Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: The replication efficiency of the HPV18-E1HA-Nluc-E2Flag genome is similar to that of the WT HPV18. (A) Map of the HPV18-E1HA-Nluc-E2Flag genome. Red indicates the position of the tags in the E1 and E2 ORFs. The sequence of Nluc is immediately after 72 nucleotides of the WT E2 ORF, which overlaps with the 3′ end of the E1-HA ORF. The full-length E2 ORF containing the Flag tag sequence follows immediately after the sequence encoding the self-processing 2A peptide situated in the 3′ end of the E2-Nluc ORF (not shown). (B) Transient replication of the HPV18 and HPV18-E1HA-Nluc-E2Flag genomes in the U2OS cells was analyzed using SB. Total DNA was treated with BglI and DpnI restriction enzymes. (C and D, left panel) SB signals corresponding to the replicated HPV18 (C) and HPV18-E1HA-Nluc-E2Flag genome (D) were quantified using ImageQuant software. The percent intensity was calculated for each sample relative to the signals obtained in the 2-day posttransfection cells. (D, right panel) The samples obtained from the cells transfected with HPV18-E1HA-Nluc-E2Flag were analyzed for Nluc activity, which was normalized by the activity of alkaline phosphatase and set as 100% for the 2-day-posttransfection cells (*, P < 0.05; **, P < 0.01; n = 3). (E) U2OS cells were transfected with the HPV18-E1HA-Nluc-E2Flag genome and incubated for 2, 3, and 4 days. Linear regression of the normalized Nluc activity and quantified SB signals was analyzed using GraphPad software. The data are expressed as percentages ± the standard deviations (SD) of the signals obtained in the cells incubated for 2 days (set as 100%).

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Sequencing, FLAG-tag, Software, Transfection, Activity Assay, Incubation

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: E1 RNAi restrains the transient replication of the modified HPV18 genomes in the U2OS cells. (A) U2OS cells were transfected with either the HPV18 or HPV18-E1HA-Nluc-E2Flag genome in the presence of E1-specific or Neg. siRNAs. The cells were incubated for 4 days. The endogenous HA-tagged E1 and Flag-tagged E2 proteins were immunoprecipitated and analyzed using WB and anti-tag antibodies. E1 was also detected in E2 immunocomplexes. GAPDH was used as a control. (B) WB signals corresponding to the E1-HA and E2-Flag protein levels were quantified and expressed as percentages of the E1-HA and E2-Flag levels, respectively, obtained in the samples transfected with Neg. siRNA (indicated as 100%). The data are expressed as average means ± the SD (***, P < 0.001; n = 4). (C) The U2OS cells were transfected with the HPV18-E1HA-Nluc-E2Flag or HPV18 WT genome, incubated for 3 days, and treated with DMSO or 9 μM CX4945 for 12, 24, or 48 h. Endogenous E1 and E2 proteins were immunoprecipitated and analyzed using WB and tag-specific antibodies. (D) WB signals corresponding to the E1-HA and E2-Flag protein levels were quantified and expressed as percentages of the E1-HA and E2-Flag levels, respectively, obtained in the samples treated with DMSO (indicated as 100%). The data are expressed as average means ± the SD (*, P < 0.05; ***, P < 0.001; n = 4). (E) U2OS cells were cotransfected with the HPV18-E1HA-Nluc-E2Flag genome, E1-specific or Neg. siRNA, and the constructs encoding either E1 or E2 of HPV18. Cells were incubated for 2, 3, or 4 days and subjected to luciferase assay. The Nluc activity was normalized to the activity of AP. The data are shown as a percentage of the normalized Nluc activity obtained in the cells transfected with the HPV18-E1HA-Nluc-E2Flag genome, Neg. siRNA and empty vector and incubated for 2 days (set as 100%) (for statistical analysis, data of each sample was compared to the data of the respective control at the same time point [**, P < 0.01; ***, P < 0.001; n = 3]). (F) U2OS cells were transfected with the HPV18-E1HA genome, E1-specific or Neg. siRNA, and E2-encoding construct. Total DNA was isolated from 2-, 3-, and 4-day-posttransfection cells, treated with BglI and DpnI restriction endonucleases and subjected to SB. (G) Cell cycle profile of the U2OS cells cotransfected with the HPV18-E1HA genome and the siRNAs was analyzed using PI.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Modification, Transfection, Incubation, Immunoprecipitation, Control, Construct, Luciferase, Activity Assay, Plasmid Preparation, Isolation

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: E1 RNAi is responsible for the suppression of the HPV18 replication. (A) U2OS cells were transfected with the constructs expressing HPV18 E1-WT, E1-siRNA1-R, and E1-siRNA2-R transcripts. E1-siRNA1-R and E1-siRNA2-R ORFs contain silent mutations to generate resistance for E1 siRNA1 and siRNA2, respectively. Housekeeping GAPDH and overexpressed E1-HA proteins were analyzed using WB. (B) U2OS cells were cotransfected with the HPV18-Nluc genome and different E1-HA expression constructs or an empty vector in quadruplicates. The cells were incubated for 2, 3, and 4 days and subjected to a luciferase assay. The Nluc activity was normalized to the total protein amount and set as 100% in the control cells cotransfected with the genome and empty vector and incubated for 2 days. The data are shown as average means ± the SD (n = 2). (C) E1-siRNA1-R and E1-siRNA2-R constructs were cotransfected with the E1 siRNAs into U2OS cells. After 3 days of transfection, GAPDH and E1-HA proteins were analyzed using immunoblotting. (D) U2OS cells were cotransfected with the HPV18-Nluc genome, E1 siRNAs, and E1-siRNA1-R and E1-siRNA2-R expression constructs in quadruplicates; incubated for 2, 3, and 4 days; and analyzed using luciferase assay. Nluc activity was normalized to the total protein amount and set as 100% in the control cells cotransfected with the genome, empty vector and Neg. siRNA and incubated for 2 days. The data are shown as average percentages of the control ± the SD (**, P < 0.01; ***, P < 0.001; n = 3). (E) U2OS cells were transfected with the indicated HPV18 genomes and incubated for 2, 3, and 4 days. Total DNA was extracted, digested with BglI and DpnI restriction enzymes, and analyzed using SB. (F) U2OS cells were cotransfected with either HPV18-E1HA-R1 or HPV18-E1HA-R2 genomes and Neg. or E1 siRNAs. Cells were incubated for 3 days. E1-HA and GAPDH proteins were analyzed using WB. (G) U2OS cells were cotransfected with either HPV18-E1HA-R1 or HPV18-E1HA-R2 genomes, E1 siRNAs, and E1-siRNA1-R or E1-siRNA2-R expression constructs, if indicated. After 3 days of incubation, total DNA was extracted, treated with BglI and DpnI restriction endonucleases, and subjected to SB analysis.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

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

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: U2OS cells support stable replication of the modified HPV18 genomes. (A) U2OS-derived HPV18-E1HA+ and HPV18-Nluc+ cells containing the stably replicating HPV18-E1HA and HPV18-Nluc genomes, respectively, were cultured for approximately two months. Total DNA was isolated at passages p2 to p12, treated with the restriction enzyme BglI linearizing the indicated HPV18 genomes, and subjected to SB. (B) Prior to SB, the LMW DNA isolated from the nonconfluent HPV18-E1HA+ and HPV18-Nluc+ cells (left and right panels, respectively) was treated with restriction endonucleases for either linearizing (lin) or not cutting (nc) the indicated modified HPV18 genomes. lin1, BglI; lin2, BglII; lin3, SdaI; nc1, HindIII; nc2, SacI; nc3, ScaI. (C) After seeding, the HPV18-E1HA+ and HPV18-Nluc+ stable cell lines were continuously propagated for 2, 4, and 7 days, resulting in nonconfluent, subconfluent, and confluent cultures, respectively. The U2OS cells were transfected with the respective modified HPV18 genomes and cultured for the indicated periods of time. Prior to SB, the isolated extrachromosomal DNA was treated with the restriction enzymes for either linearizing (BglI) or not cutting (HindIII) the indicated HPV18 genomes and with DpnI in the case of the transiently replicating genomes. Numbers indicate different forms of the uncut modified HPV18 DNA detected in the LMW DNA pool isolated from the confluent stable cell lines. ccc, covalently closed circular DNA; lin, linear DNA; oc, open circular DNA. (D) SB signals corresponding to the bands numbered in panel C were quantified. The sum of the pixels obtained from each sample was set as 100%, and the intensity of each particular band was calculated relative to 100%. The data are presented as average means ± the SD of at least five different samples.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Modification, Derivative Assay, Stable Transfection, Cell Culture, Isolation, Transfection

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: The HPV18-E1HA+ and HPV18-Nluc+ cells carry head-to-tail concatemeric episomes that replicate via non-theta-type intermediates. (A) LMW DNA was extracted via Hirt lysis from the subconfluent HPV18-E1HA+ and HPV18-Nluc+ cells. Prior to SB, equal amounts of LMW DNA (20 μg for HPV18-E1HA+ cells and 15 μg for HPV18-Nluc+ cells) were treated with 1 μl of HindIII and the indicated amounts of BglI restriction endonucleases, which serve as a noncutter and a single cutter, respectively. Reaction mixtures were incubated at 37°C for 30 min. DNA molecules with sizes corresponding to single, dimeric, trimeric, and tetrameric linear forms are indicated as 1 lin, 2 lin, 3 lin, and 4 lin, respectively. ccc, covalently closed circular DNA; oc, open circular DNA. (B) LMW DNA was isolated from the nonconfluent HPV18-Nluc+ cells and was subjected to neutral-neutral 2D analysis, using 0.4% agarose in Tris-borate-EDTA (TBE) in the first dimension and 1.2% agarose plus ethidium bromide in TBE in the second dimension. Viral DNA was detected using SB. Different forms of viral episomes from monomeric to hexameric genomes were detected. ccc, covalently closed circular DNA; oc, open circular DNA. (C) LMW DNA was isolated from the U2OS cells transiently transfected with the HPV18-Nluc genome (left panel) and from the HPV18-Nluc+ cells (right panel). DNA was digested with BglI, which cuts the viral genomes once at the origin of replication, and subjected to neutral-neutral 2D analysis as in panel B. Both the theta type (arrowhead) and the non-theta type of replication intermediates (arrows) were observed in the transiently transfected cells, while the non-theta type of replication intermediates prevailed in the HPV18-Nluc+ cells. Asterisks depict the almost fully replicated HPV DNA.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Lysis, Incubation, Isolation, Transfection

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a , Equal distribution of 32 NUP96 molecules between the cytoplasmic (maroon) and nucleoplasmic (orange) rings of human NPCs. Adapted from the electron microscopy density map EMD-2444 (refs. , , Springer Nature, and ref. , Cell Press). b – e , MINFLUX imaging of NPCs in permeabilized U2OS cells containing NUP96–mEGFP. The confocal image of eGFP fluorescence identifies the outline of a cell nucleus and a gold bead (100 nm) used for image stabilization (lower left corner, b ). A section of the bottom of the nucleus in b ( c ), and 3D MINFLUX imaging of NPCs ( d , e ) are also shown. Anti-GFP nanobodies (Nb GFP ) modified with the HMSiR blinking dye were used to visualize the NPCs within the region shown in c via the stochastic blinking of the dye. In d , the curvature of the nuclear envelope is apparent from the layers defined by the cytoplasmic and nucleoplasmic rings of the NPCs (see a ). In e , all NPCs identified by confocal imaging in c were detected. Coloration shows the z scale. During data collection, the cytoplasm was on the bottom, but images throughout this article were flipped to place the cytoplasm on top for consistency with convention. f , MINFLUX images of single NPCs (more examples in Extended Data Fig. ). g , h , Composite 2D histogram images of an averaged NPC obtained by aligning individual pores on the basis of their centroids and rotated on the basis of their expected eightfold rotational symmetry (see and Extended Data Fig. ; 37 cells, 541 NPCs, n = 82,331 localizations). The scale is percent of maximum. i , Localization precision determined from centroid deviations within HMSiR ‘trajectories’ (20 points or more per trajectory, 37 cells, 269 clusters; n = 32,184 localizations; σ x = 6.5 ± 0.1 nm (black), σ y = 7.0 ± 0.1 nm (red) and σ z = 4.2 ± 0.1 nm (blue)). The values σ x /σ y = 0.93 and σ x /σ z = 1.55 were assumed throughout this article. j , Jump step histogram analysis of localization precision. The predicted distribution assuming the localization precision values determined in i (blue curve) fits the experimental data (black) poorly, thus indicating that the method in i overestimates the localization precision. A simulation model assuming diffusional drift (red; n = 96,000 jump steps, 25 localizations per trajectory; σ x = 4.1 nm = 0.93σ y = 1.55σ z ; D x , D y and D z = 0.00072, 0.00083 and 0.0003 µm 2 s −1 , respectively) agrees with the data and yields the same centroid deviations as determined in i (see Extended Data Fig. ). See and Extended Data Fig. for a description of the analytical approach and a fit with no diffusional drift.

Article Snippet: For MINFLUX imaging, U2OS-CRISPR–NUP96–mEGFP clone #195 (300174, CLS GmbH) cells were grown in Dulbecco’s modified eagle medium (11880028, Thermo Fisher Scientific) supplemented with 1× MEM non-essential amino acids solution (11140050, Thermo Fisher Scientific), 1× GlutaMAX solution (35050061, Thermo Fisher Scientific), 1× ZellShield (13-0050, Minerva Biolabs) and 10% (v/v) fetal bovine serum (F7524, Sigma) in 5% (v/v) CO 2 enriched air at 37 °C.

Techniques: Electron Microscopy, Imaging, Fluorescence, Modification

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a , MINFLUX images of single NPCs. Images are 2D histograms of localizations for individual NPCs from permeabilized U2OS cells containing NUP96-mEGFP labeled with Nb GFP -HMSiR. b , Scatter plot of HMSiR localizations. Only high-density circular clusters were analyzed further ( squares ); deformed or incomplete clusters were rejected. c , Double-circle fitting. High quality localization clusters were fit to a double-circle model , reflecting the double-ring structure of the NPC. d , Rotation phase angle. The angles in the xy plane of the individual localizations in c relative to the centroid of the double-circle fit were binned (0–45°; assumes an eightfold periodicity), normalized, and fit to y = 1/9 + (1/20.6)*sin(8( x -ϕ)), as described previously . The 1/9 term reflects the average frequency expected for the 9 bins (5° each), and the sine scaling factor is a reasonable average based on simulations. Note that improving the chi-square of the fit by allowing for an adjustable scaling factor does not change the estimated phase angle due to the orthogonality of frequency and angle. e , 2D histogram of aligned NPC scaffolds. Localization clusters were rotated by a phase angle, as determined in d , and aligned based on their centroids, as determined in c (37 cells, 541 NPCs, N = 82,331 localizations). This is the same image as in Fig. . f , Angular distribution of rotationally corrected localizations. The angle distribution for the individual localizations in e was fit to y = 1/180 + c sin(8( x -ϕ)), where c and ϕ are fit parameters, and the 1/180 term reflects the average frequency expected for the 180 bins (2° each). g , The number of HMSiR localizations obtained per NPC scaffold. As few as ten localizations were used earlier to identify an NPC scaffold , but here more than 10-fold more localizations were obtained, on average.

Article Snippet: For MINFLUX imaging, U2OS-CRISPR–NUP96–mEGFP clone #195 (300174, CLS GmbH) cells were grown in Dulbecco’s modified eagle medium (11880028, Thermo Fisher Scientific) supplemented with 1× MEM non-essential amino acids solution (11140050, Thermo Fisher Scientific), 1× GlutaMAX solution (35050061, Thermo Fisher Scientific), 1× ZellShield (13-0050, Minerva Biolabs) and 10% (v/v) fetal bovine serum (F7524, Sigma) in 5% (v/v) CO 2 enriched air at 37 °C.

Techniques: Labeling

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: A description of the MINFLUX parameters reported in this figure and what they mean are described in the . a , The c enter f requency r atio (CFR) values obtained for the HMSiR channel (EX = 642 nm). The CFR is a measure of localization quality. The primary reason for a high CFR in these measurements is the contribution from a second fluorophore, which leads to inaccurate localization values. Thus, an upper bound cutoff of 0.8 was used for HMSiR localizations during acquisition (see ). b , The e ffective f requency at o ffset (EFO) for the HMSiR channel. Background, i.e., low-level emission from the permeabilized cells, was partially eliminated during acquisition of HMSiR dataset 1 by using an EFO > 25 kHz so as not to spend acquisition time on weak signals. A lower threshold of 15 kHz was used during the iteration sequence (Supplementary Table ), which still captures background (peak at ~20 kHz), but the 25 kHz threshold during pattern repeats (25 photons/ms) eliminated most of these weak signals. Post-acquisition, a trajectory length of ≥ 5 localizations reduced background contributions further. Also post-acquisition, an upper threshold of 60 kHz was used to eliminate localizations contaminated by on-switching of a second fluorophore. Due to the higher EFO values for HMSiR dataset 2 (see Extended Data Fig. , inset ), an EFO range of 50–100 kHz was used for this dataset. c,d , Detector channel ratio (DCR) for 561 nm excitation in the absence ( c ) and presence ( d ) of Imp α-JF549. Some background fluorescence in the JF549 channel was detectable within permeabilized U2OS NUP96-mEGFP cells that had been treated with Nb GFP -HMSiR but without addition of ‘transport mix’ (see ). This background signal ( c ) was identified and filtered out based on its DCR, which was generally higher than that of JF549 fluorescence. DCR is defined as the emission frequency in detector 1 divided by the emission frequencies measured for detector 1 + detector 2. Here, detector 1 = 650–685 nm and detector 2 = 580–630 nm. For cells without the transport mix ( c ), the DCR was mostly > 0.5 (40 min acquisition), whereas in the presence of the transport mix ( d ), most DCR values were <0.5, with the values above 0.5 likely reflecting the background. Thus, the data filtration criterion for tracking Imp α-JF549 was DCR < 0.5. e , CFR for the JF549 channel (EX = 561 nm). The data filtration criterion for JF549 was CFR < 0.8 and implemented post-acquisition. Unlike for HMSiR localizations where the CFR check during imaging was set to a low value to select for high quality localizations at the time of acquisition, for tracking Imp α-JF549 the CFR ratio was set to a large cut-off ( > 2.0) to avoid rejecting tracks that were temporarily interrupted. f , EFO for the JF549 channel (EX = 561 nm). This EFO distribution has peaks at ~37, 42, and 83 kHz. The distribution underlying the first peak represents background noise, and the latter two peaks result from the photon emission streams generated by one or two JF549 dyes. Imp α has four reactive cysteine residues and, while the protein was under-labeled with dye, some dual labeling could not be avoided. Since trafficking behavior was not expected to be influenced by the number of dyes on Imp α, the data filtration criterion was an EFO of 40–150 kHz. Of the 225 tracks used in the analysis, 19 had an EFO > 80 kHz (two JF549 dyes). Note that the clear third peak observed here is not present in b , indicating that simultaneous detection of more than one HMSiR dye was infrequent.

Article Snippet: For MINFLUX imaging, U2OS-CRISPR–NUP96–mEGFP clone #195 (300174, CLS GmbH) cells were grown in Dulbecco’s modified eagle medium (11880028, Thermo Fisher Scientific) supplemented with 1× MEM non-essential amino acids solution (11140050, Thermo Fisher Scientific), 1× GlutaMAX solution (35050061, Thermo Fisher Scientific), 1× ZellShield (13-0050, Minerva Biolabs) and 10% (v/v) fetal bovine serum (F7524, Sigma) in 5% (v/v) CO 2 enriched air at 37 °C.

Techniques: Sequencing, Fluorescence, Filtration, Imaging, Generated, Labeling

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a,b , Peripheral translocation of Imp α in MINFLUX experiments. All localizations from 225 MINFLUX tracks with | z | ≤ 25 nm ( N = 2,015) are shown in xy and xz representations. Points from trajectories are identified as import ( purple ), export ( black ), and abortive and undecided ( green ). c,d , Scatterplots of Imp β1-mEosEM localizations at the bottom of a permeabilized U2OS cell. Under constant low-level 408 nm illumination, mEosEM was stochastically photoconverted to the ‘red’ form (Supplementary Video ) and then localized by 3D astigmatism imaging (EX = 561 nm). The nuclear envelope-bound Imp β1-mEosEM was localized to distinct clusters, indicating that Imp β1 was mostly distributed within or near an NPC scaffold. e,f , Volume corrected radial density maps of Imp β1-mEosEM ( e ) and Imp β1/Imp α/NLS-BFP-mEosEM complexes ( f ) showing peripheral binding within the pore. These are the full scale representations of the images shown in Fig. . g , Volume corrected radial density map of Imp β1/Imp α/NLS-2xBFP (dyes on Imp α) and Imp β2/M9-βGal (dyes on βGal) complexes undergoing nuclear import. Data are from Chowdhury et al. ( N = 4,665 localizations, 20 cells, 257 NPCs). h , Localization densities for bound Imp β1 and transport complexes (Imp β1/Imp α/NLS-BFP-mEosEM), and actively transiting Imp α and βGal. Average localization densities were calculated for |z| ≤ 15 nm. Reproduction of Fig. with the data from g added. i-l , RanGTP wash of Imp β1-mEosEM decorated NPCs. i , Confocal image of a permeabilized U2OS NUP96-mEGFP cell nucleus after incubation with 0.5 µM Imp β1-mEosEM (EX = 478 nm). j , Confocal image of the cell nucleus in i after ‘Ran mix’ wash (see ). k , Quantification of the mEosEM fluorescence loss between i and j , corrected for ~1.6% photobleaching ( N = 10 cells, 223 NPCs). l , Volume corrected radial density map of Imp β1-mEosEM after ‘Ran mix’ wash ( N = 1,875 localizations, 24 cells, 343 NPCs). m-p , ‘Transport mix – high α’ wash of Imp β1/Imp α/NLS-BFP-mEosEM decorated NPCs. m , Confocal image of a permeabilized U2OS NUP96-mEGFP cell nucleus after incubation with Imp β1/Imp α/NLS-BFP-mEosEM complexes (EX = 478 nm). n , Confocal image of the cell nucleus in m after ‘transport mix – high α’ wash (see ). Note that this ‘transport mix– high α’ wash is the same composition of proteins used in the simultaneous import/export MINFLUX experiments (‘transport mix’), except with a higher Imp α concentration (unlabeled). o , Quantification of the mEosEM fluorescence loss between m and n , corrected for ~1.6% photobleaching ( N = 10 cells, 223 NPCs). p , Volume corrected radial density map of NLS-BFP-mEosEM after ‘transport mix – high α’ wash ( N = 2,731 localizations, 18 cells, 637 NPCs). For b, e, f, g, l , and p , the two lines around ±20 nm joined by a central curve represent the approximate position of the nuclear envelope.

Article Snippet: For MINFLUX imaging, U2OS-CRISPR–NUP96–mEGFP clone #195 (300174, CLS GmbH) cells were grown in Dulbecco’s modified eagle medium (11880028, Thermo Fisher Scientific) supplemented with 1× MEM non-essential amino acids solution (11140050, Thermo Fisher Scientific), 1× GlutaMAX solution (35050061, Thermo Fisher Scientific), 1× ZellShield (13-0050, Minerva Biolabs) and 10% (v/v) fetal bovine serum (F7524, Sigma) in 5% (v/v) CO 2 enriched air at 37 °C.

Techniques: Translocation Assay, Imaging, Binding Assay, Incubation, Fluorescence, Concentration Assay

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a , Volume-corrected radial density map for all MINFLUX localizations of Imp α–JF549 (2,678 tracks, n = 48,603 localizations). b , Confocal image of a permeabilized U2OS cell nuclear envelope decorated with Imp β1–mEosEM (excitation = 478 nm). The punctate pattern indicates localization to NPCs. Typical result from n = 41 cells. c , Volume-corrected radial density map of Imp β1–mEosEM showing peripheral binding within the pore. Reference NPC scaffolds were identified as described earlier , and mEosEM was localized by astigmatism imaging (excitation = 561 nm) after photoactivation (excitation = 408 nm; n = 5,500 localizations, 48 cells, 412 NPCs; see Extended Data Fig. and Supplementary Video ). d , Volume-corrected radial density map for translocation-arrested import complexes (Imp β1/Imp α/NLS–BFP–mEosEM). Binding within the pore was largely at the periphery, yielding a largely vacant centre ( n = 4,361 localizations, 39 cells, 391 NPCs), similar to Imp β1 alone ( c ). The white curved lines in a , c , d approximate the locations of the nuclear envelope. The image in a is scaled from 0 (black) to maximum density (yellow); c , d are scaled to 70% and 60% of maximum, respectively, due to the hotspots at approximately 100 nm and approximately 170 nm above the pore centres (see Extended Data Fig. for full scale). Average localization densities from a , c , d were calculated for | z | ≤ 15 nm. e , Localization densities for bound Imp β1, translocation-arrested import complexes and actively transiting Imp α. f , Annular zone model. Three distinct binding behaviours for Imp β1 were observed: (I) an empty centre (no binding); (II) a transport active zone (low-affinity binding of import complexes); and (III) a high-affinity binding zone for Impβ1 and import complexes. INM, inner nuclear membrane; ONM, outer nuclear membrane. The NPC structure was adapted from ref. , Cell Press.

Article Snippet: For MINFLUX imaging, U2OS-CRISPR–NUP96–mEGFP clone #195 (300174, CLS GmbH) cells were grown in Dulbecco’s modified eagle medium (11880028, Thermo Fisher Scientific) supplemented with 1× MEM non-essential amino acids solution (11140050, Thermo Fisher Scientific), 1× GlutaMAX solution (35050061, Thermo Fisher Scientific), 1× ZellShield (13-0050, Minerva Biolabs) and 10% (v/v) fetal bovine serum (F7524, Sigma) in 5% (v/v) CO 2 enriched air at 37 °C.

Techniques: Binding Assay, Imaging, Translocation Assay, Membrane

Journal: bioRxiv

Article Title: Deep learning enables fast and dense single-molecule localization with high accuracy

doi: 10.1101/2020.10.26.355164

Figure Lengend Snippet: a) DECODE can reduce acquisition times by one order of magnitude. The same sample of microtubules, labeled with anti- α tubulin primary and AF647 secondary antibodies, imaged with different UV activation intensities to result in different emitter densities between 0.08 and 0.86 emitters per frame per μm 2 and acquisition times between 93 and 1120 s, while keeping the total number of localizations the same. For high-density activation, we show a comparison with CSpline. b) Fourier Ring Correlation curves for DECODE and CSpline for different emitter densities. c) Resolution estimates obtained using the Fourier Ring Correlation and 0.143 criterion across densities for both methods. d) Fast live-cell SMLM on the nuclear pore complex protein Nup96-mMaple acquired in 3 seconds. e) DECODE enables ultra-high labeling densities. Microtubules labeled with a high concentration of anti- α and anti- β tubulin primary and AF647 secondary antibodies. e1, e2) Magnified regions as indicated in a. Data acquired with high-density labeling shows continuous structures. As a comparison, the same sample was acquired after pre-bleaching of the fluorophores to reach the single-molecule blinking regime. Here, single labels are resolved in the superresolution reconstruction and lead to a sparse decoration of the microtubules. e3, e4) Side view reconstructions of regions as indicated in e1, e2 resolving the hollow, cylinder-like structure of immunolabeled microtubules. f) Representative raw camera frames for the high-density and single-emitter acquisitions, respectively. Scale bars: 10 μm (d inset, f), 1 μm (a, d, e, e1, e2), 100 nm (e3,e4).

Article Snippet: For imaging of live cells, coverslips containing Nup96-mMaple cells (catalog no. 300461, CLS Cell Line Service, Eppelheim, Germany) were rinsed twice with warm PBS before they were mounted onto a custom manufactured sample holder in 1 mL growth medium containing 20 mM HEPES buffer and imaged directly.

Techniques: Labeling, Activation Assay, Concentration Assay, Immunolabeling

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Representative images show DAPI (blue), EdU (green), pATM (magenta), SFPQ (cyan), and merged (right) staining in siNTC-treated DIvA U2OS cells under break and no break conditions. Breaks were induced with 4-hydroxytamoxifen for 24 hours, and images were captured using a 10X magnification. Cells (∼20,000 per well) were imaged across three wells per condition (16 fields per well; 48 images total) and quantified in Cell Profiler for nuclear intensity, foci count, and cell-cycle stage based on EdU/DAPI. Data are representative of n=48 images. (B) Quantification of SFPQ–pATM and pATM–γH2AX co-localization in G2-phase cells. Violin plots show correlation coefficients of SFPQ and pATM (left) and pATM and γH2AX (right) in G2 cells with or without DNA breaks. (C) ChIP-seq data representing SFPQ-bound chromatin at 122 defined AsiSI sites under uncut (noDSB) and cut (+4OHT, 4 hours) conditions (left). ChIP-seq data representing SFPQ bound to RNU sites (right). Immunoprecipitation was performed using SFPQ polyclonal antibody. Normalized ChIP-seq signal was plotted for ±1.5 kb around AsiSI sites. SFPQ occupancy profiles are shown for two independent replicates with DSB induction (dark blue and light blue) and for the noDSB control (yellow).

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Staining, ChIP-sequencing, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) (Top) SFPQ mean intensity: Violin plots (with embedded boxplots) show the single-cell distribution of nuclear SFPQ mean fluorescence intensity in DIvA U2OS cells under no break (untreated) and break (4-hydroxytamoxifen, 4-OHT) conditions. Each dot is one nucleus; boxplots denote median and interquartile range. Cell-cycle phase (G1, S, G2) was assigned per cell using EdU incorporation (green) and DAPI DNA content (blue). (Bottom) SFPQ foci per cell: Violin plots (with embedded boxplots) show the number of SFPQ nuclear foci per cell under the same conditions and cell-cycle stratification. Quantification: Cells were left untreated or treated with 4-OHT to induce AsiSI-mediated DSBs, then stained for SFPQ (cyan), EdU, and DAPI. Images were analyzed in Cell Profiler to segment nuclei, call SFPQ foci, compute per-nucleus mean intensity and foci counts, and assigned cell-cycle stage from EdU/DAPI features. (B) Non–pre-extracted immunofluorescence staining of pATM and SFPQ in DIvA U2OS cells with or without DSB induction. Cells were left untreated or treated with 4-hydroxytamoxifen (4-OHT) to induce AsiSI-mediated DSBs and stained for DNA (DAPI, blue), EdU incorporation (green), phosphorylated ATM (pATM, magenta), and SFPQ (cyan). Images were acquired without cytoskeletal (CSK) pre-extraction to visualize total nuclear staining patterns. Merged images show nuclear co-localization of pATM and SFPQ signals in the presence and absence of DNA damage.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Fluorescence, Staining, Immunofluorescence, Extraction

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) mRNA-seq log₂ fold changes of RAD51 paralogs and pooled transcripts in the indicated Gene Ontology (GO) categories in DIvA U2OS cells treated with siSFPQ compared to siNTC control for 72 hours in the absence of DSBs. Data represent the mean of three biological replicates. Individual p-values were adjusted for multiple comparisons. Aggregate p-values were combined by Fisher’s method. (B) Differential transcript utilization analysis for RAD51 paralogs. mRNA-seq data from siNTC versus siSFPQ DIvA U2OS cells were analyzed for transcript isoform usage. Bars represent the likelihood ratio statistic for each gene, with blue bars indicating genes showing significant shifts in transcript utilization (RAD51B, RAD51C) upon SFPQ depletion. Grey bars indicate genes without significant changes. (C) Western blot analysis of SFPQ and RAD51 protein levels of the three biological replicates used for mRNA-seq following siNTC or siSFPQ treatment. Total protein staining is shown as a loading control. (D) Representative images show DAPI (blue), EdU (green), RAD51 (magenta), SFPQ (cyan), and merged (right) staining in siNTC-treated DIvA U2OS cells under break and no break conditions, pre-extracted with CSK. Breaks were induced with 4-OHT for 4 hours. Cells (∼20,000 per well) were imaged across four wells per condition (16 fields per well; 64 images total) and quantified in Cell Profiler for nuclear intensity, foci count, and cell-cycle stage based on EdU/DAPI. (E) Quantification of SFPQ and RAD51 foci per cell in DIvA U2OS cells following siRNA treatment and DNA damage induction. Violin plots show the distribution of foci counts across conditions with or without 4-hydroxytamoxifen (4-OHT) treatment and following transfection with non-targeting control (NTC), RAD51-targeting, or SFPQ-targeting siRNAs. Data are representative of n=64 images. (F) Violin plots showing correlation coefficients of SFPQ and RAD51 in G2 cells with or without DNA breaks. Quantification of SFPQ-RAD51 foci co-localization in G2-phase cells was performed using Cell Profiler analysis of single-cell fluorescence signals.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Control, Western Blot, Staining, Transfection, Fluorescence

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Differential expression analysis of mRNA-seq data comparing DSB versus no-DSB conditions in siNTC-treated DIvA U2OS cells (n=3 biological replicates). Mean log₂ fold change for the same targets is shown as . No significant expression differences were detected for these targets upon DSB induction in control cells. (B) ChIP-seq data showing SFPQ abundance at sites upstream and downstream of RAD51-paralog genes both without (noDSB) or with (+DSB) 4 hours of DSB induction. Data displayed is the average signal across all 6 RAD51 paralogs. (C) mRNA-seq log₂ fold changes of transcript expression of the indicated gene or GO category in DIvA U2OS cells treated with siSFPQ compared to siNTC control for 72 hours in the absence of DSBs. Data represent the mean of three biological replicates. Individual p-values were adjusted for multiple comparisons. Aggregate p-values were combined by Fisher’s method. (D) Western blot of DIvA U2OS cells treated with siSFPQ with or without p53 inhibition by PFT-α (30 µM) for 24 hours. Lysates were blotted for SFPQ, HSP70, MDM2 and RAD51. (E) (Left) Western blot of p53-null K562 cells treated with siSFPQ. Total protein staining is shown as a loading control. (Right) Quantification of SFPQ and RAD51 normalized band intensities relative to total protein is graphed.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Quantitative Proteomics, Expressing, Control, ChIP-sequencing, Western Blot, Inhibition, Staining

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Cycloheximide (CHX) ± carfilzomib (Carf) protein stability assay in DIvA U2OS cells. Cells were transfected with either non-targeting control (siNTC) or SFPQ-targeting (siSFPQ) siRNAs for 72 h, then treated with CHX alone or CHX + Carf to inhibit protein synthesis and proteasomal degradation, respectively. Lysates were collected at 0-, 2-, and 4-hours post-drug treatment from three independent biological replicates. (B) RAD51 abundance from normalized to total protein and then to 0 hr. condition. Data points represent individual replicates; lines indicate the mean. (C) RIP-seq analysis of SFPQ binding across RAD51 family paralogs in melanoma cells. Read coverage tracks show SFPQ-associated RNA fragments aligned to the genomic loci of RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. Peaks indicate regions of enriched SFPQ binding, with annotations of exon–intron structure shown below each track. Model for SFPQ-mediated stabilization of RAD51 mRNA and its impact on homologous recombination (HR). In the presence of SFPQ, the protein binds to RAD51 mRNA, promoting transcript stabilization. Stable RAD51 mRNA ensures sufficient RAD51 protein production, enabling efficient RAD51 filament formation on DNA and supporting robust HR (left). Upon SFPQ loss, RAD51 family mRNAs are destabilized, leading to reduced RAD51 protein abundance. This reduction impairs HR efficiency (right).

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Stability Assay, Transfection, Control, Binding Assay, Homologous Recombination, Quantitative Proteomics

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Representative time-lapse images of primary MEFs derived from wildtype (top) and Gravin knockout (bottom) cells. Cells transiently expressing GFP-H2B were monitored through mitosis. ( B ) Quantification of time-lapse experiments depicts duration of mitosis from nuclear envelope breakdown to the onset of anaphase; Wildtype, n = 199, Gravin KO, n = 121, ****p<0.0001. ( C, D ) Structured illumination microscopy (SIM) of representative mitotic HEK293 cells stably expressing Control or Gravin shRNA. Composite images (left) depict cells stained for α-tubulin (green) and DNA (blue). Immunofluorescence of pT210-Plk1 ( C ) and pT288-AurA ( D ) as an index of kinase activity (mid) and 5X magnification of centrosomal pT210-Plk1 ( C ) and pT288-AurA ( D ) signals (insets). ( E, F ) Quantification of centrosomal pT210-Plk1 ( E ) and pT288-AurA ( F ) immunofluorescence. Points represent individual cells ( n ). Data are normalized to Con shRNA; ( E ) Con shRNA, n = 45, Gra shRNA, n = 46, **p=0.0036; ( F ) Con shRNA, n = 62, Gra shRNA, n = 66, ****p<0.0001. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. ( G ) SIM micrographs of Gravin (top, gray and magenta) in interphase and pT766-Gravin (bottom, gray and magenta) in mitotic U2OS cells. Composite images (right) also depict α-tubulin (green) and DNA (blue). ( H ) Schematic of global drug distribution (gray) vs drug targeting to centrosomes (green). Gravin scaffolds centrosome-localized pools of Plk1 and AurA.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Derivative Assay, Knock-Out, Expressing, Microscopy, Stable Transfection, Control, shRNA, Staining, Immunofluorescence, Activity Assay, Two Tailed Test

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Immunoblot confirming Gravin expression (top) in wildtype (WT) but not Gravin knockout (KO) primary MEFs. GAPDH loading controls (bottom). ( B ) Matched controls pertaining to . SIM micrographs of Gravin (top, gray and magenta) in mitotic and pT766-Gravin (bottom, gray and magenta) in interphase U2OS cells. Composite images (right) also depict α-tubulin (green) and DNA (blue).

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Western Blot, Expressing, Knock-Out

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Schematic of a centrosome-directed LoKI platform. SNAP-PACT fusion proteins conjugate CLP-linked Plk1 inhibitors at centrosomes. Inset depicts BI2536 in the ATP-binding pocket of Plk1. ( B ) Chemical structure of CLP-BI2536. ( C ) Dose-response curve of in vitro Plk1 inhibition with CLP-BI2536. ( D ) Structured illumination microscopy (SIM) of a LoKI-on U2OS cell labeled with CLP-fluorescein. Immunofluorescent detection of α-tubulin (green), DNA (blue), mCherry-SNAP-PACT (magenta) and CLP-fluorescein (yellow). Magnification of SNAP and CLP-fluorescein co-distribution at a centrosome (inset). ( E ) SIM micrographs of LoKI-off (left) and LoKI-on (right) U2OS cells. SNAP expression (top, magenta), CLP-fluorescein conjugation (mid, yellow) and composite images (bottom) are depicted. ( F ) Pulse-chase experiments measuring CLP-BI2536’s ability to block CLP-rhodamine conjugation to LoKI-on. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). ( G, H ) Immunofluorescence of representative mitotic LoKI-off ( G ) and LoKI-on ( H ) U2OS cells treated with DMSO or 250 nM CLP-BI2536 for 4 hr. Composite images (left) show α-tubulin (green), DNA (blue), and SNAP (magenta). Immunofluorescent detection of pT210-Plk1 (mid, gray) as an index of kinase activity. 5X magnification of centrosomal pT210-Plk1 signals and surface plots measuring integrated intensity of pT210-Plk1 signal (insets). ( I, J ) Quantification of centrosomal pT210-Plk1 immunofluorescence for LoKI-expressing cells. Points represent individual cells ( n ). Data normalized to DMSO. Application of DMSO or CLP-BI2536 for 4 hr, ( I ) 100 nM, LoKI-off, n = 46, LoKI-on, n = 59, **p=0.0059; 250 nM, LoKI-off, n = 46, LoKI-on, n = 46, ****p<0.0001 and drug treatment followed by 1 hr washout ( J ) 250 nM, LoKI-off, n = 24, LoKI-on, n = 42, ****p<0.0001. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. Source files for analysis of pulse-chase experiments are available in and for quantification of pT210-Plk1 are available in . Figure 2—source data 1. Analysis for pulse-chase experiments with CLP-BI2536 in SNAP-PACT cells. Figure 2—source data 2. Raw analysis for pT210-Plk1 signal.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Binding Assay, In Vitro, Inhibition, Microscopy, Labeling, Expressing, Conjugation Assay, Pulse Chase, Blocking Assay, Fluorescence, Western Blot, Immunofluorescence, Activity Assay, Two Tailed Test

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Full chemical structure of CLP-BI2536. ( B ) Dose-response curve depicting in vitro Plk1 inhibition with increasing concentrations of CLP-BI2536 conjugated to purified SNAP. ( C ) Schematic of LoKI viral construct with mCherry-SNAP-PACT under control of a doxycycline-inducible promoter. ( D ) Immunoblot confirming SNAP-PACT (top) expression after induction with doxycycline for 72 hr and GAPDH loading controls (bottom). ( E ) Immunoblot of SNAP-PACT (top) expression at selected time points after removal of doxycycline and GAPDH loading controls (bottom). Quantification of amalgamated data is presented below. ( F ) Immunofluorescent detection of interphase (top) and mitotic (bottom) U2OS cells showing α-tubulin (left and green), DNA (mid and blue), and SNAP (right and magenta). ( G, H ) Diagram of centrosomal LoKI-on ( G ) platform with drugs conjugated and LoKI-off ( H ) platform containing a mutation that occludes CLP binding. Experiments were conducted at least two times (N = 2–3). Data are mean ± s.e.m.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: In Vitro, Inhibition, Purification, Construct, Control, Western Blot, Expressing, Mutagenesis, Binding Assay

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A, B ) Pulse-chase experiments carried out in U2OS cells after 1 hr ( A ) or 2 hr ( B ) treatment with CLP-BI2536. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). Experiments were conducted at least three times (N = 3). Data are mean ± s.e.m. Source files for analysis of pulse-chase experiments are available in . Figure 2—figure supplement 2—source data 1. Analysis for pulse-chase time course experiments with CLP-BI2536 in SNAP-PACT cells.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Pulse Chase, Fluorescence, Western Blot

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Immunofluorescence detection of pT210-Plk1 as an index of kinase activity in parental U2OS cells treated with DMSO or unconjugated BI2536 for 4 hr. ( B ) Quantification of centrosomal pT210-Plk1 immunofluorescence collected from parental U2OS cells. ( C ) Quantification of total Plk1 immunofluorescence at centrosomes in LoKI-expressing cells after 4 hr CLP-BI2536 treatment; 250 nM, LoKI-off, n = 55, LoKI-on, n = 47, *p=0.0217; 500 nM, LoKI-off, n = 52, LoKI-on, n = 51, *p=0.0295. ( D ) Quantification of pT210-Plk1 immunofluorescence in control cells lacking SNAP expression (not induced with doxycycline) after 4 hr CLP-BI2536 treatment. ( E ) Immunoblot detection of pT210-Plk1 (blot 2) and pT288-AurA (blot 4) in LoKI-off and LoKI-on expressing cells collected via mitotic shake-off. Cells were treated for 16 hr with nocodazole and 4 hr with nocodazole plus DMSO, 250 nM CLP-BI2536, or 100 nM CLP-MLN8237. Total Plk1 (blot 3), AurA (blot 5), SNAP-PACT (blot 1), and GAPDH (blot 6) are also depicted. ( F ) Immunoblot confirming SNAP-PACT (top) expression after induction with doxycycline for 72 hr in RPE and HeLa cells and GAPDH loading controls (bottom). ( G–J ) Immunofluorescence detection of pT210-Plk1 at centrosomes in LoKI-off (top) and LoKI-on (bottom) RPE ( G ) and HeLa ( I ) cells treated with 250 nM CLP-BI2536 for 4 hr. Quantification of pT210-Plk1 immunofluorescence at centrosomes in RPE ( H ) 250 nM, LoKI-off, n = 70, LoKI-on, n = 47, ****p<0.0001 and HeLa ( J ) 250 nM, LoKI-off, n = 42, LoKI-on, n = 46, ****p<0.0001 LoKI-expressing cells after 4 hr CLP-BI2536 treatment. Points represent individual cells ( n ). Data normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Immunofluorescence, Activity Assay, Expressing, Control, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: Quantification of non-normalized pT210-Plk1 immunofluorescence signal at centrosomes in U2OS ( A ) RPE ( B ) and HeLa ( C ) LoKI-expressing cells after treatment with indicated concentrations of CLP-BI2536 for 4 hr. Points represent individual cells ( n ). Experiments were conducted at least three times (N = 3). Data are mean ± s.e.m.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Immunofluorescence, Expressing

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Schematic depicting bipolar (top), abnormal bipolar (mid), and monopolar (bottom) mitotic spindle classifications. ( B ) Representative composite (left) images show α-tubulin (green), DNA (blue), and SNAP (magenta) or α-tubulin (right, gray) staining alone for each spindle type in U2OS cells. ( C–E ) Spindle profile measurements of U2OS ( C ) RPE ( D ) and HeLa ( E ) cells treated with 250 nM CLP-BI2536 for 4 hr. Spindle profiling indicates the % of each spindle type in drug-treated LoKI-on and LoKI-off cells. Number of cells analyzed per condition are indicated; ( C ) n = 3, *p=0.0214; ( D ) n = 3, *p=0.0269. ( F ) Schematic depicting that Plk1 phosphorylation of pericentriolar substrates facilitates accumulation of γ-tubulin at centrosomes and microtubule nucleation. ( G, H ) Representative composite (left) images show γ-tubulin (yellow) and DNA (blue) in U2OS cells expressing LoKI-off ( G ) and LoKI-on ( H ) treated with 250 nM CLP-BI2536 for 4 hr, followed by a 1 hr washout. 5X magnified grayscale images of centrosomal γ-tubulin (right). ( I ) Quantification of centrosomal γ-tubulin immunofluorescence in LoKI-expressing cells. Points represent individual cells ( n ). Data normalized to DMSO. A ROUT (Q = 1%) outlier test was performed and two values were removed prior to performing statistical tests; LoKI-off, n = 66, LoKI-on, n = 74, ****p<0.0001. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. Source files for spindle profile analyses are available in . Figure 3—source data 1. Spindle profile analyses.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Staining, Phospho-proteomics, Expressing, Immunofluorescence, Two Tailed Test

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A–C ) Mitotic spindle classifications for LoKI-expressing U2OS ( A ) RPE ( B ) and HeLa ( C ) cells treated with DMSO for 4 hr. Profiling indicates the % of each defined spindle type in drug treated LoKI-on and LoKI-off cells. Number of cells analyzed per condition are indicated. ( D ) Quantification of γ-tubulin immunofluorescence at centrosomes in LoKI-expressing U2OS cells treated with DMSO, followed by a 1 hr washout. Points represent individual cells ( n ). Data normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. Source files for control spindle profile analyses are available in .

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Expressing, Immunofluorescence, Two Tailed Test, Control

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Chemical structure of AurA kinase inhibitor CLP-MLN8237. ( B ) Dose-response curve of in vitro AurA kinase inhibition with CLP-MLN8237. ( C ) Pulse-chase experiments measuring CLP-MLN8237’s ability to block CLP-rhodamine conjugation to LoKI-on. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). ( D ) Immunofluorescence of representative mitotic LoKI-on U2OS cells treated with DMSO or 100 nM CLP-MLN8237 for 4 hr. Composite images (left) show α-tubulin (green), DNA (blue), and SNAP (magenta). Immunofluorescent detection of pT288-AurA (right, gray) as an index of kinase activity is depicted. ( E ) Quantification of centrosomal pT288-AurA immunofluorescence after 4 hr CLP-MLN8237 treatment; 50 nM, LoKI-off, n = 18, LoKI-on, n = 53, **p=0.0014; 100 nM, LoKI-off, n = 18, LoKI-on, n = 48, **p=0.0026. ( F ) Schematic of dual LoKI-on moiety conjugated to CLP-BI2536 and CLP-MLN8237. Inset depicts MLN8237 in the ATP-binding pocket of AurA. ( G ) Representative time-lapse images of mitotic LoKI-off (top) and LoKI-on (bottom) cells transiently expressing GFP-H2B. Cells were treated with a combination (both drugs) of 250 nM CLP-BI2536 and 100 nM CLP-MLN8237 and monitored for 18 hr. ( H ) Quantification of time-lapse experiments depicts duration of mitosis from nuclear envelope breakdown to the onset of anaphase; DMSO, LoKI-off, n = 264, both drugs, LoKI-off n = 353, ****p<0.0001; DMSO, LoKI-on, n = 292, both drugs, LoKI-on n = 331, ****p<0.0001; both drugs, LoKI-off, n = 353, both drugs, LoKI-on, n = 331, ****p<0.0001. Points in ( E ) and ( H ) represent individual cells ( n ). Points in ( E ) are normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. Source files for analysis of pulse-chase experiments are available in . Figure 4—source data 1. Analysis for pulse-chase experiments with CLP-MLN8237 in SNAP-PACT cells.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: In Vitro, Inhibition, Pulse Chase, Blocking Assay, Conjugation Assay, Fluorescence, Western Blot, Immunofluorescence, Activity Assay, Binding Assay, Expressing, Two Tailed Test

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Immunofluorescence detection of pT288-AurA as an index of kinase activity in parental U2OS cells treated with DMSO or unconjugated MLN8237 for 4 hr. ( B ) Quantification of centrosomal pT210-AurA immunofluorescence collected from parental U2OS cells. Data normalized to DMSO. Points represent individual cells ( n ). Experiments were conducted at least three times (N = 3). Data are mean ± s.e.m. ( C ) Immunofluorescence of representative mitotic LoKI-off U2OS cells treated with DMSO or 100 nM CLP-MLN8237 for 4 hr. Composite images (left) show α-tubulin (green), DNA (blue), and SNAP (magenta). Immunofluorescent detection of pT288-AurA (right, gray) as an index of kinase activity is depicted. ( D ) Representative time-lapse images of mitotic LoKI-off (top) and LoKI-on (bottom) cells transiently expressing GFP-H2B. Cells were treated with DMSO and monitored for 18 hr.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Immunofluorescence, Activity Assay, Expressing

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Schematic of microtubule association with centromeric chromatin at kinetochores. Diagram of LoKI-Mis12 securing CLP-linked inhibitors to kinetochores (inset). ( B ) Immunoblot confirming SNAP-Mis12 (top) expression after induction with doxycycline for 72 hr and GAPDH loading controls (bottom). ( C ) Representative SIM micrograph depicting α-tubulin (green), mCherry-SNAP-Mis12 (magenta) and centromeric DNA marker ACA (cyan) in U2OS cells. Composite image depicts co-distribution of LoKI-Mis12 with ACA. ( D ) SIM micrographs of LoKI-off (left) and LoKI-on (right) U2OS cells. SNAP expression (top, magenta), CLP-647 conjugation (mid, yellow) and composite images (bottom) are depicted. Line on CLP-dye was used to generate panel E plot. ( E ) Line plot of CLP-dye signal from a representative kinetochore in panel D. ( F ) Pulse-chase experiments measuring CLP-MLN8237’s ability to block CLP-rhodamine conjugation to LoKI-on. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). ( G, H ) Immunofluorescence of representative mitotic LoKI-off ( G ) and LoKI-on ( H ) U2OS cells treated with DMSO or 100 nM CLP-MLN8237 for 4 hr. Composite images (left) show α-tubulin (green) and DNA (blue). Immunofluorescence detection of pS69-Hec1 (mid and top right insets, gray). Heat maps (bottom right) depict maximum intensity measurements of pS69-Hec1 signal from representative insets. ( I ) Quantification of pS69-Hec1 immunofluorescence at kinetochores after 4 hr CLP-MLN8237 treatment; 100 nM, LoKI-off, n = 40, LoKI-on, n = 39, ****p<0.0001; 175 nM, LoKI-off, n = 29, LoKI-on, n = 29, *p=0.0212. Points represent individual cells ( n ). Data normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. ( J ) Implementation of LoKI at other subcellular locations shows SNAP targeting to plasma membrane via AKAP79 and mitochondrial localization via d-AKAP1. Source files for analysis of pulse-chase experiments are available in . Figure 6—source data 1. Analysis for pulse-chase experiments with CLP-MLN8237 in SNAP-Mis12 cells.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Western Blot, Expressing, Marker, Conjugation Assay, Pulse Chase, Blocking Assay, Fluorescence, Immunofluorescence, Two Tailed Test, Clinical Proteomics, Membrane

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet: ( A ) Quantification of pS69-Hec1 immunofluorescence at centrosomes after 4 hr CLP-MLN8237 treatment. ( B ) Immunoblot detection of pS69-Hec1 (blot 2) and pT288-AurA (blot 3) in Mis12-LoKI-off and Mis12-LoKI-on expressing cells collected via mitotic shake-off. Cells were treated for 16 hr with nocodazole and 4 hr with nocodazole plus DMSO or 100 nM CLP-MLN8237. Total AurA (blot 3), SNAP-PACT (blot 1), and GAPDH (blot 5) are also depicted. ( C ) Quantification of pS69-Hec1 immunofluorescence at kinetochores after 4 hr CLP-MLN8237 treatment in SNAP-PACT expressing U2OS cells. Points in ( A ) and ( C ) represent individual cells ( n ). Data normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant.

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Immunofluorescence, Western Blot, Expressing, Two Tailed Test

Journal: eLife

Article Title: Subcellular drug targeting illuminates local kinase action

doi: 10.7554/eLife.52220

Figure Lengend Snippet:

Article Snippet: Cell line ( H. sapein ) , U2OS , ATCC , HTB-96 , Maintained in the Scott Lab in DMEM supplemented with 10% FBS.

Techniques: Control, shRNA, Stable Transfection, Selection, Knock-Out, Transfection, Bicinchoninic Acid Protein Assay, DNA Purification, Recombinant, Plasmid Preparation, Modification, Software, Cloning

Journal:

Article Title: General Transcriptional Coactivator PC4 Activates p53 Function

doi: 10.1128/MCB.24.5.2052-2062.2004

Figure Lengend Snippet: PC4 directly interacts with p53 in vitro and in vivo. (A) Induction of p53 expression in the U2OS cell line. The levels of p53 and PC4 present in adriamycin (2 μg/ml)-treated U2OS cells were assessed by Western blotting using anti-p53 (upper panel) and anti-PC4 (lower panel) antibodies. (B and C) In vivo interaction of PC4 with p53. (B) Lane 1, an adriamycin (2 μg/ml)-induced U2OS cell extract was immunoblotted with polyclonal PC4 antibody N17. Lane 2, immunoprecipitation of endogenous PC4 from an induced U2OS cell lysate was performed using anti-p53 monoclonal antibody DO1 followed by immunoblotting with anti-PC4 polyclonal antibody. Lane 3, immunoprecipitation using mouse preimmune serum used as a control. (C) Lane 1, immunoprecipitation of endogenous p53 from an induced U2OS cell extract, using anti-PC4 polyclonal antibody N17 followed by immunoblotting with anti-p53 monoclonal antibody DO1. Lane 2, adriamycin-induced U2OS cell extract immunoblotted with monoclonal p53 antibody DO1. Lane 3, immunoprecipitation reaction with goat preimmune serum used as a control. (D, E, and F) Interaction of PC4 with p53 in an in vitro GST pulldown assay. (D) Schematic representation of GST and GST-p53 fusion proteins. ++, strong interaction of PC4 with respective GST-p53 fusion protein; +, weaker interaction; −, no interaction. (E) SDS-PAGE (10%) and Coomassie blue R250 staining of immobilized GST-p53 fusion proteins. Lane 1, GST-p53 (full length); lane 2, GST-p53(1-73), lane 3, GST-p53(120-290); lane 4, GST-p53(284-330), lane 5, GST-p53(328-368); lane 6, GST-p53(364-393). GST fusion proteins predominantly contain intact proteins (indicated with asterisks) with minimum low-molecular-weight breakdown products. (F) One microgram of GST (lane 2) or GST-p53 fusion proteins (lanes 3 to 8) was incubated with bacterial extract containing 200 ng of PC4 and analyzed by immunoblotting with anti-PC4 N17 antibody. Lane 1, 5% input of bacterial cell lysate. IP, immunoprecipitation; IB, immunoblot; WCE, whole-cell extract; AD, activation domain; DBD, DNA binding domain; OD, oligomerization domain.

Article Snippet: Immunoprecipitation was performed by incubating the drug-treated U2OS cell lysate with protein A-agarose beads conjugated to either anti-p53 mouse monoclonal antibody DO1 (Oncogene) or anti-PC4 goat polyclonal antibody N17 (Santa Cruz).

Techniques: In Vitro, In Vivo, Expressing, Western Blot, Immunoprecipitation, GST Pulldown Assay, SDS Page, Staining, Molecular Weight, Incubation, Activation Assay, Binding Assay