Journal: PLoS ONE

Article Title: The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure

doi: 10.1371/journal.pone.0193921

Figure Lengend Snippet: BEAS-2B cells were pretreated with or without 100 μM BSO in the presence or absence of 3% CSE. After 24 hours, localization of SIRT1, SIRT6, HDAC2 and CRM1 in cytoplasm (A) and nuclei (B) were examined by SDS-PAGE / WB. (C) SIRT1 localization after CSE exposure for 24 hours in the presence of BSO in BEAS-2B cells were detected by immunocytochemistry (× 40 in BD Pathway 435 bioimager). (D-G) In the 3% CSE exposure with 100 μM BSO pretreatment model, the SIRT1 shuttling was examined in the human primary bronchial epithelial cells (D, E) or air-liquid interface cultured human bronchial epithelial cells (F, G). (H, I) After CSE exposure with BSO pretreatment in BEAS2B, the effect of PIK75 (0.1 μM) on the SIRT1 shuttling was examined. (J) SIRT1 in nuclear fraction after nicotine (10 μM) or anisomycin (10 μg / ml) treatment for 6 hours in BEAS-2B. (K) Effects of Akt inhibitor (10 μM) on SIRT1 protein in nuclei from cells stimulated with 3% CSE for 24 hours. (L) Effects of N-acetyl cysteine (NAC, 10 mM) and SP-600125 (SP, 10 μM) on SIRT1 protein in nuclei from cells stimulated with 3% CSE for 24 hours. All values were mean value ± SEM of at least three experiments. * p < 0.05, ** p < 0.01, compared with the values of non-treatment group; †† p < 0.01 between the two groups.

Article Snippet: Commercially available reagents were obtained as follows: RPMI medium 1640 (RPMI 1640) (#11875), polymerase chain reaction (PCR) primer for SIRT1 (Hs01009005) and GNB2L1 (Hs00272002) were from Life Technologies (Carlsbad, CA, USA); fetal bovine serum (FBS), complete protease inhibitor cocktail (#11836153001), anti-SIRT6 antibody (#S4197), anti-HDAC2 antibody (#H2663), thiazolyl blue tetrazolium bromide for MTT assay (M2003), L-buthionine-sulfoximine (BSO) (#B2515), AS605240 (#A0233), rapamycin (#R8781), dimethyl biguanide hydrochloride (metformin) (#D150959), NU7026 (N1537), Z-Leu-Leu-Leu-al (MG-132) (#C2211), cycloheximide (CHX) (#C7698), resveratrol (#R5010) and sirtinol (#S7942) were from Sigma-Aldrich Co. LLC (St Louis, MA, USA); anti-SIRT1 antibody (sc-15404), PIK75 (#sc-296089), IC87114 (#sc-364509), anti-Nrf2 antibody (#sc-13032), anti-α-tubulin antibody (#sc-5286), anti-Lamin A/C antibody (#sc-7292) and anti-chromosomal region maintenance 1 (CRM1) antibody (#sc-5595) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA); anti-β-actin antibody (#ab6276) was from Abcam plc. (Cambridge, UK); goat-derived peroxidase-conjugated anti-mouse (#P0447) or anti-rabbit (#P0448) secondary antibodies were from Dako (Cambridge shire, UK); anti-phospho-SIRT1 (Ser47) antibody (#2314), anti-phospho-Akt (Ser47) (p-Akt) antibody (#4060) and anti-total Akt antibody (#4691) were from Cell Signaling Technology (Danvers, MA, USA); GSK2636771 (#S8002), BIRB796 (#S1574) and U1026 (#S1102) were from Selleck biochemicals (Houston, TX, USA); N-acetyl-Leu-Leu-Norleu-al (ALLN) (#208750) and Akt inhibitor (Akt Inhibitor II) were from Millipore (U.K.) Limited (Hertfordshire, U.K.).

Techniques: SDS Page, Immunocytochemistry, Cell Culture

Journal: Scientific Reports

Article Title: Dual mechanisms regulate the nucleocytoplasmic localization of human DDX6

doi: 10.1038/srep42853

Figure Lengend Snippet: The diagram summarized the two proposed mechanisms governing the nucleocytoplasmic distribution mechanism of DDX6 in this study. (a) The “hitchhiking” model refers to that DDX6 can enter cell nuclei as a passenger on the mitotic chromosomes. (b) The “piggyback” model refers to that 4E-T is sufficient to transport DDX6 via the importin-α/β and the CRM1 pathways in a piggyback manner.

Article Snippet: Primary antibodies recognizing GFP-tag (Living Colors, Clontech), myc-tag (clone 4A6, EMD Millipore), DDX6 (A300–460A, Bethyl Labratories, Inc), LMNA (ab108595, Abcam), GAPDH (GTX100118, GeneTex), CALR (GTX111627, GeneTex; or, 06–661, EMD Millipore), AGO2 (H00027161-M01, Abnova), 4E-T (A300–706A, Bethyl Laborator), EIF4E (#9742, Cell Signaling), POLR2A p-Ser2 (A300–654A, Bethyl Laboratory), CRM1 (sc-5595, Santa Cruz) and HRP-conjugated secondary antibodies (SC-2004 or SC-2005, Santa Cruz) were used to detect the proteins of interest.

Techniques:

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

doi: 10.1038/s41467-022-31865-5

Figure Lengend Snippet: a Confocal micrographs (maximum intensity projections) of Leptomycin B, Selinexor and control DMSO treated U2OS cells, stained for CIP2A. b Quantification of nuclear CIP2A staining of the experiment in a . Each data point corresponds to one cell (DMSO: n = 173, LeptomycinB: n = 150, Selinexor: n = 178, pooled from two independent experiments). Bars and error bars represent mean and SD. Statistical significance was calculated using one-way ANOVA and Dunnett’s multiple comparison test (α = 0.05). c Confocal micrographs (maximum intensity projections) of RPE-1 ΔCIP2A cells and RPE-1 ΔCIP2A cells stably transduced with Flag-tagged full-length CIP2A (WT) and CIP2A deletion mutant (human CIP2A amino acids 561–625; ΔNES), stained for CIP2A. d Quantification of nuclear CIP2A staining of the experiment in c . Each data point corresponds to one cell (+Epmpty: n = 199, +Flag-WT: n = 155, +Flag-ΔNES: n = 165, pooled from two independent experiments). Bars and error bars represent mean and SD. Statistical significance between +Flag-WT and +Flag-ΔNES was calculated using two-sided Welch’s t -test (α = 0.05). e Flag-immunoprecipitation from 293FT cells either Mock transfected (Mock) or transfected with a Flag-tagged full-length CIP2A wild type (WT), and Flag-tagged CIP2A lacking amino acids 561-625 (ΔNES). Relative intensities of co-immunoprecipitated CRM1 bands are indicated. f Confocal micrograph (maximum intensity projection) of Hela cells expressing endogenous Clover-tagged LMNA and stained for CIP2A. g Quantification of CIP2A-TOPBP1 proximity by in situ PLA in U2OS cells transfected with control siRNA (siCtrl) and siRNA against TOPBP1 (siTOPBP1), arrested in mitosis by Nocodazole and treated with 1 Gy of IR. Interphase cells (I) were separated from mitotic cells (M) with an automatic image analysis pipeline, based on DAPI mean intensities. Each data point represents one cell (siCtrl I: n = 92, siCtrl M: n = 63, siTOPBP1 I: n = 138, siTOPBP1 M: n = 121; pooled from two independent experiments), and bars represent median. Statistical significance was assessed by Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). Source data are provided as a Source Data file.

Article Snippet: Antibodies against the following proteins were used at the indicated dilutions: GFP (Mouse, Roche, 11814460001, 1:5000), GFP (Rabbit, Abcam, ab290, 1:1000), HA (Rabbit, Abcam, ab9110, 1:4000), MDC1 (Rabbit, Abcam, ab11171, 1:5000), MDC1 pSer168 (Rabbit, AMS Biotechnology, custom-made, 1:500) , MDC1 pSer196 (Rabbit, 21 st Century, custom-made 1:200) , TOPBP1 (Rabbit, Abcam, ab2402, 1:1500), CIP2A (Rabbit, Cell Signaling, 14805, 1:1000), CIP2A (Mouse, Santa Cruz, sc-80659, 1:1000), CRM1 (Rabbit, Atlas Antibodies, HPA042933, 1:1500), Tubulin (Mouse, Sigma, T6199, 1:5000), Tubulin hFABTM rhodamine (Bio-Rad, 12004166, 1:5000), FLAG M2 (Mouse, Sigma, F1804, 1:1000), pSer10 Histone H3 (Rabbit, Millipore, 06-570, 1:1000), Histone H3 (Rabbit, Abcam, ab1791, 1:10000).

Techniques: Control, Staining, Comparison, Stable Transfection, Transduction, Mutagenesis, Immunoprecipitation, Transfection, Expressing, In Situ

Journal: Nature Communications

Article Title: Specific inhibition of the Survivin–CRM1 interaction by peptide-modified molecular tweezers

doi: 10.1038/s41467-021-21753-9

Figure Lengend Snippet: a 293T cell lysate with overexpressed Survivin142-HA was preincubated with either unmodified tweezer (TW), TW-ELTL, TW-ELTLGEFL, or a scrambled peptide-modified tweezer, TW-LFEEGLLT, at concentrations ranging from 0.01 to 200 µM. GST-CRM1 was mixed with either non- or preincubated cell lysates in the presence of recombinant RanQ69L and dGTP to allow complex assembly. GST-CRM1 and interacting proteins were pulled by GSH-Sepharose beads. Proteins in input and beads samples were analyzed via immunoblotting with antibodies specific for GST or HA. For each tweezer, samples derive from the same experiment and gels/blots were processed in parallel. Direct comparison for this exact concentration range was performed once for TW, TW-ELTL, and TW-ELTLGEFL and for TW-LFEEGLLT in three technical replicates. b , c Atto488-labeled Survivin120 was preincubated with CRM1_1-1062VLV430AAA mutant in a ratio of 1:5 and titrated with supramolecular tweezers up to approx. 180 µM. Fluorescence anisotropy was measured ( n = 1) ( b ), and IC50 values were determined from the resulting curves ( c ). TW light blue/ triangles, TW-ELTL blue/squares, TW-ELTLGEFL dark blue/circles. Source data are provided as a Source Data file.

Article Snippet: Next, membranes were incubated in primary antibodies against HA (anti-HA, mouse monoclonal, BioLegend, Covance MMS-101R, 1:1,000), GST (anti-GST, mouse monoclonal, Santa Cruz Biotechnology Inc., sc-57753, 1:1000) or CRM1 (anti-CRM1, rabbit polyclonal, Novus Biologicals Ltd., NB100-79802, 1:10,000) overnight at 4 °C.

Techniques: Modification, Recombinant, Western Blot, Comparison, Concentration Assay, Labeling, Mutagenesis, Fluorescence

Journal: Nature Communications

Article Title: Specific inhibition of the Survivin–CRM1 interaction by peptide-modified molecular tweezers

doi: 10.1038/s41467-021-21753-9

Figure Lengend Snippet: Titration of 300 μM TW ( a ) in the cell with 321 µM Survivin120 K90/103T in the syringe. Titration of 100 µM TW-ELTL ( b ) and TW-ELTLGEFL ( c ) in the cell with 2.5 mM Survivin120 K90/103T in the syringe. All titrations were performed in PBS, pH 7.4 at 25 °C. Graphs represent one representative example each from three independent experiments ( n = 3). The black lines in the bottom panels are the best fit of the data to a one set of sites model. The heat of dilution was subtracted as constant. For thermodynamic data derived from the graphs see SI . FAM-labeled unmodified tweezer molecule ( d ), TW-ELTL ( e ), and TW-ELTLGEFL ( f ) (0.2 µM) were titrated with either Survivin120 (200 µM, circles) or Survivin120 K90/103T (400 µM, squares). Survivin120 K90/103T showed greatly reduced tweezer affinities (lower curves). d – f Data are presented as mean values ± SD with n = 3 independent experiments. g Pull-down results after immunostaining. GST-Survivin120-WT or GST-Survivin120-K90/103T were incubated with 50 µM respective tweezer molecule or ELTL/ ELTLGEFL peptides w/o tweezer. GST-Survivin120- or GST-Survivin120-K90/103T-loaded beads were mixed with CRM1 and RanQ69L prey proteins as well as dGTP to allow export complex assembly. Proteins in input and bead samples were analyzed via immunoblotting with antibodies specific for CRM1 or GST. WT, wildtype. One representative example of two independent biological replicates is shown. Samples derive from the same experiment and gels/blots were processed in parallel. h Quantification of two independent pull-down experiments. After subtraction of the CRM1 negative control from the pulled CRM1 intensity, the latter is normalized by the GST–Survivin intensity and afterwards normalized by the CRM1 intensity without tweezer incubation. Export complex assembly is only compromised by peptide tweezers in the wildtype Survivin120, but not in the mutant. No ligand: black; TW: light blue; TW-ELTL: blue; TW-ELTLGEFL: dark blue; ELTL peptide: light gray; ELTLGEFL peptide: dark gray. Source data are provided as a Source Data file.

Article Snippet: Next, membranes were incubated in primary antibodies against HA (anti-HA, mouse monoclonal, BioLegend, Covance MMS-101R, 1:1,000), GST (anti-GST, mouse monoclonal, Santa Cruz Biotechnology Inc., sc-57753, 1:1000) or CRM1 (anti-CRM1, rabbit polyclonal, Novus Biologicals Ltd., NB100-79802, 1:10,000) overnight at 4 °C.

Techniques: Titration, Derivative Assay, Labeling, Immunostaining, Incubation, Western Blot, Negative Control, Mutagenesis