Journal: Development (Cambridge, England)

Article Title: IRX3/5 regulate mitotic chromatid segregation and limb bud shape

doi: 10.1242/dev.180042

Figure Lengend Snippet: IRX3 associates with SMC1 and CUX1 in vivo. (A) Lysate from 30 WT E10.5 hindlimb buds was immunoprecipitated (IP) with anti-IRX3 and immunoblotted against SMC1, SMC3 or CUX1. Three separate co-IP experiments were performed using multiple limb buds on each occasion. Representative full-length western blots (WB) are shown, and arrows indicate expected band size. (B) IRX3 protein localised in anterior-proximal (AP), but not posterior-distal (PD), nuclei in mesoderm of 29 som. limb bud. (C) STED microscopy revealed that IRX3 puncta contacted those of SMC1 (anterior versus posterior, P=0.008) and CUX1 (anterior versus posterior, P=0.005) in anterior mesoderm of the hindlimb field at 29-32 som. (25-30 cells, n=3 embryos, unpaired, two-tailed t-test, error bars indicate s.e.m.). (D,E) PLA in vivo demonstrated physical association of IRX3 with SMC1 (8/82, 9.7%; D) and CUX1 (8/64, 12.5%; E) in anterior half mesoderm at 29-30 som. (n=3 embryos per condition). Scale bars: 400 µm (B, left); 20 µm (B, right); 5 µm (C-E).

Article Snippet: Antibodies company and catalogue number IRX3 [Novus Biologicals, 1D7, H0079191-M05, mouse, 1:250 – used for immunofluorescence (IF)]; IRX3 (Santa Cruz Biotechnology, G-6, 166877, mouse, 1:100 – used for co-IP in vivo ); SMC1 (Abcam, ab21583, rabbit, 1:250 IF, 1:1000 – used for immunoblotting (IB); SMC3 (Novus Biological, NB100-207 IF, in vitro IB; Abcam, ab9263, 1:1000 in vivo IB); NIPBL (Santa Cruz Biotechnology, C-9, sc-374625, 1:1000); CUX1 (Abcam, ab140042, mouse, 1:250 IF, in vitro IB; Abcam, ab230844, rabbit, 1:2500 in vivo IB); IgG (NA931V, GE Healthcare, 1:100) or donkey anti-rabbit IgG (NA934V, GE Healthcare, 1:100) co-IP in vitro , IgG (Santa Cruz Biotechnology, 2343, 1:100) co-IP in vivo ; Caspase-3 (BD Biosciences, 559565, 1:250); F-Actin (Alexa Fluor 633 Phalloidin, Thermo Fisher Scientific, A22284, 1:1000).

Techniques: In Vivo, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Microscopy, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: IRX3/5 regulate mitotic chromatid segregation and limb bud shape

doi: 10.1242/dev.180042

Figure Lengend Snippet: IRX3/5 maintain cohesin subunits and CUX1 in vivo. (A,B) Immunofluorescence intensities of SMC1, NIPBL, SMC3 and CUX1 were diminished in the absence of Irx3/5 in anterior (A), but not posterior (B), limb bud mesoderm. (C) Quantification of A and B (29-32 som.; n=3 embryos per condition; *P=0.008 for anterior SMC1, *P=0.003 for anterior NIPBL, *P=0.004 for anterior SMC3, *P=0.004 for anterior CUX1; unpaired, two-tailed t-test, error bars indicate s.e.m.). (D,E) Transcription of Smc1a, Smc1b and Cux1 were unchanged by real time RT-PCR in anterior and posterior limb bud tissue in Irx3/5+/− and Irx3/5−/− embryos (29-30 som., n=3 embryos for each of three independent experiments). Error bars indicate s.e.m. Scale bars: 20 µm.

Article Snippet: Antibodies company and catalogue number IRX3 [Novus Biologicals, 1D7, H0079191-M05, mouse, 1:250 – used for immunofluorescence (IF)]; IRX3 (Santa Cruz Biotechnology, G-6, 166877, mouse, 1:100 – used for co-IP in vivo ); SMC1 (Abcam, ab21583, rabbit, 1:250 IF, 1:1000 – used for immunoblotting (IB); SMC3 (Novus Biological, NB100-207 IF, in vitro IB; Abcam, ab9263, 1:1000 in vivo IB); NIPBL (Santa Cruz Biotechnology, C-9, sc-374625, 1:1000); CUX1 (Abcam, ab140042, mouse, 1:250 IF, in vitro IB; Abcam, ab230844, rabbit, 1:2500 in vivo IB); IgG (NA931V, GE Healthcare, 1:100) or donkey anti-rabbit IgG (NA934V, GE Healthcare, 1:100) co-IP in vitro , IgG (Santa Cruz Biotechnology, 2343, 1:100) co-IP in vivo ; Caspase-3 (BD Biosciences, 559565, 1:250); F-Actin (Alexa Fluor 633 Phalloidin, Thermo Fisher Scientific, A22284, 1:1000).

Techniques: In Vivo, Immunofluorescence, Two Tailed Test, Quantitative RT-PCR

Journal: Cancer Discovery

Article Title: Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia

doi: 10.1158/2159-8290.CD-22-0424

Figure Lengend Snippet: XPO1 nuclear export inhibition displaces NPM1c from chromatin. A, The binding profiles of NPM1c, XPO1, and H3K27ac at NPM1c binding genes in OCI-AML3 cells from −3 kb TSS to +3 kb transcriptional end site (TES). B, The peak overlap between NPM1c and XPO1 peaks (left) and genes with TSS peak (right) in OCI-AML3 NPM1c degron 2 cells. C, Coimmunoprecipitation (IP) of NPM1c and XPO1 in OCI-AML3 cells. D, Confocal immunofluorescence image of colocalization of NPM1ca-eGFP and XPO1 in NPM1ca-eGFP HOXB8 cells. DAPI was used to stain the DNA of the nucleus. Scale bar = 10 μm. E and F, The NPM1c ( E ) and XPO1 ( F ) binding profiles in 6,312 NPM1c binding genes with 500 nmol/L dTAG-13 and 25 nmol/L selinexor treatment for 24 hours in OCI-AML3 NPM1c degron 2 cells (−5 kb TSS to + 5 kb TES). G, Integrative Genomics Viewer view of NPM1c and XPO1 enrichment and distribution at the HOXA cluster, MEIS1, and the HOXB cluster in OCI-AML3 NPM1c degron 2 cells with 500 nmol/L dTAG-13 and 25 nmol/L selinexor treatment. H, Venn diagram showing the overlap between the downregulated genes in dTAG-13 and selinexor treatment. The representative overlapping genes are listed. P value is calculated by the Fisher exact test for 2 × 2 contingency table. I, The volcano plot shows the differential transcription genes on the gene body Bru-seq reads with 12 hours of 25 nmol/L selinexor treatment in OCI-AML3 NPM1c degron 2 cells. The horizontal dashed line indicates the cutoff for P = 0.01. The vertical dashed line indicates the 1.5-fold cutoff for fold change in gene expression. J, The Bru-seq reads of nascent transcription at the MEIS1 (left) and LHX2 (right) locus. RPKM, reads per kilobase million. K, Two color confocal fluorescence images of a U2OS LacO array cell cotransfected with EYFP-LacI with mCherry-XPO1 (top), and images of cells cotransfected with EYFP-NPM1c-LacI and mCherry-XPO1 (bottom). The LacO array locus is circled, and magnified LacO array locus images are shown at the lower left part of the image. The nucleus (nuc) is annotated in the bottom images. L, Enrichment of mCherry signal quantification at the LacO array locus in the cells transfected in K . Mean ± SD is shown. P value is calculated by the Student t test.

Article Snippet: Antibodies used in Co-IP included NPM1c (Novus Biologicals, Rabbit Polyclonal, NB110-61646, RRID:AB_964800) and XPO1 (Novus Biologicals, Rabbit Polyclonal, NB100-79802, RRID:AB_2215823).

Techniques: Inhibition, Binding Assay, Immunofluorescence, Staining, Gene Expression, Fluorescence, Transfection

Journal: Cancer Discovery

Article Title: Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia

doi: 10.1158/2159-8290.CD-22-0424

Figure Lengend Snippet: XPO1 inhibition synergizes with Menin inhibitor in NPM1c leukemia treatment. A, HOXA9 , HOXA10 , and HOXB4 relative expression levels after treatment with 50 nmol/L MI-3454, 25 nmol/L eltanexor, and combination of the two in OCI-AML3 cells. n = 3; mean ± SEM is shown. P value is calculated by one-way ANOVA test with Tukey test on all pairwise comparisons between treatment groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, Differentiation (CD11b flow cytometry) induced by 7-day treatment with 50 nmol/L MI-3454, 25 nmol/L eltanexor, and combination of the two in OCI-AML3 cells. C, In vitro colony-forming assay of NPM1c AML blasts with DMSO, 50 nmol/L MI3454, 25 nmol/L eltanexor, and the combination. n = 3; mean ± SD is shown. P value is calculated by one-way ANOVA test with Tukey test on all pairwise comparison between treatment groups. n = 2 for AML-5317. n = 3 for AML-557. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, Leukemic burden assay scheme with NSGS mice (left). Leukemia burden after vehicle, MI-3454, eltanexor, and combination treatment (right). Mean ± SD is shown. The number of mice in each group is listed. P value is calculated by t test of hCD45 engraftment at day 84 after transplantation comparing between the combination group and all other groups. E, The normal differentiation process in hematopoiesis. During the normal hematopoiesis process, the HOXA/B cluster and MEIS1 are stepwise silenced through chromatin modification. First, in the HSPCs ready to differentiate, active histone acetylation marks will be removed by HDACs. Then during terminal differentiation, heterochromatin marks like H3K27me3 will accumulate in the HOXA/B cluster and MEIS1 to fully silence the HOXA/B cluster and MEIS1. This will ensure the proper shutdown of stem cell self-renewal genes and prevent the ectopic expression of the HOXA/B cluster and MEIS1. F, When NPM1c mutation occurs in the leukemia cell of origin (likely HSPCs), the NPM1c protein will form multicomponent condensate through multivalent heterotypic interactions. This formation of condensate will lead to transcriptional amplification of the HOXA/B cluster and MEIS1 genes and prevent the initial step during normal hematopoiesis to silence the gene by HDACs. This transcriptional hijacking by NPM1c will not only lead to the arrest of gene expression at the HSPC stage but also block the differentiation initiated by HDACs. TF, transcription factor.

Article Snippet: Antibodies used in Co-IP included NPM1c (Novus Biologicals, Rabbit Polyclonal, NB110-61646, RRID:AB_964800) and XPO1 (Novus Biologicals, Rabbit Polyclonal, NB100-79802, RRID:AB_2215823).

Techniques: Inhibition, Expressing, Flow Cytometry, In Vitro, Comparison, Transplantation Assay, Modification, Mutagenesis, Amplification, Gene Expression, Blocking Assay

Journal: Aging Cell

Article Title: Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

doi: 10.1111/acel.13002

Figure Lengend Snippet: Mislocalization of NES‐containing proteins is caused by progerin‐mediated overexpression of CRM1. (a) Wild‐type (WT) and HGPS cells, treated for 24 hr with 10 nM LMB or vehicle alone, were immunolabeled for the indicated proteins (bar, 10 µm). The Fn/c ratio was calculated ( n = 50 cells), and significant differences were determined by Mann–Whitney U test (graphs). (b) Protein levels of STAT3, B23, ZO‐2, and actin (loading control) were analyzed, and typical gels from two independent experiments are shown. (c) WT and HGPS cells were immunolabeled for CRM1 and analyzed by CLSM. Typical images from 3 independent assays are shown (bar 10 µm). (d) Lysates from WT and HGPS cells were subjected to Western blot using antibodies against CRM1, lamin A/C, or actin (loading control), and relative CRM1 levels were obtained (graph). (e) CRM1 messenger RNA expression was examined by qRT‐PCR. (d‐e) Significant differences were determined by unpaired t test. (f) WT and HGPS cells were transfected with both Luciferase reporter construct containing the human CRM1 promoter and Renilla luciferase vector, which was used to normalize transfection efficiency. Enzymatic activities were estimated after incubation for 48 hr as described in Methods. Data represent mean ± SEM of three independent experiments (unpaired t test). (g) Lysates from HeLa cells stably transfected to express GFP‐lamin A or GFP‐progerin were subjected to Western blotting using antibodies against CRM1, GFP, or actin (loading control). (h) HeLa cells expressing GFP‐lamin A or GFP‐progerin were subjected to chromatin immunoprecipitation (ChIP) with anti‐NF‐YA antibodies followed by PCR for the CRM1 promoter region. Data correspond to two independent experiments in triplicate

Article Snippet: CRM1 , Novus Biologicals, (Cat: NB100−79802) , Rabbit polyclonal , 1:4,000 , 1:250.

Techniques: Over Expression, Immunolabeling, MANN-WHITNEY, Western Blot, RNA Expression, Quantitative RT-PCR, Transfection, Luciferase, Construct, Plasmid Preparation, Incubation, Stable Transfection, Expressing, Chromatin Immunoprecipitation

Journal: Aging Cell

Article Title: Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

doi: 10.1111/acel.13002

Figure Lengend Snippet: LMB‐mediated inhibition of CRM1 prevents cellular senescence by improving lamin B1 levels. (a) WT and HGPS fibroblasts were treated for 2 days with 1 nM LMB or vehicle alone. The activity of β‐galactosidase was assessed, and representative images are shown. Bar = 100 µM. The percentage of senescent cells was calculated from 3 separate experiments ( n = 300 cells for each condition). (b) Lysates from WT and HGPS fibroblasts, previously treated for 6 days with 50 nM LMB or vehicle, were analyzed by Western blotting using antibodies against lamin B1 and actin (control). Lamin B1 levels were assessed (bottom panel). (c) WT and HGPS fibroblasts treated with 50 nM LMB or vehicle alone for 3 days were immunostaining for lamin B1, and typical images are shown. Bar, 10 µm. (d) WT and HGPS fibroblasts treated with 50 nM LMB or vehicle for 6 days were analyzed by CLSM, using antibodies against H3K9me3 and lamin A/C. Representative images are shown. B Bar, 10 µm. Bottom. Line profile analysis showing H3K9me3 fluorescence pattern. (e) Lysates from HGPS cells treated as peer C were analyzed by Western blotting using antibodies against H3K9me3 and actin (control). Relative H3K9me3 levels are shown (right graph). (b and e) Significant differences were determined by unpaired t test

Article Snippet: CRM1 , Novus Biologicals, (Cat: NB100−79802) , Rabbit polyclonal , 1:4,000 , 1:250.

Techniques: Inhibition, Activity Assay, Western Blot, Immunostaining, Fluorescence

Journal: Aging Cell

Article Title: Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

doi: 10.1111/acel.13002

Figure Lengend Snippet: LMB‐mediated inhibition of CRM1 alleviates aging features of HGPS cells. (a) WT and HGPS fibroblasts were treated for 3 days with 50 nM LMB or vehicle alone. Cells were immunolabeled for lamin A/C before being analyzed by CLSM, and representative images are shown. Bar, 10 µm. Right. Nuclear morphometric analysis was carried out as described in Methods ( n = 200 nuclei for each condition), with significant differences determined by Mann–Whitney U test. (b) WT and HGPS fibroblasts, treated as peer A, were immunostained for fibrillarin to decorate nucleoli. Bar, 10 µm. Right. Nucleolar area was determined as described in Methods ( n > 600 nucleoli per condition). Number of nucleoli per cell was estimated (bottom panel; n > 80 cells per sample), and significant differences were determined by Mann–Whitney U test. (c) Lysates from WT and HGPS‐1 fibroblasts, previously treated for 0, 3, or 6 days with 1 nM LMB or vehicle, were analyzed by Western blotting using antibodies against the indicated proteins. Relative protein levels from three independent experiments are shown (bottom graphs), with significant differences determined by unpaired t test. (d) WT and HGPS fibroblasts were treated as peer A. Cells were labeled with phalloidin to visualize actin cytoskeleton. Bar, 50 µM. Right. The cellular area was estimated ( n > 300 cells), with significant differences determined by Mann–Whitney U test

Article Snippet: CRM1 , Novus Biologicals, (Cat: NB100−79802) , Rabbit polyclonal , 1:4,000 , 1:250.

Techniques: Inhibition, Immunolabeling, MANN-WHITNEY, Western Blot, Labeling

Journal: Aging Cell

Article Title: Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

doi: 10.1111/acel.13002

Figure Lengend Snippet: Ectopic overexpression of CRM1 induces premature aging phenotype in normal primary fibroblasts. Human primary fibroblasts derived from a healthy donor were stably transfected to express Flag‐CRM1 or Flag alone. (a) Lysates were subjected to Western blotting using antibodies against CRM1, Flag, or actin (loading control). Relative CRM1 levels are shown (right panel). (b) Subcellular distribution of STAT3 was evaluated by CLSM and its n/c ratio estimated (graph). Bar, 10 µM. (c) The activity of β‐galactosidase was measured, and the percentage of senescent cells was estimated from 3 independent experiments ( n = 300 cells for condition). Bar, 100 µM. (a‐c) Significant differences were determined by unpaired t test. (d) Transfected fibroblasts were labeled with phalloidin to visualize actin. Right . Flag‐CRM1‐expressing fibroblasts were treated with 50 nM LMB or vehicle alone for 3 days, prior to being stained with phalloidin and analyzed by CLSM. Bar, 50 µM. The cellular area was determined ( n > 150 cells for condition), and significant differences were determined by Mann–Whitney U test. (e) Lysates from fibroblasts expressing Flag or Flag‐CRM1, which were previously treated with 50 nM LMB or vehicle alone for six days, were analyzed by Western blotting with antibodies against lamin B1 and actin (control). Bottom . Relative lamin B1 levels are shown. Significant differences were determined by unpaired t test. (f) Transfected fibroblasts with or without LMB treatment were immunostained for lamin B1, and typical images are shown. Bar, 10 µm. Bottom. Line profile analysis showing lamin B1 fluorescence pattern. (g) WT fibroblasts stably expressing Flag or Flag‐CRM1 were treated as peer (e) prior to be analyzed by Western blotting for H3K9me3. Bottom. Relative H3K9me3 levels are shown, and significant differences were determined by unpaired t test. (h) Nuclear distribution of H3K9me3 was analyzed in the indicated transfected fibroblasts with or without LMB treatment. Bottom . Line profile analysis showing the H3K9me3 fluorescence pattern

Article Snippet: CRM1 , Novus Biologicals, (Cat: NB100−79802) , Rabbit polyclonal , 1:4,000 , 1:250.

Techniques: Over Expression, Derivative Assay, Stable Transfection, Transfection, Western Blot, Activity Assay, Labeling, Expressing, Staining, MANN-WHITNEY, Fluorescence

Journal: Aging Cell

Article Title: Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

doi: 10.1111/acel.13002

Figure Lengend Snippet: (a‐b) Enhanced nuclear export activity due to CRM1 overexpression overcomes deficient Ran gradient in HeLa cells. Cells were double‐transfected to stably expressed Flag‐CRM1 or Flag alone, and a shRNA against NTF2 gene or a shRNA control. (a) Lysates from the transfected cells were analyzed by Western blotting using antibodies against CRM1, NTF2, and actin (control). Middle. Relative protein levels were assessed from three independent experiments (unpaired t test). Right . Distribution of STAT3 was analyzed in the indicated transfected cells. Bar, 20 µM. (b) Transfected cell lysates were analyzed by Western blotting with antibodies against lamin B1, H3K9me, and actin (control). Middle. Data correspond to 3 independent experiments (unpaired t test). Right . Distribution of H3K9me3 was analyzed in the indicated transfected cells. Bar, 20 µM. (c–e) Restoration of lamin B1 expression in HGPS cells (c) HGPS‐1 cells were transiently transfected to express GFP‐lamin B1 or GFP alone. Transfected cells were immunolabeled for lamin A/C (d) and H3K9m3 (e) to estimate the percentage of cells with aberrant nuclear morphology and heterochromatin loss, respectively. Bar, 10 µM

Article Snippet: CRM1 , Novus Biologicals, (Cat: NB100−79802) , Rabbit polyclonal , 1:4,000 , 1:250.

Techniques: Activity Assay, Over Expression, Transfection, Stable Transfection, shRNA, Western Blot, Expressing, Immunolabeling

Journal: Aging Cell

Article Title: Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

doi: 10.1111/acel.13002

Figure Lengend Snippet: CRM1 expression and activity increased during normal aging. (a) Primary human fibroblast from healthy individuals of varying ages or HGPS‐1 fibroblasts were analyzed by Western blotting using antibodies against CRM1, lamin A/C, and actin (control). CRM1 expression is shown (bottom panel; unpaired t test). (b) Localization of the NES‐containing proteins STAT3, Z0‐2, and B23 was evaluated in the indicated fibroblast cultures, treated with LMB or vehicle alone for 24 hr. Typical images are shown. Bar, 20 µM. (c) The n/c ratio of STAT3, Z0‐2, and B23 was calculated as peer Methods ( n = 50 cells; Mann–Whitney U test)

Article Snippet: CRM1 , Novus Biologicals, (Cat: NB100−79802) , Rabbit polyclonal , 1:4,000 , 1:250.

Techniques: Expressing, Activity Assay, Western Blot, MANN-WHITNEY

Journal: Aging Cell

Article Title: Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

doi: 10.1111/acel.13002

Figure Lengend Snippet: List of antibodies and plasmids used in this study

Article Snippet: CRM1 , Novus Biologicals, (Cat: NB100−79802) , Rabbit polyclonal , 1:4,000 , 1:250.

Techniques: