EMBO/SourceData huggingface dataset:
Figure 2A. Unified cell cycle map of unperturbed (gray) and serum-starved cells (1d: light blue, 7d: dark blue, N = 3007 cells). The proliferative cell cycle (dotted gray line) and the hypomitogenic arrest trajectory (black dotted line) are indicated on the map. Inset: Each treatment condition is shown individually on the unified map (other conditions are shown in lighter gray).(B) Diffusion pseudotime, (C) p21, (D) cyclin D1, (E) DNA content of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.(F) phospho/total RB of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.G. Time-lapse imaging of cyclinD1-mVenus intensity in unperturbed (control, green) and serum-starved cells (blue). Cells were serum-starved for at least 8h prior to imaging. The solid line represents the population median and the shaded area indicates the 95% confidence interval. N = 105 control cells and N = 111 starved cells.I. Median nuclear p27 abundance in serum-starved cells is plotted on the map.
Figure 3Unified cell cycle map of unperturbed (gray) and etoposide-treated cells (1 μM; 1d: light green, 2d: green, 3d: dark green, 4d: darker green - see inset, N = 4315 cells). The unperturbed cell cycle trajectory (dotted gray line) and two arrest trajectories (into G02C and G04C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).B-E. (B) Diffusion pseudotime, (C) DNA content, (D) phospho/total RB and (E) p21 of unperturbed (left panels) or etoposide-treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown.F. Time-lapse imaging of CDK2 activity (DHB-mCherry, gray) and p21-YPet (green) intensity in etoposide-treated cells. Schematic shows the two arrest trajectories observed following etoposide treatment. Cells that successfully complete G2 ("Mothers", N = 32 cells) but arrest following cell division ("Daughters", N = 45 cells) are shown in the two leftmost panels. Cells that arrest in G2 (N = 40 cells) are shown in the rightmost panel. The solid lines represent population medians and the shaded area indicates the 95% confidence interval.
Figure 4A. Arrest architecture of replicative stress. 2C and 4C arrest trajectories are shown, the regions that correspond to mitotic skipping and the senescent state are annotated and the arrest trajectory of 8C polyploid cells is indicated. N = 4315 cellsB. Progression of arrest states over time following etoposide treatment. Etoposide-treated cells (upper panel: colored by condition, lower panel: colored by DNA content) populating 2C, 4C, 8C and senescent arrest states at each day of treatment (1-4 d).C. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide treatment (1 μM, 0-4 d).D-K. (D) GSK3β, (E) phospho(Thr157)-p27, (F) p27, (G) CDK4, (H) cyclin D1, (I) cyclin E, (J) cell area and (K) DNA:cytoplasm ratio of etoposide-treated cells are plotted on the map. Median nuclear values are shown in D-I. Area indicated with a dotted line represents the senescent region of the map.L-O. (L) Cyclin A, (M) cyclin B1, (N) CDH1 and (O) SKP2 of etoposide-treated cells are plotted on the map. Median nuclear values are shown. Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence.
Figure 5A. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide or palbociclib treatment (1 μM, 4/8 d). Control: N = 6050 cells, etop 4d: N = 409 cells, etop 8d: N = 732 cells, palbo 4d: N = 1837 cells, palbo 8d: N = 1677 cells. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.0001).B. Unified cell cycle map of the unperturbed (gray), palbociclib- (palbo, 1 μM, 4/8 d, blue) and etoposide- (etop,1 μM, 4/8 d, green) treated cells. Control: N = 10499 cells, etop: N = 2692 cells, palbo: N = 4931 cellsC-K. (C) DNA content, (D) phospho/total RB, (E) p16, (F) TGFβ1, (G) IL-6, (H) IL-8, (I) phospho-p38, (J) phospho-STAT3 and (K) phospho-STAT5 are plotted on the map. Median nuclear values are shown.
Figure 6D. Representative single-cell trace demonstrating mitotic skipping and endoreduplication following etoposide treatment (1 μM) by time-lapse imaging. CDK2 activity (DHB-mCherry, blue), cell cycle phase (PCNA-mTq2, S phase shown as gray shaded area) and p21-YPet intensity (green) are plotted versus time of etoposide treatment.E. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following siRNA-mediated knockdown of cyclin D in control and etoposide-treated cells. Boxplots show data from four independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.F. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin D1 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.G. Cyclin A abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin A2 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.
Figure 1(E) Representative images of the pilot validation screen for the negative control (no siRNA) and for five sets of siRNA-treated cells with reduced TGN retrieval ratio. Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text. Scale bar, 100 μm (top two rows) and 38 μm (bottom row).
Figure 3(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and SFT2D2 KD cells (bottom).(B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset).(C) Cells stably expressing SFT2D2-Myc were costained with antibodies against various post-Golgi SNARE proteins. Colocalization was quantified by Pearson's correlation coefficient (right-hand graph) and indicates very extensive colocalization with STX6, STX7, and VAMP8. The image panels illustrate colocalization of SFT2D2-Myc and STX5 at the Golgi (top, arrowhead) but much more extensive colocalization of SFT2D2-Myc and STX6 (bottom, arrowheads). In (B) and (C), the white dashed box delineates the area magnified in the insets.(D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells.(E) Control and SFT2D2 KD HeLa cell lysates were separated by LDS-PAGE and blotted for the indicated SNARE proteins or actin.
Figure 4(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and ZDHHC5 KD cells (bottom).(B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset).(C) Control and ZDHHC5 siRNA-treated SFT2D2-Myc cells were mixed and stained for ZDHHC5, Myc, and VPS35. KD cells are marked by an asterisk.(D) Control and ZDHHC5 siRNA-treated HeLa cells were mixed and stained for ZDHHC5, α5-integrin, and VPS35. KD cells are indicated with an asterisk.(E) Control (top) and ZDHHC5 KD (bottom) HeLa cells were fixed and stained for TGN46 and β1-integrin.
Figure 5(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom).(B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1).(C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35.(D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells.(E-G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell's TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.
Figure 6(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown.(B-E) Quantitative analysis of SFT2D2, ZDHHC5, and GRINA KD cells immunofluorescence using automated microscopy (see Experimental Procedures). (B) Representative images showing VPS35, TGN46, and CIMPR staining. Scale bar, 50 μm. (C) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2, ZDHHC5, or GRINA KD cells. (D) SFT2D2, ZDHHC5, and GRINA KD increase the Pearson's correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130. In some cases, identical correlation coefficients were measured in the replicate experiments. (E) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells.
Figure 1D: Co-immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels. The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP: LETM1) using a LETM1 monoclonal antibody and Protein G magnetic beads (ProtG). ProtG beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. 10% of the input was used for immunoblotting. Prohibitin (PHB) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes. The middle and right panel of the coIPs illustrates the converse experiment, in the middle in TMBIM5WT and right TMBIMKO, using TMBIM5 as bait (right panel, IP: TMBIM5). The last two right panels show blots from BN-PAGE conducted in TMBIM5WT and KO.
Figure 2A: Western blot analysis of TMBIM5 in control and targeted HeLa and HEK293 clones.E: Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls. F: Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual experiments (biological replicates), mean ± SD, One-way ANOVA with Bonferroni's multiple comparisons test performed against TMBIM5WT *p<0.05, **p<0.008, Two-way ANOVA with Bonferroni's multiple comparisons test performed for the OPA1 statistics against TMBIM5WT, ***p=0.0009, ****p<0.0001.
Figure 7(A) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods.
Figure 1(C) Acoustic startle reflex test to compare hearing ability between Wt (N = 7) and Hpn-/- (N = 4) mice.(F) Immunoblot analysis of phospho-Smad2/3 (TGFβ pathway signaling marker) and total Smad2/3 in lysates from indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mice. GAPDH was used as the loading control. The histogram depicts quantification of pSmad2/3 compared to Wt normalized to total Smad2/3.
Figure 2(B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression
Figure 3(D) Immunoblot analysis of phospho-Smad2/3, total Smad2/3, and GAPDH (loading control) expression in Wt tumors transplanted into either Wt or Hpn-/- recipients (n = 10 tumors each).(E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated.(G) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A-pIND20-HPN pL6-TGFβ1 cells with (DOX+) or without (DOX-) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns).(H) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM).
Figure 4(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels.(D) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N-terminal fragment of fibronectin (lower panel). Full-length fibronectin (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin.
Figure 5(A) Immunoblot analysis of fibronectin in indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mouse (representative of at least three repeats). GAPDH blot is used here as the loading control.(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in prostate lysates of control mice or mice with prostate-specific hepsin overexpression N=2 mice each, the two prostate lobes were run separately (a and b).
Figure 6(A) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells without hepsin overexpression. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats). (B) Immunoblot analysis of cell lysates (pSmad2/3, total Smad2/3 and vinculin as loading control) and concentrated cell culture supernatant (anti-V5 for TGFβ) from MCF10A-pIND20-HPN pL6-TGFβ1-V5 cells without hepsin overexpression. Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats).(E) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) and without (DOX-) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels (N = 3 biological repeats).
Figure 1.(A-C) 10X phase contrast images of vero monolayers at 3 days post-inoculation. Panels shown are (A) mock, (B) NP, and (C) OP.(D) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids, seen as black dots.
Figure 2.A) Two SARS-CoV-2 passage 4 stocks (black and gray) were quantified utilizing plaque assay at day two (closed circles) and day three (open circles) post infection of Vero E6 and Vero CCL81.B) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation.C) Cell monolayers two days post infection in Vero E6 (top) and Vero CCL81 (bottom row) at multiple dilutions.
Figure 3.Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation.Protein lysates were probed by western blot with B) anti-SARS nucleocapsid (N) antibody or C) anti- SARS-CoV spike (S) antibody.
Figure 1A. NIH3T3 cells transfected with KV10.1*EGFP did not show primary cilia. Cells were transiently transfected, after 24 h serum was removed for additional 24 hours to induce ciliogenesis, and finally cells were stained with anti-acetylated α-tubulin. While most cells were ciliated, those showing green fluorescence were devoid of cilia. Scale bar: 10µm.B. NIH3T3 cells transfected with KV10.1 (red bars) showed markedly less cilia than control cells (empty vector, white bars). Subconfluent cultures grown in the presence of FCS were serum starved for 24 hours to induce ciliogenesis. Cilia were stained with anti acetylated α-tubulin antibody. To determine ciliary disassembly, cells were starved for 24 hours and then incubated for 4h in FCS to induce cell cycle re-entry and ciliary resorption.C. Similarly, hTERT-RPE1 cells transfected with KV10.1 also showed less cilia. Ciliogenesis and ciliary disassembly were induced as in B and cilia were stained using anti acetylated α-tubulin and quantified as in A. The Inset shows the equivalent experiment using the structurally related potassium channel KV10.2, which did not alter the frequency of expression of cilia.D. Examples of fields of view of hTERT-RPE1 cells transfected with KV10.1, serum starved for 24 hours and cilia were revealed with anti Arl13B antibody (arrows). A majority of control-transfected cells showed cilia, while KV10.1 transfected did not. Scale bar: 10µm.
Figure 2A. Representative images showing hTERT-RPE1 grown in the presence of serum and stained with anti acetylated α-tubulin antibody. There were more ciliated cells after treatment with siRNA against KV10.1 (KV10.1 KD), as compared to scrambled siRNA-transfected cells, which were devoid of cilia (control). Scale bar: 10 µm.B. Quantification of the effect depicted in A for the number of images indicated at the base of the columns.C. Time course of ciliary disassembly. hTERT-RPE1 cells were serum starved for 24 hours, fixed at the times indicated after serum reintroduction, and stained with anti acetylated α-tubulin to reveal cilia. While in the presence of scrambled siRNA the abundance of cilia decreased rapidly (black line), it remained virtually unchanged in cells where KV10.1 was knocked down (red). N=8-13.D. Time course of ciliary shortening after serum reintroduction. In the same experiments as in C, the length of the remaining cilia was measured automatically using ImageJ (FIJI). While ciliary length decreased with a similar time course in the siRNA-treated cells as in controls, cilia were longer in KV10.1-knockdown cells. A re-elongation of cilia was evident in both populations after 6 hours. Values are mean ± SEM for a number of cilia between 62 (control, 4.5 h) and 308 (both groups, 1.5 h).E. Blockade of permeation through the K+ channel altered the speed of deciliation. After serum starvation, medium containing serum and Astemizole (10 µM) was added, and cells were fixed and stained at he indicated time points (N=12).
Figure 3A. Quantification of the amount of ciliated cells in primary embryonic fibroblasts from KV10.1 knockout mice. Actively growing cultures, cultures after 24 h serum starvation, and four hours after subsequent serum reintroduction were stained for the presence of cilia using anti-acetylated α-tubulin. An increase in frequency of cilia in the knockout cells was evident under all conditions, but especially marked in the ciliary resorption test (4h after serum reintroduction), indicating an influence of KV10.1 expression on deciliation.B. Exogenous expression of human KV10.1 reverted the phenotype in MEFs. Cells obtained from three animals were transfected with KV10.1 or empty vector (mock), serum starved for 48 h and cilia were revealed by immunocytochemistry using anti-acetylated α-tubulin in the indicated number of images. KV10.1 expression decreased the frequency of ciliated cells.C. MEFs from knockout mice did not show primary cilia when transfected with KV10.1. Cells were co-transfected with human KV10.1 and mVenus, serum starved for 48 hours and then fixed and stained for cilia (AcTub). Venus-positive cells did not show cilia. Scale bar: 25 µm.D. In wild type MEFs, transfection with KV10.1 reduced the presence of primary cilia. When a gain-of-function mutant (L352V) that originates developmental defects in human patients was transfected, the frequency of cells with cilia was further reduced.E. Sensitivity to SHH signaling was significantly increased in primary fibroblasts from KV10.1 knockout mice, in agreement with the higher abundance of primary cilia. Cells were serum starved and SHH was stimulated after 24 hours by addition of culture supernatant of SHH-expressing HEK293 cells. Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT-PCR.