted in a downregulation of p16 INK4a ( middle lane ). Data for the Western blot analysis represents three biologically independent experiments (n=3). B . SA-β-gal ( upper ) and γ-H2AX ( lower ) staining showing the restoration of replicative senescence in the CUX1 shRNA knockdown and p16 INK4a over-expressed ECs ( right panel ). Quantitative plots for both β-gal positive cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) in γ-H2AX staining are shown on the right side of the panel. Data for SA-β-gal and γ-H2AX staining represents three biologically independent experiments (n=3). C . qPCR analysis of the SASP genes IL-6 , IL1β and ICAM1 showing that an over-expression of p16 INK4a in the CUX1 shRNA knockdown human ECs rescued the expression of IL-6 , IL1β and ICAM1 ( right lane ). Data for qPCR analysis represents three biologically independent samples (n=3), each performed in duplicate.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Western Blot, Over Expression, Plasmid Preparation, shRNA, Knockdown, Staining, Expressing

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: A . Western blot analysis and qPCR showing an over-expression of CUX1 in human ECs ( middle lane ) resulted in an upregulation of p14 ARF and p15 INK4b . The upregulation of p14 ARF , and p15 INK4b can be blocked by RNAi knockdown ( right lane ). Data for the Western blot analysis represents three biologically independent experiments (n=3). Data for qPCR analysis represents three biologically independent experiments (n=3). B . qPCR showing an over-expression of CUX1 in human ECs ( middle lane ) resulted in an upregulation of ANRIL. The upregulation of ANRIL can be inhibited by RNAi knockdown ( right lane ). Data for qPCR analysis represents three biologically independent experiments (n=3). C . SA-β-gal ( upper ) and γ-H2AX ( lower ) staining demonstrating that the increase in cellular senescence in the CUX1 over-expressed human ECs cannot be inhibited by the downregulation of p14 ARF , p15 INK4b or ANRIL. D . Quantitative plots for both β-gal positive cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) in γ-H2AX staining are shown on the right side of the panel. Data for SA-β-gal and γ-H2AX staining represents three biologically independent experiments (n=3). E . qPCR analysis of the SASP genes IL-6 and ICAM1 showing that a knockdown of p14 ARF , p15 INK4b , and ANRIL did not decrease the CUX1-induced expression of SASP genes. Data for qPCR analysis represents three biologically independent samples (n=3), each performed in duplicate. pLVX: lentiviral vector for gene expression.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Western Blot, Over Expression, Knockdown, Staining, Expressing, Plasmid Preparation, Gene Expression

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , Immunoblot and qPCR analysis showing induction of CUX1 and p16 INK4a in response to bleomycin (BLEO) activation in human ECs (middle). p53 expression was also induced by bleomycin activation, but in a post-transcriptional fashion as evidenced by qPCR. Downregulation of CUX1 by shRNA in human ECs blocked the induction of p16 INK4a in response to bleomycin activation, but not p53 (right). Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. b , SA-β-gal (top) and γ-H2AX (bottom) staining demonstrating increase in cellular senescence in bleomycin-treated ECs (middle) and a reduction in senescence in bleomycin-treated and CUX1 shRNA knockdown ECs (right). Right: quantitative plots for both β-gal + cells (%) with SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining are shown. Data for SA-β-gal and γ-H2AX staining represent n = 3 biologically independent experiments. c , qPCR analysis showing increased expression of IL-6 and in bleomycin-treated ECs (middle) and restoration of their expression following CUX1 shRNA knockdown (right) ( n = 3). d , e , Decrease in both BrdU incorporation ( d ) and percentage of S/G2/M cell numbers ( e ) in bleomycin-treated ECs (middle) demonstrated an increase in bleomycin-induced senescence. Knockdown of CUX1 by shRNA in bleomycin-treated ECs (right) resulted in recovery from decreased BrdU incorporation and reduced percentage of S/G2/M cell numbers, indicating blockage of senescence in bleomycin-treated and CUX1-downregulated human ECs. shCUX1, shRNA for CUX1. Data for BrdU incorporation represent n = 12 biologically independent samples; data for cell cycle analysis represent n = 3 biologically independent samples. P values were calculated using two-tailed Student’s t -test, and all data are presented as mean ± s.e.).

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Western Blot, Activation Assay, Expressing, shRNA, Staining, Knockdown, BrdU Incorporation Assay, Cell Cycle Assay, Two Tailed Test

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: A . Western blots and qPCR analysis showing an induction in CUX1 and p16 INK4a expression in response to 200 µM H 2 O 2 exposure for 4 hrs in human ECs ( middle lane ). p53 expression was also induced by H 2 O 2 activation, but in a post-transcriptional fashion. A downregulation of CUX1 by shRNA in human ECs blocks the induction of p16 INK4a in response to the H 2 O 2 treatment, but not affect the p53 expression ( right lane ). Data for Western blot analysis represents three biologically independent experiments (n=3). Data for qPCR analysis represents three biologically independent experiments (n=3), each performed in duplicate. B . SA-β-gal ( upper ) and γ-H2AX staining ( lower ) showing an increase in cellular senescence in H 2 O 2 -treated ECs ( middle panel ) and the restoration of senescence following a CUX1 knockdown ( right panel ) by comparing to the scrambled control ( left panel ). Quantitative plots for both β-gal positive cells (%) in SA-β-gal staining (upper) and γ-H2AX foci/cells (%) in γ-H2AX staining ( lower ) are presented on the right side of the panel. Data for SA-β-gal staining and γ-H2AX staining represents three biologically independent experiments (n=3). C . qPCR analysis showing an increased expression of the SASP genes IL-6 and IL-1β in the H 2 O 2 -treated ECs ( middle lane ) and the restoration of the expression of these genes upon a CUX1 shRNA knockdown ( right lane ) by comparing to the scrambled control (left lane ). Data for qPCR analysis represents three biologically independent experiments (n=3), each performed in duplicate.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Western Blot, Expressing, Activation Assay, shRNA, Staining, Knockdown, Control

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: A . Heatmap showing the top 20 upregulated and downregulated DEGs including DDAH1 and DNMT3b 48 hrs after CUX1 was knockdown by siRNA. Gene expression is shown in normalized log2 counts per million. DEGs were selected based on a more than 1.5 FC between CUX1 siRNA-treated samples versus scrambled siRNA-treated samples and adjusted P -value less than 0.05. CUX1 and p16 INK4a , p14 ARF , and p15 INK4b are among the top 20 downregulated DEGs. B . Volcano plot showing 471 DEGs with the log 2 FC in the CUX1 siRNA-treated human ECs. The x-axis displays logFC between CUX1 siRNA-treated samples versus scrambled siRNA-treated samples(logFC.E2D) and the y-axis displays −log10 adjusted P -value. Genes with log 2 FC outside the range −2 to +2 were squished to this range. DEG2 in blue including CDKN2A , TNFRSF10B , CXCL1 , CXCL12 and CCL2 were previously identified as endothelial senescence-related genes 67 . The top 5 upregulated and downregulated DEGs were also listed, they are CDKN2A , CUX1 , DDAH1 , GNAT1 , CDKN2B , RP11-301G19.1 , MET , MX1 , IFI44L , NRK . C . List of the 29 upregulated and 5 downregulated pathways with NOM p -value < 0.05 in the CUX1 siRNA-treated human ECs.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Knockdown, Gene Expression

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , b , qPCR showing significant increase in expression of CUX1 ( P = 0.036) ( a ) and p16 INK4a ( P = 0.011) ( b ) in plaque versus normal-appearing zones obtained from patients with carotid artery atherosclerosis. Data for qPCR analysis represent n = 11 plaque zones and n = 9 normal-appearing zones. c , d , Nonparametric Spearman correlation analysis ( c ) and trend analysis ( d ) showing significant correlation between the expression levels of CUX1 and p16 INK4a ( P = 0.047 and P = 0.005, respectively; n = 13). e , Immunocytochemical staining with antibodies specifically against CUX1 (green) and p16 INK4a (red) in plaque and normal-appearing zones from patients with carotid artery atherosclerosis. Data were generated by staining of n = 8 plaque zone and n = 8 normal-appearing zones in two independent experiments. DAPI (blue) was applied to stain fixed cells. f , Statistical analysis of immunocytochemical staining showing significant induction of CUX1 ( P = 0.0025) and p16 INK4a ( P = 0.0006) in plaque zones compared to normal-appearing zones. g , qPCR showing a trend of increase with no statistical significance in the expression of SASP genes IL-6 (left; P = 0.502), IL-1β (middle; P = 0.255) and ICAM1 (right; P = 0.17). Data for qPCR analysis represent n = 11 plaque zones and n = 9 normal-appearing zones. a – d , g , Data presented as mean ± s.e. P values were calculated using two-tailed Student’s t -test. f , Non-normally distributed data are presented as median ± interquartile range, and P values were calculated with the nonparametric Mann–Whitney test.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Expressing, Staining, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , Immunoblot analysis showing passage-dependent induction of p53 expression in p10 compared to p5 ECs. qPCR analysis showing that induction of p53 was not at the transcriptional level. b , Immunoblot (left) and qPCR analysis (right) showing no significant change in p53 expression following shRNA-mediated CUX1 knockdown in p10 ECs. c , d , Immunoblot and qPCR analysis showing no significant change in CUX1 expression ( d ) after p53 siRNA-mediated knockdown ( c ) in human ECs. e , f , SA-β-gal ( e ) and γ-H2AX ( f ) staining showing no significant change in EC senescence by comparison of scrambled siRNA control ECs with p53 siRNA knockdown ECs. Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. Data for SA-β-gal and γ-H2AX staining represent n = 3 biologically independent experiments. P values were calculated using two-tailed Student’s t -test, and all data are presented as mean ± s.e.).

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Western Blot, Expressing, shRNA, Knockdown, Staining, Comparison, Control, Two Tailed Test

Journal: Nature Aging

Article Title: Post-GWAS functional analysis identifies CUX1 as a regulator of p16 INK4a and cellular senescence

doi: 10.1038/s43587-022-00177-0

Figure Lengend Snippet: a , The mechanism underlying the contribution of atherosclerosis-associated fSNP rs1537371 to susceptibility to age-related disease. Increased binding of CUX1 to the A allele (risk allele) versus the C allele (nonrisk allele) resulted in a higher level of p16 INK4a expression which, in turn, augments senescence. b , The mechanism underlying the contribution of the CUX1/p16 INK4a pathway to cellular senescence. Increased expression of CUX1 in response to telomere shortening, DNA damage and oxidative stress resulted in upregulated expression of p16 INK4a and induction of cellular senescence.

Article Snippet: For overexpression of human CUX1 p200, human CUX1 cDNA from pXJ42-p200 CUX1 (Addgene) was cloned into the lentiviral expression vector pLVX puro using Xho I and Xba I cutting sites (Takara Bio) and confirmed by sequencing. p16 INK4a was overexpressed using lentiviral expression vector p156RRL (Addgene).

Techniques: Binding Assay, Expressing

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: Expression profiling was performed on microdissected tumor cells from 17 mammary tumors that developed in MMTV-p110 or p75 CUX1 transgenic mice, including 8 solid carcinomas (p110-416, p110-314, p110-360, p75-534, p75-150, p110-143, p75-129, p110-225), 4 adenosquamous carcinomas (p75-101, p75-94, p75-80, p75-47), 1 adenoma (p110-98), 2 adenomyoepitheliomas (p75-90, p110-374), and 1 adenocarcinoma (p75-216). The complete expression profiles are available on the Gene Expression Omnibus (GEO) repository under accession number GSE54804. The figure shows a heatmap of mammary tumors grouped according to an unsupervised hierarchical clustering of Wnt gene expression. Expression of Glis1 , as well as Vim , Cdh1 , Cdh2 , Snai1 , Snai2 , Ocln and Twist1 in each tumor is shown below. The cluster of “high-Wnt” tumors is boxed in yellow and the cluster of “low-Wnt” tumors is boxed in blue. Each column represents one tumor, with the transgene (p110 or p75 CUX1) and mouse ID number indicated below.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Expressing, Transgenic Assay, Gene Expression

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: Cell lines were established from two mammary tumors that developed in transgenic mice carrying a MMTV-p75 CUX1 transgene: an adenosquamous carcinoma from mouse #p75-80 and a solid carcinoma from mouse #p75-534. The two cell lines were analyzed for Wnt gene expression (A), nuclear β-catenin expression (B), and TCF/β-catenin transcriptional activity (C). (A) mRNA levels of Wnt ligand genes were measured by RT-qPCR. Results of triplicate experiments are shown. * P Value<0.05 on a Welch corrected T test. Und: Undetected. (B) Cells were analyzed by indirect immunofluorescence using antibodies specific for the active, non-phosphorylated form of β-Catenin. Histogram shows the relative mean nuclear signal for β-Catenin as measured using the ImageJ software. The value for p75-534 was set to 1. * P Value<0.05 on a Welch corrected T test. A representative image of each cell line is shown on the right. Scale bar: 20µm. (C) The TOP/FOP luciferase reporter assay was performed in the p75-534 and p75-80 mammary tumor cell lines. In addition, p75-80 cells were cotransfected with a vector expressing either sFRP1, sFRP2 or SOST or an empty vector. Where indicated, niclosamide was added to the cells at the time of transfection. Results of 3 independent transfections are shown. * P Value<0.05 on a Welch corrected T test. A schematic representation of the reporter constructs is shown.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Transgenic Assay, Gene Expression, Expressing, Activity Assay, Quantitative RT-PCR, Immunofluorescence, Software, Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Construct

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: CUX1 specific or scrambled siRNA were transfected in a panel of 6 human cancer cell lines from breast (A), ovarian (B) and lung (C) cancers. 3 days later total mRNA was isolated and quantitative RT-PCR was performed using HPRT1 mRNA as a control. The values represent fold difference in mRNA expression between cells treated with CUX1 or scrambled siRNA. (Und: undetected). * P Value<0.05 on a Welch corrected T test.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Transfection, Isolation, Quantitative RT-PCR, Control, Expressing

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: (A) HEK293 cells were infected with retroviruses to establish cells stably carrying a vector that expresses p110 CUX1 or nothing (vector). Nuclear and cytoplasmic extracts were analyzed by immunoblotting with the indicated antibodies. (B) The TOP or FOP luciferase reporters were introduced into HEK293 cells together with 500 ng of a vector expressing p110 CUX1 or an empty vector. Luciferase activity was measured 36 hours after transfection. Where indicated, a vector expressing either sFRP1, sFRP2, SOST or DKK1 was transfected as well, or niclosamide or IWP-2 was added to the cells at the time of transfection. Results of 3 independent transfections are shown. * P Value<0.05 on a Welch corrected T test. (C) Hs578t and MCF7 breast cancer cells were stably infected with either a lentivirus expressing p110 CUX1 or an empty lentivirus (vector). Nuclear and cytoplasmic extracts were analyzed by immunoblotting with the indicated antibodies. (D) The TOP or FOP luciferase reporters were introduced into Hs578t and MCF7 cells together with 500 ng of a vector expressing p110 CUX1 or an empty vector. Luciferase activity was measured 36 hours after transfection.* P Value<0.05 on a Welch corrected T test.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Infection, Stable Transfection, Plasmid Preparation, Western Blot, Luciferase, Expressing, Activity Assay, Transfection

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: (A) Heatmap of a human breast tumor dataset sorted according to WNT genes expression using the BreSAT algorithm. Expression of CUX1 , GLIS1 , VIM , TWIST1 , SNAI1 , CDH2 , SNAI2 , OCLN and CDH1 in each tumor is shown below. (B) CUX1 and GLIS1 expression in the top 25% and bottom 25% samples sorted according to WNT genes expression. * indicates p<0.05, ** <0.01, *** <0.001 on a Welch-corrected student's T test. (C) CDH1 , OCLN , CDH2 , SNAI1 , SNAI2 , TWIST1 and VIM expression in the top 25% and bottom 25% samples sorted according to Wnt genes expression. * indicates p<0.05, ** <0.01, *** <0.001 on a Welch-corrected student's T test.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Expressing

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: We established populations of MCF10A cells stably carrying lentiviral vectors that express nothing (vector), GLIS1, p110 CUX1, or both GLIS1 and p110 CUX1. (A) Wnt gene mRNA levels were measured by RT-qPCR. Und: Undetected. * P Value<0.05 on a Welch corrected T test when comparing the p110+Glis1 samples vs. each of the other 3. (B) β-Catenin protein levels were measured by immunofluorescence using antibodies specific for the active, non-phosphorylated form of the protein. Histogram shows the mean nuclear signal for β-Catenin for at least 50 cells per condition. * P Value<0.05 on a Welch corrected T test. A representative image of each condition is shown below. Scale bar: 20µm. (C) β-Catenin protein levels were measured by immunoblotting using antibodies specific for the active, non-phosphorylated form of the protein. γ-Tubulin is shown as a loading control. (D) MCF10A cells were transiently transfected with either the TOP of the FOP luciferase reporter, a vector expressing p110 CUX1 or an empty vector, and a vector expressing GLIS1 or the corresponding empty vector. Luciferase activity was measured in 3 independent transfections. * P Value<0.05 on a Welch corrected T test. (E) MCF10A cells ectopically expressing GLIS1, p110 CUX1 or both were imaged by time-lapse video microscopy and their speed of migration was quantified. The average of 3 independent experiments is shown with error bars representing standard error. ** P Value<0.01, *** <0.001 on 3 combined Welch corrected T tests using the Fisher method. (F) Time-lapse video microscopy was carried out as in E and distance migrated from the point of origin was quantified. (G) MCF10A cells overexpressing GLIS1 and p110 CUX1 alone or both were subjected to inverted Boyden chamber invasion assays. The percentage of cells able to migrate more than 20 µm into the matrix is shown for each line. The average of 3 independent experiments is shown. (H) Confocal Z-Stacks acquired 10 µm apart are aligned to demonstrate invasion of MCF10A-derivative cells into the matrix. Representative Z-stack for each condition is displayed. The leftmost image represents cells at the surface of the matrix (Depth = 0).

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Western Blot, Control, Transfection, Luciferase, Expressing, Activity Assay, Microscopy, Migration

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: We established populations of Hs578t cells stably carrying lentiviral vectors that express nothing (vector), GLIS1, p110 CUX1, or both GLIS1 and p110 CUX1. (A) Wnt gene mRNA levels were measured by RT-qPCR. Und: Undetected. * P Value<0.05 on a Welch corrected T test. (B) β-Catenin protein levels were measured by indirect immunofluorescence using antibodies specific for the active, non-phosphorylated form of the protein. Histogram shows the mean nuclear signal for β-Catenin for at least 50 cells per condition. * P Value<0.05 on a Welch corrected T test. A representative image of each condition is shown below. Scale bar: 20µm. (C) Hs578t cells were transiently transfected with either the TOP of the FOP luciferase reporter, and suboptimal amounts (50 ng) of effector plasmids coding for nothing (−), p110 CUX1 or GLIS1. Luciferase activity was measured in 3 independent transfections. * P Value<0.05 on a Welch corrected T test. (D) Hs578t cells ectopically expressing GLIS1, p110 CUX1 or both were imaged by time-lapse video microscopy and their speed of migration was quantified. The average of 3 independent experiments is shown with error bars representing standard error. ** P Value<0.01, *** <0.001 on a Welch corrected T test. (E) Time-lapse video microscopy was carried out as in D and distance migrated from the point of origin was quantified.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Transfection, Luciferase, Activity Assay, Expressing, Microscopy, Migration