EMBO/SourceData huggingface dataset:
Figure 6Representative blots showing expression levels of LATS1/2, CCNE1, CCND1 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage four (P4) and Passage seven (P7). Actin was used as internal control.mRNA levels of LATS1, LATS2 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage seven and protein levels were analyzed using quantitative real-time PCR.Representative gel photographs showing mRNA levels of LATS1, LATS2, and YAP in the LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells examined using semi-quantitative RT-PCR at passage seven.Representative images showing the morphologic changes and SA-β-gal staining in control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.Quantitative data showing the ratio of SA-β-gal positive cells in the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the seventh passage.Growth curves of the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.
Figure 7Representative images showing cells with ectopic expression of LATS2, which is labelled with GFP (the control is empty vectors).Relative mRNA levels of LATS1, LATS2, and YAP in the control and LATS2-expressing hOSEs at the 4th passage. Relative mRNA levels were determined with RT-PCR.Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2.Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage.Growth curves of control and LATS2-overexpressing hOSE cells.Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2.Representative images showing the effect of LATS2 on the morphology and β-galactosidase activities in cultured primary hOSE cells at the 9th passage.Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2.mRNA levels of major components of the DREAM complex (E2F4, E2F5, RBBP4, TFDP2) in the control and LATS2-overexpressing hOSEs at the 9th passage. Relative mRNA levels were examined by real-time PCR.Growth curves of the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells at their 16th passages.Representative images showing cellular morphology and β-galactosidase activities in control and LATS2-overexpressing hOSEs (K). Scale Bar: 50 µm. Right bar graph (L) showing quantitative results of SA-β-gal positive cells in the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells.
Figure 8Representative images showing colony formation in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 knockout. hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging.Quantitative data showing colony numbers in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope.Representative images showing tumorigenesis (red circle area) of hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. Tumors formed by hOSE-YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area. Right panel showing representative tumors formed by hOSE-YAPS127A-LATS2KO and hOSE-YAPS127A-shRB1 cells. Please note that no tumor xenografts formed in hOSE-YAPS127A alone group.Representative images showing expression of YAP, Ki67 and LATS2 in hOSE-LATS2KO-YAPS127A cell-derived tumors examined by immunohistochemistry.
Figure 1(A) Alcian blue‐stained sections of descending colons from control and Atg5VC mice. The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside. Bars=200 μm.(B) Quantification of average mucin area/goblet cell in control and Atg5VC mice (n=5-7 mice/group from 3 independent experiments; 100 goblet cells measured/mouse). **P0.01 as determined by the Student's t‐test. This analysis was performed on upper crypt goblet cells (above the upper dashed line in A).(C) Quantification of the average number of goblet cells per crypt (n=3 mice/group; over 100 crypts measured/mouse).(D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm.(E, F) Quantification of (E) the average granule density and (F) the average number of mucin granules/goblet cell (n=3 mice/group and 20 goblet cells evaluated/mouse). ***P0.001 as determined by the Student's t‐test.(G, H) Whole‐mount images of the mucosal surface from control and Atg5VC transverse colons. Whole‐mount colonic samples were either stained with Ulex europaeus agglutinin‐1 (UEA) that labelled mucus (G) or analysed by the scanning electron microscopy (H). Yellow arrows indicate the crypt opening orifices. Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice (insets in H show higher power image of the latter). Bars=100 μm.
Figure 2(B) Representative whole‐mount images of single spheroids cultured in either 50% conditioned media (CM) or 5% CM+5 μM DAPT and stained for Muc2 (green) to visualize goblet cells. Bars=20 μm.(C, D) Graph of relative expression (as measured by qRT‐PCR) of Atoh1 (C) and Muc2 (D) mRNAs comparing spheroids grown in either 5% CM or 5% CM+ DAPT to spheroids grown in 50% CM (n=4-5 samples/group). Error bars indicate s.e.m. *P0.05; **P0.01 as determined by a two‐tailed Student's t‐test.(E, F) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5% CM+DAPT and stained for Muc2 (green: E) or with the lectin, UEA (red: F). Bars=20 μm.(G) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 as determined by a two‐tailed Student's t‐test).(H) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice as measured by qRT-PCR (n=4-5 samples/group). No statistically significant differences were found in Muc2 expression as determined by the Student's t‐test.(I, K) Images of FIP200f/f (I) and Atg14f/f (K) colonic epithelial spheroids treated with recombinant Tat‐Cre and stained with UEA (red).(J, L) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 and ***P0.001 as determined by a two‐tailed Student's t‐test).
Figure 3(A) Images of colonic sections stained for LC3β (green) from control and Atg5VC mice. The white‐dashed lines indicate the border between the basal surface of the crypt epitheliumand the mesenchyme. White asterisks denote non‐specific plasma cell staining from the mouse monoclonal anti‐LC3β antibody. Bars=50 μm.(B) Immunogold transmission electron microscopic image for LC3β in a colonic goblet cell from a wild‐type mouse. The inset is a low power image of the entire goblet cell. The black box within the inset indicates the area shown in this panel. Note this area is not associated with mucin granules. Bar=500 nm.(C) Enriched KEGG pathways for Atg5‐deficient epithelial cells obtained from a microarray comparing control and Atg5VC colonic crypt base epithelium. P‐values were determined by modified Fisher's exact test.(D-G) Double label immunogold transmission electron microscopic images from wild‐type mice for LC3β (12 nm particles) and EEA1 (D), Rab7 (E), Rab11 (F), or Lamp1 (G) (18 nm particles). Bars=200 nm.
Figure 4(B) Colonic epithelial monolayers from wild‐type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β (green) and EEA1 (red). Bars=5 μm.(C, D) Quantification of average number of LC3β dots per cell (C) and the percentage of LC3β dots that co‐localized with EEA1 per cell (D) (n=3 experiments/group; 25-30 cells/experiment).(E) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated. GAPDH is a loading control. Representative image is shown from n=3 experiments.(F) Images of wild‐type colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 100 μM Dynasore. Bars=20 μm.(G) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ***P0.001 as determined by the Student's t‐test.
Figure 5(A) Colonic epithelial monolayers from a wild‐type and p22 mut mouse stained with LC3β (green) and EEA1 (red). Bar=5 μm.(B, C) Quantification of average number of LC3β dots per cell (B) and the percentage of LC3β dots that co‐localized with EEA1 per cell (C) (n=3 experiments/group; 25-30 cells/experiment).(D) LC3 immunoblots of either wild‐type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated. Representative image is shown from n=3 experiments.(E) Double label immunogold transmission electron microscopic images for LC3β (12μnm particles) and p22phox (18 nm particles) showing a p22phox‐positive single membrane cytoplasmic vesicle (D) and a p22phox‐, LC3β‐positive vacuole (E). Bars=200μnm.(F) Colonic epithelial monolayers from a wild‐type mouse stained for LC3β (green) and p22phox (red) and quantification of percent LC3β dots co‐localized with p22phox per cell, Bar=5 μm.(G) Colonic epithelial cellspheroids from control and p22 mut mice stained with TRITC‐UEA to label goblet cellmucin. Bars=50 μm.(H) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. **P0.01; ****P0.0001 as determined by the Student's t‐test.
Figure 6(A) Colonic epithelial cell spheroids from p22phox−/−, Atg5VC and wild‐type mice. Wild‐type spheroids were treated with vehicle (control), 10 μM DPI (NADPH oxidase inhibitor) or 100 μM Dynasore. Intracellular ROS in live cells were labelled by 5 μM DCF. Bars=100 μm.(B) Quantification of fluorescent DCF normalized to control (n=3 independent experiments/group; 30 spheroids quantified/experiment).(C) Quantification of average area of cytoplasmic mucin from wild‐type mice treated with 3% NAC as compared to controls (n=6-7 mice/group). Error bars indicate s.e.m. *P0.05; **P0.01; ****P0.0001 as determined by the Student's t‐test.(D) TEM images of colonic epithelial monolayers treated with cerium chloride. The presence of electron dense precipitate is indicative of ROS production. Bar=500 nm.
Figure 7Exogeneous ROS can restore mucingranule accumulation defects. (A) Quantification of fluorescent DCF normalized to controls (n=3 independent experiments/group; 10-30 spheroids quantified/culture). *P0.05 as determined by ANOVA with Tukey's multiple post‐test comparisons(B) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (C) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). ***P0.001, ****P.0001 as determined by ANOVA with Tukey's multiple post‐test comparisons.(D) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT-PCR. No statistically significant differences were found in either group as determined by ANOVA.(E, G) Images of p22 mut or Dynasore‐treated colonic epithelial spheroids (mucin is labelled with TRITC‐UEA; red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (F, H) Quantification of average mucin area/goblet cell (n=6 sample/group; 30-60 cells were quantified/sample) corresponding to experiments performed using p22 mut (E) and Dynasore‐treated wild‐type spheroids (G)Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA‐AM (I) or PMA/ionomycin (J). (I, J) Quantification of average mucin area/goblet cell (n=5 sample/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ****P0.0001 as determined by the Student's t‐test (I) or ANOVA with Tukey's multiple post‐test comparisons (J).
Figure 1B. In vitro modification reactions containing the six fragments and HeLa NE were performed. After nuclease P1 digestion, the products were analyzed by 1D-TLC with pU and pψ as controls.C. WT and mutant 7SK fragment 5 containing U250G were analyzed as in B.E. 7SK RNA, generated by either in vitro transcription (IVT) or purified from HeLa total RNA and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.F. Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation (IP) with the indicated antibodies.G. 2D-TLC analysis of 32p-labeled mononucleotides derived from IVT or CDK9-bound 7SK RNA, with the first panel depicting a schematic migration pattern of the indicated 5'-phosphorylated mononucleotides.H. 7SK RNA, obtained by either IVT or anti-CDK9 IP and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.
Figure 2C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag or immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag--tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated HA-tagged proteins into HeLa cells. Anti-HA immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.R. The indicated 7SK were in vitro transcribed in the presence of 32p-UTP and subjected to in vitro modification reactions. The nuclease P1-digested products were analyzed by 1D-TLC with pU and pΨ as controls.
Figure 3A. Anti-V5immunoprecipitates (IP) from HeLa cells expressing nothing (-) or V5-DKC1 were analyzed by immunoblotting for V5-DKC1 and qRT-PCR for the bound 7SK RNA. The error bars represent mean ± SD from three independent experiments.B. Anti-FlagIP from NE of HeLa or the HeLa-derived F1C2 cells expressing CDK9-F were analyzed by Western blotting (WB) for the indicated proteins and primer extension (PEx) for 7SK RNA.C. WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline (DOX) treatment for 5 days.D. The 7SK RNA levels in these two clones were detected by qRT-PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels. The error bars represent mean ± SD from three independent experiments.E. 1D-TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment.F. NE and anti-CDK9IP from NE of inducible DKC1 KD clone 1, which was untreated or treated with DOX, were analyzed by WB for the indicated proteins and PEx for 7SK RNA.G. Clone 1 was treated with DOX for 3 days and then transfected with the Tat-Flag cDNA (20 ug/150-mm dish). Two days later, NE and anti-FlagIP from NE were analyzed as in F.
Figure 4A. Luciferase activities were measured in HeLa cells transfected with the indicated reporter constructs, an empty vector (-) or the Tat cDNA, and either siDKC1 or a non-target siRNA (siNT). For each reporter, the activity in cells expressing siNT was set to 1.B, C, and E. The mRNA levels of luciferase (B) or Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.D. NH1 cells containing the integrated HIV-1 LTR-luciferase reporter and expressing Tat were transfected with siDKC1 or siNT. ChIP-qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV-1 promoter. The signals were normalized to those of input and plotted.B, C, and E. The mRNA levels of Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.F. Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10-derived clone inducibly expressing shDKC1 upon exposure to DOX.G and H. The inducible DKC1 KD clone, treated or untreated with DOX for 4 days, was incubated with the indicated concentrations of JQ1 (G) or prostratin (H) for 24 h. The GFP expression was measured by flow cytometry and expressed as percentages of GFP(+) cells of the entire population. The error bars in all panels represent mean ± SD from three independent experiments, and the p values determined by the Student's t-test.
Figure 1A. Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) treatment as indicated.B. Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP. **, P<0.01 by unpaired two-tailed t test (n=3 biological repeats in one experiments).C. Size distributions for vesicles as analyzed in D.D. Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells.F. Flow cytometry analysis for CD63+ exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants. Quantification was done by mean fluorescence intensity. **, P<0.01 by unpaired two-tailed t test (n=4).
Figure 2B. Co-immunoprecipitation of GSDMD with full-length and deletion mutants of IQGAP1 tested in HEK293 cells.C. Co-immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells.D. Co-immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant (D276A) restored GSDMD KO YAMC cells. Cells were treated as indicated.E. Co-immunoprecipitation of C-terminal GSDMD (CT) with IQGAP1 overexpressed in HEK293 cells.F. Co-immunoprecipitation of pyroptosis mutants full-length (I105N FL) and N-terminal GSDMD defective of pyroptosis (I105N-NT) with IQGAP1.
Figure 3A. Imaging analysis for co-localization of CD63 with IQGAP1 in IQGAP1 (FL) and IQ domain truncation (ΔIQ) restored YAMC cell. Cells were treated with LPS plus ATP. The IQGAP1-CD63 co-localization dots (shown by yellow) were counted, and Pearson's correlation coefficient was calculated for the quantification of co-localization.B. Imaging analysis for co-localization of CD63 with GSDMD in IQGAP1 full length (FL) and IQ domain truncation restored YAMC cell (ΔIQ) after LPS plus ATP treatment. The GSDMD-CD63 co-localized dots were counted and Pearson's correlation coefficient was calculated for quantification.C. Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells.D. Western blots analysis for supernatants collected from IQGAP1 KO YAMC cells, IQGAP1 full length and IQ domain truncation restored IQGAP1 KO YAMC cells. Cells were treated as indicated and the supernatants were subjected to IP with anti-IL-1β. E. Western blots analysis for whole cell lysate collected from panel D.F. ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated.G. Vesicle size distributions for exosome samples collected from panel E.
Figure 4A. Imaging analysis for early endosome (shown by marker EEA1) change in WT and IQGAP1 KO YAMC cells upon indicated treatments. Quantification was done by counting the EEA1 puncta from each cell. Five field views of cells' puncta were counted. Three independent experiments were repeatedB. Co-localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells. Yellow dots indicated the co-localization signal.C. Co-localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells.D. Co-localization of CD63 with IQGAP1-GSDMD complex, as measured by PLA, shown by the yellow dots after LPS and ATP treatment.E. Co-localization of CD63 and pro-IL-1β in YAMC and IQGAP1 KO YAMC cells.F. Western blot analysis of extracellular vesicle secretion from WT and TSG101 knockdown cells upon indicated treatments. Secreted proteins were pulled down by IL-1β in the supernatant and the indicated proteins were probed for.G. Total particle count and size distribution for exosomes collected from WT and TSG101 knockdown cell supernatants with indicated treatment.H. Western blot analysis for interaction of TSG101 with IQGAP1 in WT and GSDMD KO cells by pulling down IQGAP1 under indicated treatments.I. Co-immunoprecipitation of GSDMD with TSG101 in WT and IQGAP1 KO cells by pulling down TSG101 under indicated treatments.
Figure 5A. Total particle count and size distribution of exosomes secreted in the supernatant from IQGAP1 KO (IQ1 KO), IQGAP1 full length restored (FL) and GRD domain truncation restored (ΔGRD) YAMC cells upon LPS or LPS plus ATP treatment.B. PLA assay for IQGAP1 and CDC42 interaction in IQGAP1 FL restored cell and ΔGRD restored cell. The green dots showed the interaction of IQGAP1 and CDC42.C. Colocalization of CD63 with IQGAP1-CDC42 complex (shown by PLA signal) in WT and GSDMD KO cells under LPS treatment. Quantification was performed by counting PLA green dots.D. Co-immunoprecipitation analysis of endogenous IQGAP1 with CDC42 in wild-type and GSDMD KO cells upon LPS or LPS plus ATP treatment.E. Co-immunoprecipitation analysis of GTP-bound CDC42 in indicated cells following LPS treatment.F. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in wild-type and IQGAP1 KO cells upon LPS or LPS plus ATP treatment.G. Western blots for exosome secretion upon CDC42 GTPase inhibition. Cells were pretreated with and without 10µM ML141 for 24 hours to fully block its GTPase activity, followed by LPS and ATP treatment as indicated. H. Particle analysis and size distribution for exosomes collected from cells with the same treatment as panel G.I. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in vehicle or ML141 (10µM for 24 hours) treated cells following LPS or LPS plus ATP treatment.
Figure 6A. Histology staining for IQGAP1 and GSDMD in naive and inflamed (3 days of DSS-challenge) colon tissues.B. Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls.C. Western blot analysis for supernatant collected from colon explants of DSS-treated wild-type (WT), Gsdmd-/-(GSDMD KO) and Iqgap1-/- (IQGAP1 KO) mice. Supernatant was first pulled down by IL-1β antibody and samples were subjected to Western blot analysis. The indicated proteins were probed.D. Immunofluorescent staining with anti-GTP-CDC42 in DSS treated colons from Gsdmd+/+ (WT) and Gsdmd-/-(GSDMD KO), IQGAP1+/+ and IQGAP1-/- mice.
Figure 1(C) Quantification of AQR position in adult WT, cst-1 and/or cst-2 mutant animals. The full length between the URXs and the end of the nematode tail is divided into 10 blocks, and the percent of AQR that stopped within each block is indicated. ***, P < 0.001 by Fisher's exact test. N = 50 - 109.(E - F) Fluorescence time-lapse images of F-actin, plasma membrane and histone during AQR/QR.ap migration in WT (E) or cst-1; cst-2 double mutant (F) animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrows, migration distance. F-actin is labeled with GFP-tagged with the actin-binding domain of Moesin (GFP::Moesin ABD), and the plasma membrane and Histone are labeled with mCherry-tagged with a myristoylation signal and a histone. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bars, 5 μm. (G - J) Quantification of the QR.ap migration angle (G H), distance (I) and speed (J). Each line in (G I) represents the measurement from one time-lapse movie. Error bars indicate SD. N = 10 - 17. Statistical significance is based on Student's t-tests, **, P 0.01; ***, P  0.001.