Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Schematic of CRISPR/Cas9-mediated PLXNB2 knockout (KO) with small guide (sg) RNA targeting second coding exon. (B) Western blots show Plexin-B2 expression in different SD2 GSCs, with β-actin as loading control. Note Plexin-B2 precursor at 240 kDa and mature form at 170 kDa. (C) IF images show Plexin-B2 expression in different SD2 GSCs, with Hoechst nuclear counterstain. (D) Left, schematic of atomic force microscopy (AFM) indentation method to probe cell stiffness by cantilever deflection. Middle, AFM indentation curves of different SD2 GSCs; right, box plots of cell stiffness, showing 25– 75% quartiles, median (line), and mean (plus sign). n= 6 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (E) Left, depiction of membrane tension measurement with optical tweezers. Middle, force measurements during tether extrusion (shaded box). Right, quantifications of tether extrusion forces. n=5 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (F) Left, schematic of FLIM of cell membranes labeled with Flipper-TR membrane dye, with low and high membrane tension associated with shorter and longer lifetimes, respectively. Middle top, representative FLIM images, with lifetime heatmap shown on right. Middle bottom, images show similar fluorescence intensities of Flipper-TR dye in WT and PB2 KO cells. Right top, violin plots show fluorescence lifetime from 3 images per group. Two-sided unpaired t-test. Right bottom, phasor plots of FLIM image data, with arrow indicating a shift to shorter lifetime values for PB2 KO cells. (G) Model of Plexin-B2 regulation of cortical contractility and membrane tension. Phalloidin staining show differences of F-actin network in WT and PB2 KO SD2 GSCs. DAPI for nuclear staining. Arrows point to stress fibers and spread-out contours of the WT GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Microscopy, Membrane, Labeling, Fluorescence, Staining

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Top, timeline for dextran uptake assay. Bottom, live-cell imaging of WT and PB2 KO SD2 GSCs labeled with SPY-Actin and exposed to dextran-Alexa488. Enlarged images of boxed areas are shown below. Quantification of the areas of dextran + clusters per cell are shown in box plots, with 25–75% quartiles, median (line), and mean (plus sign). n=85 cells for WT, n=44 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (B) Top, live cell confocal plane images of WT and PB2 KO GSCs with side views of z-stacks showing intracellular localization of diffuse dextran-Alexa 488 signals in PB2 KO cells in addition to dextran endosome signals. In contrast, WT cells contained only dextran + endosomes. Bottom, histograms show fluorescence profiles showing bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n=177 cells for WT, n=161 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (C, D) Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell fluorescence imaging at 72 hr after transfection shows internalization of myr-palm-GFP or -CPF on endomembranes (arrow) in WT GSCs, in contrast to membrane retention of the probes (arrowhead) in PB2 KO GSCs. (E) Left, schematic of TauSTED super-resolution microscopy of GSCs labeled with MemGlow. Middle, TauSTED live-cell images show reduced endosomes (arrowheads) in PB2 KO cells compared to WT. Right, box plots show areas of MemGlow clusters in each cell. n=26 cells for WT, n=13 cells for PB2 KO. Two-sided unpaired t-test. (F) Working model of regulation of cortical and membrane tension by Plexin-B2, affecting endocytosis and membrane permeability in GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Labeling, MANN-WHITNEY, Fluorescence, Membrane, Imaging, Transfection, Super-Resolution Microscopy, Permeability

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Left, schematic of PH(PLCδ1)-GFP PIP2 probe. Right, live-cell imaging at 72 hr post transfection reveals that the PH(PLCδ1)-GFP probes were largely internalized in WT GSCs (arrow), but retained on membrane of PB2 KO GSCs (arrowhead). (B) Left, still images of videography show accumulation of the PH(PLCδ1)-GFP probes (arrow) in front of the nucleus (NucSpot) of migrating WT SD2 GSCs in tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Dashed lines delineate cell boundary. Long arrow denotes direction of migration. Right, quantifications of the ratio of PH(PLCδ1)-GFP fluorescence intensity at front vs. rear of GSCs during passage. n=13-16 cells per condition. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (C) Left, schematic of R(+8)-pre-GFP probe for negative surface charge of inner plasma membrane. Right, live-cell imaging at 72 hr post-transfection shows internalization of the probes (arrow) in WT GSCs, in contrast to the predominant membrane localization in PB2 KO GSCs (arrowhead). (D) Left, still images of videography show accumulation of the R(+8)-pre-GFP probes (arrow) at front zone of WT GSCs when traversing the 3 µm tunnel, but not in PB2 KO cells. Right, bar graphs show the ratio of R-pre-GFP fluorescence intensity at rear vs. front of GSCs when passing through tunnels. n=22 cells for WT, n=27 cells for PB2 KO. Mann–Whitney–Wilcoxon test. Data represent mean ± SEM. (E) Diagram illustrating voltage sensitive FluoVolt membrane dye, with fluorescent intensity quenched by voltage-sensitive electron transfer from electron-rich donor mediated by “molecular wire” in plasma membrane. (F) Left, FluoVolt live-cell imaging shows reduced FluoVolt fluorescent intensity in cell membrane of Plexin-B2 KO cells, consistent with higher negative charges of inner membrane. Right, box plots of membrane FluoVolt intensity. n=25 cells for WT, n=27 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (G) Left, still images from videography show higher FluoVolt fluorescent signals at rear zone (arrowhead) of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs show the ratio of FluoVolt intensity at rear vs. front during confined migration. n=15 cells per group. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (H) Live-cell images and quantifications show the effects of constitutive active (CA) RAP1B-V12 or dominant-negative (DN) RAP1B-N17 on FluoVolt intensity in WT or PB2 KO GSCs. n=25 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (I) Left, still images capture calcium localization (Fluo4-AM fluorescence, arrowhead) at the rear of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs showing Fluo4-AM intensity ratio at rear vs. front in GSC during passage through tunnels. n=15-16 cells. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (J) Left, still images from videography show that calcium chelator BAPTA-AM disrupted the pattern of high FluoVolt signals at the rear of WT GSCs (arrowhead) during confined migration. Right, bar graphs show FluoVolt intensity ratio at rear vs. front of GSCs when traversing tunnels. n=21 cells for WT, n=16 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (K) Model of Plexin-B2 signaling affecting membrane surface charge and electric field during polarized confined migration, with PIP2 enrichment at cell front and Ca 2+ at rear zone, leading to asymmetry of FluoVolt and R(+8)-pre-GFP.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Transfection, Membrane, Migration, Fluorescence, Comparison, Clinical Proteomics, MANN-WHITNEY, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Structure model of the extracellular domain of human Plexin-B2 show the locations of lock1 and lock2 mutations predicted to form disulfide bridges that lock the ring structure. (B) Western blots show absence of mature Plexin-B2 (170 kDa) in PB2 KO GSC, and expression of lock mutants in PB2 KO SD2 and SD3 GSCs. β-actin serves as a loading control. (C) Still images from videography show passage of GSCs (nuclei visualized by NucSpot) through microchannels with PB2 wildtype rescue construct but not lock mutants, nor PB2 with deletion of extracellular domain (dECTO). Chevrons point to 3 µm constrictions. (D) Box plots show velocity through constrictions, stalling time at constrictions, and sum of forward and backward movements, with 25–75% quartiles, minimal and maximal values (whiskers), median (line), and mean (cross). For velocity and sum of movements: n=17-20 cells per condition. For stalling time at constriction: n=14-28 cells per condition. One-way ANOVA followed by Dunnett’s multiple comparisons test. (E) Still images from videography show F-actin assembly (SPY-actin, arrowhead) at cell rear and MemGlow + endosomes (arrow) at cell front of SD3 GSCs with Plexin-B2 WT rescue but not mutant rescues when traversing 3 µm constrictions (chevrons). (F) Bar graphs showing fluorescence intensity ratio of SPY-actin and MemGlow at rear vs. front of GSCs during confined migration. n=10-18 cells per condition. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data represent mean ± SEM. (G) Model of Plexin-B2 signaling and mechano-electrical regulation of membrane tension and membrane surface charge during polarized confined migration. Regionalized enrichment of endocytosis/PIP2 at cell front and F-actin/Ca 2+ at rear zone lead to asymmetry of FluoVolt and R(+8)-pre-GFP membrane probes.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Western Blot, Expressing, Control, Construct, Mutagenesis, Fluorescence, Migration, Membrane

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Confocal microscopic analysis of unfixed HeLa cells penetrated by Tween20 and immunostained with anti-PQBP1, antifibrillarin, and antinucleolin antibodies. Nuclei were costained with Hoechst 33342. The signals of PQBP3/NOL7 were distributed in the peripheries of nucleoli. In addition, smaller speckles were observed in the nucleoplasm and cytoplasm. ( B ) Super-resolution microscopy images of HeLa cells after fixation. The distribution pattern was similar to that observed by standard confocal microscopy. The speckle diameters were ~260 nm in the nucleoplasm and ~300 nm in the cytoplasm. Staining localized to the outer shell of the nucleolus resembled a chain or cluster of similarly sized speckles. ( C ) Super-resolution microscopy images of normal iPSC-derived neurons after fixation. The relationship of PQBP3/NOL7, nucleolin, and fibrillarin was similar to that in HeLa cells. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Super-Resolution Microscopy, Confocal Microscopy, Staining, Derivative Assay

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) PQBP3/NOL7 immunocytochemistry of HeLa cells at various cell densities. High signal intensities of PQBP3/NOL7 were observed in cells at low cell densities, while the signals were dispersed into the cytosol in cells at medium densities and eliminated in confluent cells (high density). ( B ) Relationship between percent cell margin in contact with neighboring cells and nucleolar distribution of PQBP3/NOL7. Localization of PQBP3/NOL7 was semiquantitated into three stages weighed by different values (1: robust nucleolar PQBP3/NOL7, 0.5: modest nucleolar PQBP3/NOL7, 0: no nucleolar PQBP3/NOL7). A Kendall’s rank correlation test revealed a negative relationship between % cell contact margin and PQBP3/NOL7 nucleolar distribution (tau = −0.786, p = 1.654 × 10 −62 ). ( C ) Western blot of PQBP3/NOL7 with nuclear fraction, cytoplasmic fraction and total cell extract of HeLa cells cultured at low and high cell densities. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. ( D ) Representative images of cells in the stages of “robust nucleolar PQBP3/NOL7” (upper panels) and “faint nucleolar PQBP3/NOL7” (lower panels). In cells classified as “faint nucleolar PQBP3/NOL7,” nuclear PQBP3 signals became obscure, though fibrillarin signals of nucleoli were robust, and abnormal protrusion of nuclear margin and extranuclear DNA stains were observed in Hoechst 33342 labeling (white arrows). ( E ) Electron microscopy of HeLa cells with faint nucleolar PQBP3/NOL7 (#1, #2) and robust nucleolar PQBP3/NOL7 (#3). Nuclear membrane of #1 and #2 cells became obscure and their protrusion contains chromatin. Nucleoli of #1 and #2 cells did not show normal substructures. The continuity of the protrusion and the nucleus excluded that such protrusions were micronuclei. Cells with robust nucleolar PQBP3/NOL7 (#3) showed normal structures of nucleoli and nuclear membrane. Experiments in this figure were technically replicated until the necessary N was acquired. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Immunocytochemistry, Western Blot, Cell Culture, Labeling, Electron Microscopy, Membrane

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Confocal microscopy of HeLa cells after ten passages (10G), which were penetrated with Tween20, immunostained with anti-PQBP1, and costained with Hoechst 33342. Red arrows indicate cells with dispersed nucleolar PQBP3/NOL7 staining. ( B ) Confocal microscopy images of HeLa cells after 20 passages (20G) stained as described above. Red arrows indicate cells with dispersed nucleolar PQBP3/NOL7 staining, and chromatin distribution (Hoechst 33342-stained area) shifted and deviated in the nucleus. Purple arrows indicate cells in which chromatin was nearly absent. ( C ) Enlarged image of the area indicated by dotted lines in ( B ). ( D – F ) Specific distributions of PQBP3/NOL7 during cell division. Foci of PQBP3/NOL7 localized to the centrosome (white arrow) were observed in addition to the diffuse cytoplasmic distribution. ( G ) Quantitative analyses of percentage of red arrow type or purple arrow type of cells in three different passage groups. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. In red arrow type of cells, statistical significance was found in comparison of <5G and >10G (#: p = 0.0415), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.0027). In purple arrow type of cells, statistical significance was found in comparison of <5G and >10G (##: p = 0.0082), <5G and >10G (###: p < 0.0001), and >10G and >20G (###: p = 0.0005). ( H – J ) Yellow arrows indicate cells exhibiting morphological changes of cell death, in which siPQBP3 signals (red) were absent or low, and PQBP3 signals (green) were relatively high. Contrastingly, siPQBP3-transfected cells with high red signals and low green signals did not exhibit such changes or apoptotic features (white arrows).

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Confocal Microscopy, Staining, Comparison, Transfection

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) PQBP3/NOL7 immunocytochemistry of HeLa cells at less than 5 passages (<5G), more than 10 passages (>10G), and more than 20 passages (>20G). Signals were detected in β-Gal staining of >10G and >20G cells in which PQBP3/NOL7 was decreased in the nucleus and shifted to the cytoplasm. ( B ) Signal intensities of β-Gal were quantified in HeLa cells (30 cells from 3 wells) and compared among three groups. Statistical significance was found in comparison of <5G and >10G (###: p < 0.0001), <5G and >20G (###: p < 0.0001), and >10G and >20G (###: p < 0.0001). ( C ) Signal intensities of PQBP3/NOL7 in nucleus, cytoplasm, or total cell were quantified and compared among three groups (30 cells from 3 wells). (Nuc) Statistical significance was found in comparison of <5G and >10G (#: p = 0.027), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.0051). (Cyt) Statistical significance was found in comparison of <5G and >10G (###: p < 0.0001), <5G and >20G (###: p < 0.0001), and >10G and >20G (###: p < 0.0001). ( D ) Schematic presentation of the method to quantify the extranuclear DNA signals stained by Hoechst 33342 (left panel). The signal intensities were compared among three groups (right graph). Statistical significance was found in comparison of <5G and >10G (###: p < 0.0001), and <5G and >20G (###: p < 0.0001). ( E ) Western blot analyses of nuclear, cytoplasmic, and total PQBP3/NOL7 in 5G, >10G, and >20G HeLa cells (left panels). Statistical comparisons of band intensities among the three groups (right graphs). (Nuclear fraction) Statistical significance was found in comparison of <5G and >10G (#: p = 0.0189), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.0015). (Cytoplasmic fraction) Statistical significance was found in comparison of <5G and >10G (#: p = 0.025), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.003). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Immunocytochemistry, Staining, Comparison, Western Blot

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) protocol of mTOR signal activation in HeLa cell culture (<5G). MHY1485, an activator of mTOR (final concentration in the medium: 10 μM) was added to the culture medium 48 h after cell seeding and cells were harvested after another 4 h. ( B ) Western blot analysis of nuclear and cytoplasmic PQBP3/NOL7 under mTOR activation. ( C ) Quantitative analyses of band intensities of western blots. Statistical analyses (Welch’s t-test) revealed decrease of nuclear PQBP3/NOL7, increase of cytoplasmic PQBP3/NOL7, and shift of PQBP3/NOL7 from nucleus to cytoplasm. ( D ) Immunocytochemistry of HeLa cells treated with MHY1485. Cytoplasmic shift of PQBP3/NOL7 and faint signals of β-GAL were detected in HeLa cells treated with MHY1485. ( E ) Quantitative analyses of cytoplasmic shift of PQBP3 (PQBP3 nuclear/cytoplasmic signal ratio), senescence (β-GAL signal intensity), and cytoplasmic shift of nuclear DNA (Hoechst nuclear/cytoplasmic signal ratio) in HeLa cells by mTOR activation with MHY1485. Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Activation Assay, Cell Culture, Concentration Assay, Western Blot, Immunocytochemistry

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Western blot analysis of PQBP3/NOL7 in total extracts of HeLa cells transfected with two types of PQBP3-siRNA or scrambled-siRNA. Statistical significance was found in comparison of non TF and si-PQBP3#1 (##: p = 0.0048), non TF and si-PQBP3#2 (##: p = 0.0019), si-PQBP3#1 and si-Scramble (##: p = 0.0033), and si-PQBP3 and si-Scramble (##: p = 0.0013). ( B ) Upper panels show three signals of immunostained PQBP3, Hoechst 33342, and fluorescence-labeled siRNA. Almost all cells were transfected with PQBP3 siRNA (#1, #2), and PQBP3 signals were accordingly reduced. Representative cells from nontransfected, PQBP3 siRNA-transfected (#1, #2), and Scrambled siRNA-transfected cells (green arrow in upper panels) are shown in middle panels. The PQBP3 siRNA-transfected cell exhibited a notched nucleus (white arrow) in which an additional large bleb (asterisk) was formed. When Hoechst 33342 signals were acquired at the same signal intensity threshold, cytoplasmic genomic DNA was present in the PQBP3 siRNA-transfected cell but not in the nontransfected or Scrambled siRNA-transfected cells. ( C ) Representative Hoechst 33342 images show a normal nucleus in a nontransfected cell (left panel) and an abnormal nucleus with a protrusion (light blue arrows) in a PQBP3-siRNA-transfected cell (middle panel), and a micronucleus (white arrow) that was detected in only a single cell among Scrambled-siRNA-transfected cells (right panel). ( D ) Quantitative analyses of frequency of cells with notched nuclei (left graph) and frequency of cells with micronuclei (right graph) from 5 to 6 randomly visual fields from independent wells containing 15–50 cells. (left) Statistical significance was found in comparison of non-transfect and si-PQBP3#1 (##: p = 0.0075), non-transfect and si-PQBP3#2 (#: p = 0.0119), si-PQBP3#1 and si-Scramble (##: p = 0.0075), and si-PQBP3#2 and si-Scramble (#: p = 0.0119). (right) Statistical significance was found in comparison of non-transfect and si-PQBP3#1 (##: p = 0.0025), non-transfect and si-PQBP3#2 (#: p = 0.0047), si-PQBP3#1 and si-Scramble (###: p = 0.0006), and si-PQBP3#2 and si-Scramble (##: p = 0.0011). ( E ) Quantitative analysis of cytoplasmic DNA signal intensity per cell in nontransfected, PQBP3 siRNA-transfected, and Scrambled siRNA-transfected cells. The original images were corrected by canceling noise signals, and signals outside of the nucleus were measured (see Methods). Cell numbers are shown in the figure, and images were captured from three wells. Statistical significance was found in comparison of non-transfect and si-PQBP3#1 (###: p = 0.0075), non-transfect and si-PQBP3#2 (#: p = 0.0119), si-PQBP3#1 and si-Scramble (##: p = 0.0075), and si-PQBP3#2 and si-Scramble (#: p = 0.0119). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Western Blot, Transfection, Comparison, Fluorescence, Labeling

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) SAHFs was analyzed in HeLa cells treated with hydrogen peroxide. Nuclear speckles reactive to anti-H3K9me3 antibody indicating SAHFs was observed. Such cells with SAHFs were reactive to β-Gal staining. ( B ) Positive stains for β-Gal were observed in PQBP3-siRNA-transfected but not Scrambled-siRNA-transfected HeLa cell. (PQBP3) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). (β-Gal) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). ( C ) SAHFs was analyzed in human normal iPSC-derived neurons treated with hydrogen peroxide. Nuclear speckles reactive to anti-H3K9me3 antibody was observed similarly to HeLa cells treated with hydrogen peroxide. Such human iPSC-derived neurons with SAHFs were reactive to β-Gal staining. ( D ) Positive stains for β-Gal were observed in human normal iPSC-derived neurons that were transfected PQBP3-siRNA but not by Scrambled-siRNA-transfected. (PQBP3) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). (β-Gal) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Staining, Transfection, Comparison, Derivative Assay

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: HeLa cells were transfected with pEGFP-C1-PQBP3, pEGFP-N1-PQBP3, or pEGFP-N1 plasmids to express EGFP-PQBP3, PQBP3-EGFP, or EGFP proteins, and following Hoechst 33342 staining without fixation, EGFP signals in live cells were observed with confocal microscopy. Similar expression patterns were observed in EGFP-PQBP3 and PQBP3-EGFP fusion proteins. EGFP protein alone did not exhibit the nucleolar pattern of the PQBP3 fusion proteins. Some cell images are redisplayed from Fig. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Transfection, Staining, Confocal Microscopy, Expressing

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Senescent HeLa cells were fixed with 10% formaldehyde and stained with antifibrillarin and DAPI. Signals of EGFP-PQBP3 fusion proteins were directly detected. Most of the nontransfected cells contained cytoplasmic genomic DNA, as revealed by DAPI (purple arrows), while a small part of nontransfected cells showed normal morphology (white arrows). Transfected cells did not contain cytoplasmic genomic DNA (green arrows). ( B ) Nonfixed HeLa cells were directly observed to evaluate the effect of EGFP-PQBP3 or EGFP expression on cytoplasmic genomic DNA. In the left panels, nontransfected cells (purple arrows) but not EGFP-PQBP3-expressing cells (green arrows) contained cytoplasmic genomic DNA. In the right panels, cytoplasmic DNA was not affected in EGFP-expressing cells (white arrows) in comparison to neighboring nontransfected cells. Lower tables show quantitative analysis of 50 cells from 5 wells, each transfected with pEGFP-C1-PQBP3 or pEGFP-C1 plasmid. A strong negative correlation between EGFP-PQBP3 expression and cytoplasmic genomic DNA was statistically confirmed by Fisher’s exact test ( p = 1.087 × 10 −7 , n = 5 wells, n = 50 cells), while no relationship was detected between EGFP expression and cytoplasmic genomic DNA. Experiments in this figure were technically replicated until the necessary N was acquired. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Staining, Transfection, Expressing, Comparison, Plasmid Preparation

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Protocol for transfection of EGFP-PQBP3 or EGFP expression plasmids and induction of senescence in HeLa cells with hydrogen peroxide (H 2 O 2 ). ( B ) Immunocytochemistry of PQBP3, PSME3, and Lamin B1 in HeLa cells with or without treatment. Upper image panels show HeLa cells with or without H 2 O 2 treatment. A portion of H 2 O 2 -treated cells (white arrows) lost the Lamin B1 ring at the nuclear membrane, and PQBP3 and PSME3 were dispersed to the cytosol in these cells. The right graphs show signal densities of PQBP3 or PSME3 on the Lamin B1 ring (upper and middle graphs) and numbers of PQBP3-PSME3 colocalized dots on the Lamin B1 ring (lower graph). The signal intensities of PQBP3 and PSME3 on the Lamin B1-positive area were measured by ImageJ. ( C ) Immunoprecipitation analysis of the interactions between PQBP3, PSME3, and Lamin B1. Left panels and right panels show input and output, respectively, of immunoprecipitations. Input panels show decreased PSME3 and Lamin B1, after H 2 O 2 treatment. Output panels show suppressed interactions between Lamin B1 and PSME3 by EGFP-PQBP3 in coprecipitation. Black and gray arrows indicate each proteins. ( D ) Qunatitative analyses of Lamin B1 protein levels normalized to GAPDH. (Lamin B1/GAPDH) Statistical significance was found in comparison of (1) and (3) (#: p = 0.0452), (2) and (4) (###: p = 0.0006), and (3) and (4) (#: p = 0.0494). (PQBP3-EGFP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (1) and (3) (###: p = 0.0005), and (3) and (4) (###: p = 0.0002). (PSME3 in EGFP-IP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (1) and (3) (#: p = 0.006), and (3) and (4) (#: p = 00252). (Lamin B1 in PSME3-IP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (2) and (4) (##: p = 0.0012), and (3) and (4) (##: p = 0.0093). (PSME3 in Lamin B1-IP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (2) and (4) (#: p = 0.0138), and (3) and (4) (#: p = 0.0111). ( E ) Immunoprecipitation analysis of the interactions between PQBP3, PSME3, and Lamin B1 was performed by these proteins endogenously expressed in HeLa cells. Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Transfection, Expressing, Immunocytochemistry, Membrane, Immunoprecipitation, Comparison

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Sequential reprobing of the same filter with anti-Ubiquitin antibody and anti-Lamin B1 antibody revealed existence of ubiquitinated Lamin B1 (black arrow), which was increased in HeLa cells treated with hydrogen peroxide despite of the decrease of total Lamin B1. ( B ) Immunoprecipitation and detection of Lamin B1 by anti-Ubiquitin, anti-SUMO1, and anti-Lamin B1 antibodies to confirm existence of ubiquitinated and SUMOylated Lamin B1. The band detected in ( A )) was confirmed by immunoprecipitation as mono-ubiquitinated Lamin B1 (black arrow). In addition, some higher bands were shown as ubiquitinated Lamin B1 in HeLa cells treated with hydrogen peroxide (red arrow). Similarly, SUMOylated Lamin B1 were confirmed. The bands of ubiquitinated Lamin B1 were also reactive to SUMO1 (red arrow), indicating that two modifications occurred simultaneously on Lamin B1. ( C ) Protein modifications were examined in GST-Lamin B1 and cell lysates from HeLa cells with or without H 2 O 2 treatment expressing EGFP-PQBP3 or EGFP by transient transfection. Considering the molecular weight 26 kDa of GST, the band indicated with balck arrow corresponds to the band in ( A ), and it is mono-Ubiquitinated Lamin B1. The band size of blue arrow in SUMO1 blot is consistent with SUMOylated and Ubiquitinated GST-Lamin B1, and corresponds to SUMOylated and Ubiquitinated Lamin B1 detected in immunoprecipitation (( B ), red arrow). Light gray arrow-indicated band in ( B ) corresponds to that in (( C )). ( D ) PSME3 knockdown by PSME3-siRNA (KD) and PSME3 overexpression by pCMV3-Myc-PSME3 revealed a reverse relationship between PSME3 and Lamin B1. Ubiquitinated and/or SUMOylated LaminB1 was examined by western blot (upper panels) or immunoprecipitation (lower panels). Red and black arrows indicate modified and non-modified Lamin B1 as described above. Right graphs show quantitative analyses of red arrow band intensities in Ub and SUMO blots subtracted by backgrounds and corrected by Lamin B1. (PSME3) Statistical significance was found in comparison of (1) and (2) (###: p = 0.0007), (1) and (3) (###: p < 0.0001), and (2) and (3) (###: p < 0.0001). (Lamin B1) Statistical significance was found in comparison of (1) and (2) (#: p = 0.0141), (1) and (3) (###: p < 0.0001), and (2) and (3) (###: p < 0.0001) (GAPDH) Statistical significance was found in comparison of (1) and (3) (##: p = 0.0014), and (2) and (3) (###: p = 0.0006). (Ub(85kDa)) Statistical significance was found in comparison of (1) and (3) (##: p = 0.009), and (2) and (3) (#: p = 0.0157). (SUMO1(85kDa)) Statistical significance was found in comparison of (1) and (3) (###: p = 0.0004), and (2) and (3) (##: p = 0.0012). (Ub-Lamin B1) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (1) and (3) (###: p < 0.0001), and (2) and (3) (###: p = 0.0003). (SUMO-Lamin B1) Statistical significance was found in comparison of (1) and (2) (#: p = 0.0182), (1) and (3) (###: p = 0.0002), and (2) and (3) (#: p = 0.0238). ( E ) Inhibitors of ubiquitination (0.1 μM TAK-243) or SUMOylation (10 μM 2-D08) was added to the culture medium of HeLa cells, and the cells were transfected by pCMV3-Myc-PSME3 6 h later. Inhibition of SUMOylation suppressed Lamin B1 decrease by PSME3-OE (black arrow), and the suppressive effect was smaller in inhibition of ubiquitination. Right panels confirm the effects of TAK-243 and 2-D08, respectively, on ubiquitination and SUMOylation, in which the Lamin B1 band reactive to both anti-Ubiquitin and anti-SUMO1 antibodies in Fig. 9B is indicated (red arrows). (Lamin B1) Statistical significance was found in comparison of (1) and (4) (#: p = 0.0178), (2) and (3) (#: p = 0.0126), (2) and (4) (###: p < 0.0001), and (3) and (4) (###: p = 0.0004). (Ub) Statistical significance was found in comparison of (1) and (4) (##: p = 0.003), (2) and (3) (##: p = 0.0047), and (3) and (4) (#: p = 0.0204). (SUMO1) Statistical significance was found in comparison of (1) and (4) (###: p = 0.0002), (2) and (4) (##: p = 0.0012), and (3) and (4) (###: p = 0.0002). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Expressing, Transfection, Molecular Weight, Knockdown, Over Expression, Western Blot, Modification, Comparison, Inhibition

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Colocalization of PQBP3/NOL7 and polyQ disease proteins in cell models. Left panels show single expression of a polyQ disease protein fused to DsRed (DsRed-Atxn1, DsRed-Atxn7, DsRed-Htt, and DsRed-AR) containing a normal or mutant length of polyQ sequence. A small portion of DsRed-Atxn1 formed nuclear speckles in both normal and mutant polyQ lengths, while most of DsRed-Atxn1 exhibited relatively homogeneous nucleoplasm distribution. DsRed-Atxn7 was dominantly distributed in nucleoli. In most cells, DsRed-Htt was predominantly distributed around the nucleus in the cytoplasm, while a part of mutant DsRed-Htt formed cytoplasmic inclusion bodies, as we reported previously (Tagawa et al, ). DsRed-AR, in the absence of androgen treatment, was distributed in the cytoplasm. ( B ) Expression of EGFP-PQBP3 changes the cellular distribution of polyQ disease proteins. EGFP-PQBP3 colocalized with Atxn1 on the nucleoli, while a portion of EGFP-PQBP3 formed nucleoli composed only of PQBP3. EGFP-PQBP3 colocalized with Atxn7 at fibrillarin-positive nucleleoli. EGFP-PQBP3 coexpression shifted localization of DsRed-Htt and DsRed-AR from the cytoplasm to the nucleoli. ( C ) Evaluation of senescence by DHB-mVenus in three types of induction, confluence quiescence, proliferation senescence, H2O2 senescence, and mutant Atxn1 expression. Representative nuclear and cytoplasmic images of DHB-mVenus and quantitative analysis of the percentage of senescence-phenotype cells, in which DHB-mVenus was distributed only in nucleus, among total pDHB-mVenus-transfected cells are shown. ( D ) Immunohistochemistry for PQBP3/NOL7 and Lamin B1 in cerebellar cortex of Atxn1-KI mice and littermate controls at 9 weeks of age. Nucleolar PQBP3/NOL7 signal was decreased in Purkinje cells of Atxn1-KI mice, and nuclear membrane Lamin B1 signal in Purkinje cells also decreased (white arrows). High-magnification original and enhanced images revealed notches and blebbing of nuclear membranes in Purkinje cells of Atxn1-KI mice (white arrow). On the other hand, Lamin B1 signals were unaltered in granule cells. ( E ) Immunohistochemistry for Atxn1/PSME3/Ubiquitin (left panels) and PQBP3/PSME3/Ubiquitin (right panels) in cerebellar cortexes prepared from Atxn1-KI mice and littermate controls at 9 weeks of age. In a portion of Purkinje cells of Atxn1-KI mice, Atxn1 formed nuclear inclusions with ubiquitin (white arrow), while nuclear PSME3 was decreased. PQBP3 was detected in Purkinje cell nuclei of control mice (white arrow) but decreased in Purkinje cell nuclei of Atxn1-KI mice. PSME3 was localized to the nucleus of Purkinje cells in control mice but dispersed to the cytoplasm in Atxn1-KI mice. These changes in expression and localization of PSME3, PQBP3, and Lamin B1 were homologous to those observed in H 2 O 2 -induced senescent HeLa cells (Fig. ). ( F ) Immunohistochemistry for PQBP3/Atxn1/Ubiquitin (left panels) in cerebellar cortexes prepared from Atxn1-KI mice and littermate controls at 9 weeks of age. PQBP3 puncta located at the periphery of nucleus and/or in the cytoplasm (white thin arrows) were costained with Atxn1 and Ubiquitin antibodies in abnormal Purkinje cells (yellow thick arrow), while nucleolus of relatively normal Purkinje cells (light blue thick arrow) was also found in Atxn1-KI mice at 9 weeks. Lower graphs show quantitative analyses of signal intensity of PQBP3 merged with Atxn1 in Purkinje cells, signal intensity of PQBP3 non-merged with Atxn1 in Purkinje cells, and signal intensity of PQBP3 in nucleoli of Purkinje cells. Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Expressing, Mutagenesis, Sequencing, Transfection, Immunohistochemistry, Membrane, Ubiquitin Proteomics, Control