Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Effects of FSH on phosphorylation and nuclear exclusion of FOXO1 in ovarian GCs. ( a ) The expression of FSHR, FOXO1, and phosphorylated FOXO1 (p-FOXO1) corresponding to the sites Thr 24 , Ser 248 , and Ser 311 in cultured GCs under treatment with FSH or/and LMB was determined via Western blotting by using the anti-FSHR, anti-FOXO1, and anti-p-FOXO1 corresponding to Thr 24 , Ser 248 , and Ser 311 , respectively. The β-actin was used as a loading control. All blots were cropped, and the gels were run under the same experimental conditions. ( b ) The expression levels of FSHR protein in cultured GCs under FSH or/and LMB treatment by Western blotting, as shown in ( a ). ( c ) Expression levels of FOXO1 under treatment the same as ( b ). ( d ) The expression levels of p-FOXO1 corresponding to the site Thr 24 . ( e ) The expression levels of p-FOXO1 corresponding to the site Thr 248 . ( f ) The expression levels of p-FOXO1 corresponding to the site Thr 311 . For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05. ( g ) The subcellular localizations of FOXO1 protein in cultured GCs under FSH or/and LMB treatment by immunofluorescence staining method. The red line segment at the bottom right corner of the image is the scale bar (Leica, 400×; scale bar = 5 µm).

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot, Control, Immunofluorescence, Staining

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: The roles of activated PI3K/Akt signaling in the FSH-induced phosphorylation of FOXO1 in GCs. ( a ) The expression of FSHR and phosphorylated Akt (p-Akt) in cultured GCs under treatment with FSH or/and Ly294002 was determined by Western blotting. ( b ) The expression levels of FSHR protein in GCs under FSH or/and Ly294002 treatment are shown in ( a ). ( c ) The expression levels of p-Akt protein in GCs under the same treatment as ( b ). ( d ) The expression of FOXO1, p-FOXO1 corresponding to the phosphorylation site, Se r248 or Ser 311 , in cultured GCs under treatment with FSH or/and Ly294002 and LMB was examined by Western blotting, respectively. ( e ) The expression levels of FOXO1 protein in cultured GCs. ( f ) The expression levels of pFOXO1 corresponding to the Ser 248 site. ( g ) The expression levels of pFOXO1 corresponding to the Ser 311 site. ( h – j ) The expression levels of PKACA and acetylated FOXO1 (Ac-FOXO1) in cultured GCs under treatment with FSH or/and KH7 were tested by Western blotting, respectively. ( k – m ) The expression levels of the pFOXO1 proteins corresponding to the site, Ser 248 or Ser 311 , in cultured GCs under treatment with Ly294002 or/and TSA were determined by Western blotting, respectively. β-actin was used as a loading control. All blots were cropped, and the gels were run under the same experimental conditions. For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot, Control

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Effects of FSH-induced FOXO1 nuclear exclusion on GC proliferation and apoptosis via PI3K/Akt signaling pathway. ( a ) The subcellular localizations of FOXO1 protein in cultured GCs under FSH or/and Ly294002 treatment by immunofluorescence assay. The red line segment at the bottom right corner of the image is the scale bar (Leica, 400×; scale bar = 5 µm). ( b ) The expression levels of BCL mRNA in cells under FSH or/and LMB treatment by RT-qPCR assay. ( c ) The expression levels of CASP3 mRNA under the same treatment as ( b ). ( d ) The expression levels of CCND1 mRNA. ( e ) The expression levels of PCNA mRNA. ( f – k ) The GC proliferation and apoptosis under FSH or/and LMB treatment by flow cytometry assay. All data are presented as means ± SEM. n = 3. For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Cell Culture, Immunofluorescence, Expressing, Quantitative RT-PCR, Flow Cytometry, Control

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Crosstalk of PI3K/Akt and P62/Keap1/Nrf2 pathways in regulating GC proliferation and apoptosis mediated by FOXO1. ( a ) Expression levels of P62 mRNA under FOXO1 overexpression or/and P62 knockdown examined by RT-qPCR assay. NC: negative control, OE: overexpression, SR: siRNA. The mRNA expression was normalized to that of the 18S rRNA gene; the values of the bar graphs represent the mean ± SEM of 3 hens ( n = 3) from a representative experiment. ( b ) The expression levels of Keap1 mRNA under the same condition as ( a ). ( c ) The expression levels of Nrf2 mRNA under the same conditions as ( a ). ( d ) The expression levels of p62 mRNA under 740-Y-P or/and LMB treatment by RT-qPCR assay. ( e ) The expression levels of BCL2 mRNA under 740-Y-P treatment or/and P62 knockdown by RT-qPCR. ( f – h ) The expression level results of CASP3 , CCND1, and PCNA mRNA under the same conditions as ( e ). ( i – m ). GC proliferation and apoptosis under treatment with FSH or/and P62 knockdown by flow cytometry assay. All data are presented as the means ± SEM. n = 3. For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Expressing, Over Expression, Knockdown, Quantitative RT-PCR, Negative Control, Flow Cytometry, Control

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Ankle1 shuttles between nucleus and cytoplasm. a Schematic representation of Ankle1’s domain organization depicting predicted ankyrin repeats, the LEM domain and a GIY-YIG nuclease domain. Putative nuclear export sequences (NES1, NES2) and nuclear localization sequences (NLS1, NLS2), identified in silico are indicated. b Immuno-fluorescence analysis of ectopic Ankle1-V5 in U2OS cells without or following a 3 h treatment with 50 nM leptomycin B, an inhibitor of CRM1-mediated export. Cells were stained with antibodies to V5, and DNA with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: In Silico, Fluorescence, Staining

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Localization of Ankle1 fragments containing different domains and export and import signals. Localization of GFP-tagged Ankle1 truncation constructs ectopically expressed in U2OS ( a ) and HeLa ( b ) cells was determined by confocal fluorescence microscopy. Molecular weights and schematic representations of domain organization of respective truncation protein constructs are indicated. Cells were fixed after 3 h of mock or leptomycin B treatment. DNA was counterstained with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Construct, Fluorescence, Microscopy

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Mutation analyses identify NES2 and NLS2 as the predominant sequences controlling nucleo-cytoplasmic shuttling of Ankle1. a , b , d , e U2OS cells were transiently transfected with Ankle1-V5 carrying point mutations in NLS or NES sequences and either mock-treated or treated with leptomycin B for 3 h and processed for confocal immunofluorescence analyses using antibodies to V5 and DAPI to detect DNA. Scale bars: 10 μm. c Mean fluorescence intensities in nuclei and cytoplasm of cells expressing wild-type Ankle1-V5, Ankle1-NES1mut-V5 or Ankle1-NES2mut-V5 were measured in original unprocessed digital images prior to contrast/brightness adjustment and nucleus to cytoplasm signal ratios were calculated. Data were obtained from three independent experiments and analyzed using Student’s t -test. Ankle1-NES1, P = 0.002; Ankle1-NES2, P = 5.4E-21; n = 50; 15–17 cells each from three independent experiments

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Mutagenesis, Transfection, Immunofluorescence, Fluorescence, Expressing

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Plac1 expression and lentivirus-mediated reduction of Plac1 in EO771 cells. ( a ) EO771 mouse mammary tumor cells expressed high levels of Plac1 in comparison to mouse placenta. ( b ) EO771 cells were transduced with lentiviruses expressing crambled RNA (Scr) or four Plac1 shRNAs designated sh81, sh187, sh300 and sh490; sh490 inhibited RNA expression >98%, and these cells were designated EO771/shPlac1. ( c ) EO771/Scr and EO771/shPlac1 cells were grown as monolayers, and the number of viable cells were quantified by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis of three samples. The growth of EO771/shPlac1 cells differed significantly ( P < 0.001) from EO771/Scr cells by the two-sided Student’s t test. ( d ) qRT-PCR analysis of immune cell-related gene expression downregulated in EO771/shPlac1 cells. Shown is the mean ± S.D. of triplicate analysis of three samples. Significant differences between EO771/Scr and EO771/shPlac1 cells were obtained for CD274 ( P < 0.01), Plac1 ( P < 0.01), Cxcl1 ( P < 0.001), Ccl5 ( P < 0.001) and Lif ( P < 0.001) using the two-tailed Student’s t test; values for Ccl7 were not significantly different ( P > 0.05). ( e ) Heatmap of gene expression as determined by Affymetrix microarray analysis of EO771/Scr (Ctl) and EO771/shPlac1 (sh) cells. Shown are immune cell-related transcripts (Table ) representing ≥3.0-fold change in expression.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Comparison, Transduction, RNA Expression, Staining, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Microarray

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771/Scr and EO771/shPlac1 cells in syngeneic and SCID mice. ( a ) Syngeneic C57BL/6 mice or ( b ) SCID mice at five weeks of age, were inoculated in the mammary gland with 1 × 10 6 cells. Tumor size was measured by calipers in two dimensions. Tumor growth for EO771/Scr and EO771/shPlac1 cells in syngeneic mice differed significantly (P = 0.040) by the unpaired Student’s t test. There was no significant difference (P > 0.05) in tumor growth between the two cell lines in SCID mice. Shown is the mean ± SD, N = 5 per group. ( c ) H&E staining and Plac1 IHC in isografts of EO771/Scr and EO771/shPlac1 cells. Magnification 400X.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Staining

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771 cells in syngeneic mice following treatment with a Cxcr 2 antagonist. ( a ) Syngeneic 57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age, and injected i.p. daily with vehicle (blue) or 2 mg/kg (red) or 20 mg/kg (green) SB225002 beginning 11 days after cell inoculation. SB225002 completely suppressed tumor growth after 14 days. Differences between vehicle- and 2 mg/kg SB225002-treated mice were not significantly different (P = 0.145); differences between vehicle- and 20 mg/kg SB225002-treated mice were significantly different (P = 0.005) by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N = 5 per group. ( b ) Immune gene expression in tumors 17 days after treatment with 20 mg/kg SB225002. Shown is the relative expression in control and SB225002-treated mice in comparison to their changes in EO771/shPlac1 cells (Table ). ( c ) FACS analysis of immune cell tumor infiltrates in isografts after treatment with vehicle or SB225002 as in ( b ). SB225002 treatment reduced the percentage of immune cell tumor infiltrates of CD11b + /Gr-1 + myeloid-derived suppressor cells ( MDSC ) and Foxp3 + /CD25 + T cells ( Treg ), and increased the percentages of CD8 + /CD4 + T cells ( T) , CD3 + /NK1.1 + NK cells ( NK ) and F4/80 + /CD80/86 + macrophages ( Mϕ ) and CD11c + /CD80/86 + dendritic cells ( DC ). Numbers in parentheses ( ) represent the percentages of each cell population. ( d ) Bar graph represents the mean±SD of the percent distribution of immune cell tumor infiltrates as in ( c ); P values were determined by the unpaired two-tailed Student’s t test, N = 4 per group. ( e ) CD8 + T cell infiltration determined by IHC in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of CD8 + T cells increased after treatment with 20 mg/kg SB225002. Magnification 600X. ( f ) Macrophage ( F4/80 ) and Treg cell ( Foxp3 ) infiltration, Plac1 expression and apoptosis by cleaved caspase-3 expression ( Caspase ) in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of macrophages and Treg cells were reduced and apoptosis was increased after treatment with 20 mg/kg SB225002. Magnification 400X

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Injection, Two Tailed Test, Gene Expression, Expressing, Control, Comparison, Derivative Assay

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Lentivirus-mediated reduction of Cxcl1 in EO771 cells. ( a ) EO771 cells were transduced with lentiviruses expressing scrambled RNA (Scr) or three Cxcl1 shRNAs designated sh118, sh174, sh218; sh174 inhibited RNA expression >99% (EO771/shCxcl1). ( b ) EO771/Scr and EO771/shCxcl1 cells were grown as monolayers, and the number of viable cells were determined by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis from three samples, which were significantly different ( P < 0.001) by the two-tailed Student’s t test. ( c ) Growth of EO771/Scr and EO771/shCxcl1 cells in syngeneic mice. Mice at five weeks of age were inoculated in the mammary gland with 1x10 6 cells, and tumor size was measured by calipers in two dimensions. Differences in tumor growth between EO771/Scr and EO771/shCxcl1 cells were significantly different (P = 0.006) by the unpaired two-tailed Student’s t test; N = 5. ( d ) qRT-PCR analysis of genes downregulated in EO771/shCxcl1 cells. Shown is the mean ± SD of triplicate analysis of 3 samples. Significant differences between EO771/Scr and EO771/shCxcl1 cells were obtained for Plau ( P < 0.02), C3 ( P < 0.01), Ly6a ( P < 0.01), Ccl7 ( P < 0.001) and Il23a ( P < 0.01) by the two-sided Student’s t test; differences for CD68 were not significantly different ( P > 0.05).

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Transduction, Expressing, RNA Expression, Staining, Two Tailed Test, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Expression of immune-related genes in E0771/shCxcl1 cells. Shown are ≥3-fold changes in gene expression with a raw score ≥300 in EO771/shCxcl1 or E0771/Scr cells.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Gene expression common to EO771/shPlac1 and EO771/shCxcl1 cells. Shown is the ratio between EO771/shPlac1 or EO771/shCxcl1 cells to EO771/Scr control cells for genes with ≥3.0-fold changes in expression and a raw score ≥300.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Gene Expression, Control, Expressing

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Cxcl1 rescue of EO771/sh490 cells. ( a ) EO771/Scr and EO771/sh490 cells expressing eGFP were transduced with a lentivirus expressing Cxcl1 and mCherry, and selected for 35 days in 3.5 mg/ml G418. The merged photo shows cells co-expressing eGFP and mCherry (yellow). Magnification 200X. ( b ) qRT-PCR for Plac1 and Cxcl1 in EO771/Scr, EO771/sh490 and EO771/sh490/Cxcl1 cells. Shown is the mean ± S.D. of triplicate determinations.( c ) EO771/sh490/Cxcl1 cells were grown in 96-well plates at an initial density of 5,000 cells per well in media supplemented with 3.5 mg/ml G418. Cell density was determined by sulforhodamine B staining. Shown is the mean ± SD of triplicate determinations. ( d ) Syngeneic C57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age. There was a significant difference in the growth EO771/sh490 cells (P = 0.021) and EO771/sh490/Cxcl1 cells (P = 0.034) vs. EO771/Scr cells by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N=6 per group.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LOUCY and FKH1 cells were treated with ( A ) LMB (20 nM, 3 h) or ( C ) KPT-185 (1 μM, 24 h) and NUP214 (green) and CRM1 (magenta) localization was studied by confocal microscopy. DNA stained with DAPI is depicted in blue. Scale bars, 10 μm. Quantitative analysis of the number of NUP214 nuclear bodies in ( B ) LOUCY and ( D ) FKH-1 cells. At least 150 cells were analyzed for each condition. Statistical differences were calculated using GraphPad Prism (v5.01) using one-way analysis of variance (ANOVA) test. No treatment vs LMB; No treatment vs KPT-185. *** p < 0.001.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: Confocal Microscopy, Staining

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LOUCY cells (SET-NUP214) were treated with ( A ) LMB (20 nM, 3 h) or ( B ) KPT-185 (1 μM, 24 h). Cells were allowed to grow in drug-free medium for up to 48 h, after clearance from the respective drug. The presence of SET- NUP214 nuclear bodies was evaluated by immunofluorescence using anti-NUP214 (green) and anti-CRM1 (magenta) antibodies. DNA stained with DAPI is depicted in blue. Scale bars, 10 μm.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LOUCY cells (SET-NUP214) were treated with ( A ) LMB (20 nM, 3 h) or ( B ) KPT-185 (1 μM, 24 h) and cells were allowed to grow in drug-free medium for up to 48 h after treatment. The formation of SET-NUP214 (green) nuclear foci is accompanied by the accumulation of CRM1 (magenta) in these structures. Shown are representative confocal images. DNA stained with DAPI is depicted in blue. Scale bars, 10 μm.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: Staining

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: Leukemia cell lines LOUCY ( A ), MEGAL ( B ), and FKH-1 ( C ) were treated with LMB (20 nM) or KPT-185 (1 μM) for the indicated time points and cell viability was measured by Trypan Blue exclusion dye. Statistical differences were calculated using GraphPad Prism (v5.01) using one-way analysis of variance (ANOVA) test. No treatment vs LMB; No treatment vs KPT-185. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques:

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: LMB or KPT-185 treated LOUCY, MEGAL, and FKH-1 cells were allowed to grow in drug-free medium for up to 48 h after drug clearance. ( A ) Cell viability was determined by Trypan Blue exclusion dye. ( B ) WST-1 assay was performed to measure cellular metabolic activity. Absorbance was measured at 450 nm. Results are normalized to untreated cells and expressed as percentage. ( C ) Colony forming assays of LMB and KPT-185 treated MEGAL and FKH-1 cells. Colonies were visualized under a 10× microscope objective after growth in drug-free Methocult™ medium for 14 days. Scale bars, 100 μm. Statistical differences were calculated using GraphPad Prism (v5.01) using one-way analysis of variance (ANOVA) test. No treatment vs LMB; No treatment vs KPT-185. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: WST-1 Assay, Activity Assay, Microscopy

Journal: Oncotarget

Article Title: Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

doi: 10.18632/oncotarget.27711

Figure Lengend Snippet: Flowcytometric analysis of LOUCY, MEGAL, and FKH-1 cells after withdrawal from LMB or KPT-185 treatment. Cell proliferation was evaluated by using FITC-Ki-67 antibodies. ( A ) Histogram representation and ( B ) quantification of FITC-Ki-67 positive cells in the population of single cells at the indicated time points.

Article Snippet: Cells were seeded at 0.8 × 10 6 cells/ml for 24 h. Next, cells were treated with 1 μM KPT-185 (Selleck Chemicals, Munich, Germany) or 20 nM LMB (Enzo Life Sciences, Brussels, Belgium).

Techniques: