human cux1 cdna  (Addgene inc)

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: Fig. 1 | Identification and characterization of fSNP rs1537371. 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 p14ARF, p16INK4a, p15INK4b 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 p16INK4a, 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 p16INK4a mRNA levels showing significantly higher expression of p16INK4a 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 p16INK4a 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. p16INK4a was overexpressed using lentiviral NATUre AGING | VOL 2 | FEBRUARy 2022 | 140–154 | www.nature.com/nataging152 ArticlesNature agiNg 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: Fig. 2 | The role of CUX1 in regulation of p14ARF, p15INK4b, p16INK4a and ANRIL expression via binding to fSNP rs1537371. 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 p14ARF, p15INK4b, p16INK4a 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 p14ARF, p15INK4b and p16INK4a 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. p16INK4a was overexpressed using lentiviral NATUre AGING | VOL 2 | FEBRUARy 2022 | 140–154 | www.nature.com/nataging152 ArticlesNature agiNg expression vector p156RRL (Addgene).

Techniques: Expressing, Binding Assay, shRNA, Knockdown, Control, Sequencing, Luciferase, Reporter Assay, Activity Assay, 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: Fig. 6 | Elevated CUX1 and p16INK4a expression in plaque zones from patients with carotid artery disease. a,b, qPCR showing significant increase in expression of CUX1 (P = 0.036) (a) and p16INK4a (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 p16INK4a (P = 0.047 and P = 0.005, respectively; n = 13). e, Immunocytochemical staining with antibodies specifically against CUX1 (green) and p16INK4a (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 p16INK4a (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. p16INK4a was overexpressed using lentiviral NATUre AGING | VOL 2 | FEBRUARy 2022 | 140–154 | www.nature.com/nataging152 ArticlesNature agiNg 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: Fig. 7 | Demonstration that CUX1 regulates replicative senescence independently of p53 expression. 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. p16INK4a was overexpressed using lentiviral NATUre AGING | VOL 2 | FEBRUARy 2022 | 140–154 | www.nature.com/nataging152 ArticlesNature agiNg expression vector p156RRL (Addgene).

Techniques: Expressing, Western Blot, 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: Fig. 8 | Models highlighting the role of CUX1 in mediation of cellular senescence by activation of p16INK4a expression. 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 p16INK4a expression which, in turn, augments senescence. b, The mechanism underlying the contribution of the CUX1/p16INK4a pathway to cellular senescence. Increased expression of CUX1 in response to telomere shortening, DNA damage and oxidative stress resulted in upregulated expression of p16INK4a 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. p16INK4a was overexpressed using lentiviral NATUre AGING | VOL 2 | FEBRUARy 2022 | 140–154 | www.nature.com/nataging152 ArticlesNature agiNg expression vector p156RRL (Addgene).

Techniques: Activation Assay, Expressing, Binding 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: The Journal of Biological Chemistry

Article Title: Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling *

doi: 10.1074/jbc.M110.188482

Figure Lengend Snippet: Fto/Rpgrip1l hypothalamic expression. A, Fto, Rpgrip1l, and Cux1 transcript levels, assessed by RT PCR, in the PVN, DMH, VMH, and arcuate hypothalamic nuclei of lean (+/+) C57BL/6J mice. *, ARH versus PVN, DMH, or VMH. B, assessment of Fto and Rpgrip1l mRNA levels in the PVN, DMH, VMH, and ARH of +/+ C57BL/6J mice compared with fasted +/+ mice, Lepob, as well as mice exposed to 4 °C. Mice were either administered leptin (fasted +/+) or saline (+/+, fasted +/+, 4 °C +/+, Lepob) intraperitoneally. Error bars represent one S.D. Asterisk indicates statistical significance (p < 0.05). Each column represents the mean of measurements from eight mice.

Article Snippet: The full-length mouse Cux1 (p200) cDNA was cloned by OriGene in pCMV6 (Rockville, MD).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling *

doi: 10.1074/jbc.M110.188482

Figure Lengend Snippet: A, CUX1 is composed of an N-terminal autoinhibitory domain (AI), DNA-interacting cut-like repeats (CR) 1–3, and cut HD, as well as two repressor domains (R1 and R2) that do not interact with DNA. Cathepsin L cleaves P200 (at a site between CR1 and CR2) to P110 (21). Modeling of P200 (B) and P110 (C) binding affinities for the A (obesity risk) or C alleles of rs8050136. Consensus recognition sequences for CR1–3 and HD were determined from previous reports of in vitro binding experiments (27, 29). CR2 and CR3 domains recognize a degenerate sequence suggesting flexibility in DNA interaction, whereas CR1 or HD determines binding specificity. DNA consensus binding site for each DNA-binding domain (except HD domain) consists of an obligatory ATA sequence (in green and circled). Thus, the rs8050136 site has either three ATA sequences, including the A obesity-risk allele (two in the top and one in the reverse strand), that align with the predicted DNA binding consensus for P200, including CR1, CR2, and CR3, or two ATA and one ATC core sequence, including the rs8050136 C protective allele that aligns with the predicted DNA binding consensus for P110, including HD, CR2, and CR3. Boxed bases show the position of rs8050136 (A/C). D, nonradioactive EMSA using SYBR Green for DNA staining, Left panel, N2a cellular extracts enriched with human P200 or P110 mixed with double-stranded (DS) oligonucleotides carrying the A or C alleles. A double-stranded oligonucleotide in which the three predicted ATA recognition sequences were replaced with GGG (“M”) was also used as a control (described in supplemental Table 1). Band intensities were measured using Image J 1.36B (National Institutes of Health). Right panel, EM supershift assay using an antibody that recognizes the HD domain present in P200 and P110. Mouse IgG was used as a negative control. *, statistically significant (p = 0.001), comparing band intensity between 1st and 2nd and 3rd and 4th gel lanes (gel on left).

Article Snippet: The full-length mouse Cux1 (p200) cDNA was cloned by OriGene in pCMV6 (Rockville, MD).

Techniques: Binding Assay, In Vitro, Sequencing, SYBR Green Assay, Staining, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling *

doi: 10.1074/jbc.M110.188482

Figure Lengend Snippet: Sequence preference of p200 (Fto transcriptional repressor) and p110 (Fto and Rpgrip1l transcriptional activator) in the mouse. A, genomic organization of the mouse Fto/Rprgrip1l interval and Cux1-binding site on chromosome 8. B, modeling of p200 binding affinity for the mouse binding site. C, EMSA using N2a cellular extracts enriched with mouse p200 or p110 mixed with double-stranded (DS) oligonucleotides carrying the A or human C [M(C)] alleles. EM supershift assay was performed using an antibody that recognizes the HD domain present in p200 and p110. Mouse IgG was used as a negative control. *, statistically significant (p = 0.003), comparing major band intensity between 2nd and 3rd gel lanes.

Article Snippet: The full-length mouse Cux1 (p200) cDNA was cloned by OriGene in pCMV6 (Rockville, MD).

Techniques: Sequencing, Binding Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling *

doi: 10.1074/jbc.M110.188482

Figure Lengend Snippet: A, luciferase assay used to measure putative minimal FTO (FTO1p and FTO2p) and RPGRIP1L (RPGRIP1Lp) promoter activity upon human P200 ((pCMV) P200) or P110 ((pCMV) P110) overexpression or transfection with empty pCMV and in the presence or absence of the putative enhancer carrying the CUX1-binding A (Enh(A)) or C (Enh(C)) alleles. To control for background, extracts from cells transfected with empty pGL3 or pGL3 carrying the putative enhancer (Enh(A)+Enh(C)) in the absence of FTO1p, FTO2p, and RPGRIP1Lp were also assayed for luciferase activity. Transfection with 20 ng of Enh(C):FTO1p, Enh(C):FTO2p, or Enh(C):RPGRIP1Lp pGL3-based plasmids resulted in off-scale fluorescence intensity; only 2 ng of the above plasmids was used in this experiment. B, expression analysis in N41 cells overexpressing p200 or p110. C, expression analysis in primary neuronal cultures treated with leptin and/or cathepsin L inhibitor I. Each bar represents n = 3. Experiments were repeated twice. *, statistically significant. Used only for comparisons of data within close range (p values <0.01). Error bars represent S.D.

Article Snippet: The full-length mouse Cux1 (p200) cDNA was cloned by OriGene in pCMV6 (Rockville, MD).

Techniques: Luciferase, Activity Assay, Over Expression, Transfection, Binding Assay, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling *

doi: 10.1074/jbc.M110.188482

Figure Lengend Snippet: A, nuclear cathepsin L activity in the arcuate nucleus of lean (+/+), fasted +/+, and mice exposed to 4 °C and Lepob mice injected with saline intraperitoneally or fasted +/+ mice administered leptin intraperitoneally. p110 protein was measured in pooled nuclear extracts of fasted +/+, Lepob, and +/+ mice and mice exposed to 4 °C by Western blotting. *, statistically significant (p < 0.01).+/+ saline, significantly higher that +/+ fasted (saline); +/+ fasted (leptin), +/+ (4 °C) (saline), or Lepob (saline). B, Western blot showing p-Stat3 levels in whole protein extracts from primary neuronal cultures treated with cathepsin L inhibitor I (Cat. Inh. I) or DMSO. We failed to detect p110 species in nuclear fractions from neuronal cultures treated with cathepsin L inhibitor I. Experiments were repeated twice. Each error bar represents one standard deviation. p200, p110, nucleolin, p-Stat3, and β-tubulin-specific bands were variably exposed to film to achieve optimal quantitation range. Therefore, no comparisons should be made between different protein species.

Article Snippet: The full-length mouse Cux1 (p200) cDNA was cloned by OriGene in pCMV6 (Rockville, MD).

Techniques: Activity Assay, Injection, Western Blot, Standard Deviation, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling *

doi: 10.1074/jbc.M110.188482

Figure Lengend Snippet: Immunofluorescence of N41 cells co-transfected with the Lepr-b::eGFP overexpression vector. A, treated with 200 ng(/ml) of leptin for 6 h after becoming quiescent; B, no leptin treatment after becoming quiescent; C, co-transfected with Fto siRNA, grown for 48 h, and treated with leptin for 6 h after becoming quiescent; D, co-transfected with Rpgrip1l, grown for 48 h, and treated with leptin for 6 h after becoming quiescent; E, co-transfected with p200 overexpressing vector, grown for 48 h, and treated with leptin for 6 h after becoming quiescent; F, co-transfected with p110-overexpressing vector, grown for 48 h, and treated with leptin for 6 h after becoming quiescent; and G, treated with 200 ng(/ml) of leptin and cathepsin L inhibitor I (CatL inhib.) (10 μm) for 6 h after becoming quiescent. In vitro assessment of leptin receptor activity was by measurement of p-Stat3 levels in N41 cells co-transfected with the Lepr-b::eGFP overexpression vector. H, Fto-specific or Rpgrip1l-specific siRNA, grown for 48 h, and treated with leptin prior to becoming quiescent; I, p200 or p110 overexpressing (over.) vector, grown for 48 h, and treated with leptin prior to becoming quiescent. Quiescent N41 cells treated with Fto-specific scrambled siRNA displayed near identical p-Stat3 levels to N41 cells treated with Rpgrip1l-specific siRNA. p-Stat3 and β-tubulin-specific bands were variably exposed to film to achieve optimal quantitation range. Therefore, no comparisons should be made between different protein species.

Article Snippet: The full-length mouse Cux1 (p200) cDNA was cloned by OriGene in pCMV6 (Rockville, MD).

Techniques: Immunofluorescence, Transfection, Over Expression, Plasmid Preparation, Inhibition, In Vitro, Activity Assay, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling *

doi: 10.1074/jbc.M110.188482

Figure Lengend Snippet: Schematic representing the proposed model of FTO/RPGRIP1L rs8050136 allele-specific transcriptional regulation in response to feeding or fasting. Increased circulating leptin upon feeding leads to increased nuclear cathepsin L (CATL) activity in arcuate neurons expressing LEPR, increased P110 levels upon P200 cleavage by CATL, and in turn increased FTO/RPGRIP1L expression. By an unknown mechanism, FTO/RPGRIP1L facilitates LEPR clustering close to the base of the cilium at a site that may be the post-Golgi network (33, 70, 71) and/or the ciliary basal body (33). LEPR trafficking to the basal body may be facilitated by RPGRIP1L that localizes to the basal body protein complex (BBsome) and by FTO that may control expression of gene(s) implicated in LEPR trafficking. LEPR may multicluster at the membrane in the region of the cilium, thus enhancing leptin signaling. In some photoreceptor connecting cilia, RPGRIP1L localizes to the basal body as well as inside the cilium (9). Although we did not see LEPR inside the cilium, it is possible that the experimental conditions employed in this study limited our ability to visualize a small number of LEPR molecules transported into the cilium by RPGRIP1L. Fasting (low-leptin ambient condition) leads to decreased nuclear cathepsin L enzymatic activity in LEPR-positive arcuate neurons, decreased P110 levels, and decreased FTO/RPGRIP1L expression, resulting in dispersal of LEPR throughout the cell. Individuals with rs8050136 A (obesity risk), as opposed to individuals with rs8050136 C (protective) allele, display lower p110 binding, and thus lower FTO/RPGRIP1L expression levels causing decreased clustering of LEPR in close proximity to the cilium, resulting in less efficient leptin signaling. Abbreviations: PGN, post-Golgi network; BB, basal body.

Article Snippet: The full-length mouse Cux1 (p200) cDNA was cloned by OriGene in pCMV6 (Rockville, MD).

Techniques: Activity Assay, Expressing, Binding Assay