Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: Identification of UCA1 as an hnRNP I-binding partner. ( a ) Enrichment of UCA1 by hnRNP I antibody, as detected by RIP assay. ( b ) Confirmation of the interaction between UCA1 and hnRNP I by RNA precipitation assay using UCA1 RNA probe. Preparation of UCA1 RNA probe and cellular extract from MCF-7 cells were detailed in the text. ( c ) The 5′ end of UCA1 is responsible for its interaction with hnRNP I, as detected by RNA precipitation. UCA1 RNA probes used in this experiment were indicated in top panel. RoR exon 4 was used as a positive control. ( d ) A putative hnRNP I-binding motif in UCA1 is critical to its interaction with hnRNP I. Description of the mutant UCA1 at the putative hnRNP I-binding site was indicated in left panel where the conserved binding motif was underlined. Y, either C or T. Detection of hnRNP I in the pellets by western blot as indicated in right panel. Error bars represent S.E.M., n =3. ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Positive Control, Mutagenesis, Western Blot

Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: Effect of hnRNP I on UCA1 expression. ( a ) Ectopic expression of hnRNP I increases UCA1, as detected by qRT–PCR. ( b ) Suppression of UCA1 by hnRNP I-siRNA. ( c ) Effect of hnRNP I-siRNA on UCA1 stability. MCF-7 cells were first transfected with control siRNA or hnRNP I-siRNA overnight and the transfected cells were treated with actinomycin D at 1 μ g/ml. RNA was isolated at 0, 6 and 12 h, respectively. Error bars represent S.E.M., n =3. * P <0.05; ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

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

Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: Induction of UCA1 through interaction with hnRNP I. ( a ) Doxo induces UCA1 independent of p53, as detected by qRT–PCR in MCF-7 and MDA-MB-231 cells. Cells were treated with doxo at 1 μ g/ml for 24 h before they were harvested for RNA extraction. ( b ) Suppression of doxo-induced UCA1 by hnRNP I-siRNA. MCF-7 cells were first transfected with control siRNA or hnRNP I-siRNA and then treated with doxo at 1 μ g/ml for 24 h before they were harvested for RNA extraction. ( c ) The phosphorylated form of hnRNP I is localized in the cytoplasm and is responsible for interaction with UCA1. RNA precipitation was used to detect hnRNP I. The same membrane was probed simultaneously with hnRNP I antibody (mouse origin) and p-hnRNP I antibody (rabbit origin), followed by secondary antibody labeled with either IRDye 680 or IRDye 800. The bottom panel shows the cytoplasmic (cyto) and nuclear (nuc) extracts used for RNA precipitation assays in the top panels. GAPDH serves as a cytoplasmic marker and NPM as a nuclear marker. ( d ) Redistribution of hnRNP I in response to doxo treatment. MCF-7 cells were first transfected with GFP–hnRNP I fusion construct, followed by doxo treatment (1 μ g/ml) for 8 h or UV treatment (20 J/m 2 ) for 3 h before fixation for fluorescent microscopy. ( e ) Colocalization of phosphorylated hnRNP I with UCA1 by IF and fluorescence in situ hybridization. ( f ) Doxo increases UCA1 pulldown by hnRNP I antibody. MCF-7 cells were treated with doxo the same way as in d , and then cytoplasmic fraction was prepared for RIP assay. Error bars represent S.E.M., n =3. ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, RNA Extraction, Transfection, Control, Membrane, Labeling, Marker, Construct, Microscopy, Fluorescence, In Situ Hybridization

Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: UCA1 suppresses p27 protein level by competing for hnRNP I. ( a ) Interaction of p27 mRNA with hnRNP I, as detected by RIP assay. ( b ) Although ectopic expression of UCA1 suppresses, UCA1-siRNA increases the p27 protein level. MCF-7 cells were transfected with vector or UCA1; control siRNA or UCA1-siRNA and then harvested for western blotting 24 h after transfection. ( c ) Suppression of the p27 protein level by hnRNP I-siRNA. The procedure was same as in b . ( d ) Effect of UCA1 and UCA1-siRNA on the p27 5′-UTR luciferase reporter activity. MCF-7 cells were transfected with the p27 5′-UTR luciferase reporter along with UCA1 or UCA1-siRNA. Luciferase assay was carried out 24 after transfection. Relative luciferase activity is Renilla luciferase activity relative to Firefly luciferase activity as an internal control. ( e ) Suppression of the interaction of p27 mRNA with hnRNP I by UCA1. MCF-7 cells were transfected with vector control or UCA1, and cellular extract was prepared for RIP assay using hnRNP I antibody 24 h after transfection. Error bars represent S.E.M., n =3. * P <0.05; ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

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

Journal: bioRxiv

Article Title: Exportin 1-mediated nuclear/cytoplasmic trafficking controls drug sensitivity of classical Hodgkin lymphoma

doi: 10.1101/2022.07.19.500622

Figure Lengend Snippet: A , Docking of selinexor on XPO1 wt and XPO1 E571K proteins. The protein is represented in the cartoon presentation except for C528, used as anchor, and the three residues made flexible during docking, that are shown as sticks and its distance to the side chain of residue 571 is reported. B , cHL cell lines were seeded and treated with various concentrations of selinexor. Cell viability was measured using an MTS assay. On the graph are presented the means ± s.d. of one representative experiment performed with triplicate samples. IC 50 were calculated with the Prism software (v8.0) and verified with the CompuSyn software. The experiment was performed three times with similar results. C , XPO1 expression was analyzed by IF in parental (p) and CRIPSR Cas9-edited (Δmut) UHO1 cells. We used a primary Ab against XPO1 (Table S1) and a goat Alexa Fluor 488-conjugated anti-rabbit IgG as secondary Ab. Slides were counterstained with DAPI and analyzed with a confocal microscope (Fluoview FV100, Olympus) (x 540, magnification). Images were processed with the ImageJ software and the curves of fluorescence intensity (FI) in arbitrary units, as a function of distance (in pixels) along the white line crossing one representative cell were exported. Due to the limited number of UHO1Δmut cells, the experiment has been done twice. D , UHO1 parental (p) and edited (Δmut) cells were treated with selinexor (40 nM) for 48 h and cell viability was assessed with an MTS assay as described. Due to the limited number of edited cells, the experiment was performed only once with triplicate samples. The means ± s.d. are indicated on the histograms. * p = 0.0182 with the t -test. E , cHL cells lines were treated with vehicle (V) for 24 h or selinexor (S, 10 or 100 nM) for 8 or 24 h. Whole-cell proteins were purified, separated on SDS-PAGE and transferred onto nitrocellulose sheets. Membranes were incubated with an anti-XPO1 Ab (Table S1). Three independent experiments were run (Fig. S3) and the levels of XPO1 and β-actin (as an internal control) proteins estimated by densitometry (ChemiDoc XRS+, ImageLab software, Bio-Rad). For each cell line and each culture condition, the level of XPO1 was calculated relative to the control condition (V) defined as 1. The corresponding values were used to draw the histograms. ns, not significant; **, p = 0.084; ***, p = 0.003 with the t -test.

Article Snippet: The Alt-R Cas9 Electroporation Enhancer (IDT) was added to the mixture to ensure an optimal delivery of the RNP/Cas9 complex during the transfection step.

Techniques: MTS Assay, Software, Expressing, Microscopy, Fluorescence, Purification, SDS Page, Incubation

Journal: bioRxiv

Article Title: Exportin 1-mediated nuclear/cytoplasmic trafficking controls drug sensitivity of classical Hodgkin lymphoma

doi: 10.1101/2022.07.19.500622

Figure Lengend Snippet: A , cHL cell lines were seeded and treated with various concentrations of ibrutinib. Cell viability was measured using an MTS assay. On the graph are presented the means ± s.d. of one representative experiment performed with triplicate samples. IC 50 were calculated with the PRISM software and confirmed with the CompuSyn software. The experiment was performed three times with similar results. B , PMBL cell lines were seeded and treated with various concentrations of ibrutinib. Cell viability was measured using an MTS assay. On the graph are presented the means ± s.d. of one representative experiment performed with triplicate samples. IC 50 were calculated with the PRISM and the CompuSyn softwares. The experiment was performed twice with similar results. C , XPO1 expression was analyzed by WB in the parental (p) U2940 cells along with edited KAS/KS and EAS/ES clones. The anti-XPO1 Ab is directed against the C-terminal part of the XPO1 protein (residues 772-1071) and detects both the wt and the mutant protein (Table S1). An anti-β-actin Ab served as a control of charge and transfer. The original blots are presented Fig. S10. D , U2940 parental cells and derivatives (K and E series) were seeded and treated with various concentrations of ibrutinib. Cell viability was measured using an MTS assay. On the graph are presented the means ± s.d. of one representative experiment performed with triplicate samples. IC 50 were calculated with the PRISM and CompuSyn softwares. The experiment was performed twice with similar results. E , MedB1 cells were edited with a CRISPR-Cas9 strategy to knock-out the wt allele (Fig. S1). The expression and localization of XPO1 were analyzed by IF as described previously. F , Parental and edited MedB1 cells were assayed for selinexor or ibrutinib sensitivity with an MTS assay. Cells were seeded and incubated with selinexor (3 μM) or ibrutinib (50 μM) for 48 h. Due to the limited number of edited cells the experiment was performed once with triplicate sample. The means ± s.d. are indicated on the histograms. ** p = 0,0016; *** p = 0.0010 with the t -test.

Article Snippet: The Alt-R Cas9 Electroporation Enhancer (IDT) was added to the mixture to ensure an optimal delivery of the RNP/Cas9 complex during the transfection step.

Techniques: MTS Assay, Software, Expressing, Clone Assay, Mutagenesis, CRISPR, Knock-Out, Incubation