EMBO/SourceData huggingface dataset:
Figure 6A. Fraction of remaining branches, over time, starting at an average of 5 and 25 minutes after branch nucleation, in the presence (blue) or absence (gray) of VCA. Lines are exponential fits, yielding debranching rates of (3.7±0.6)x10-4 s-1 and (20±8)x10-4 s-1 without VCA for 5- and 25-minute-old branches, respectively; and debranching rates of (3.8±1.3)x10-3 s-1 and (8.2±2.8)x10-3 s-1 with 0.5 µM VCA for 5- and 25-minute-old branches, respectively.B. Fraction of attached SPIN90-nucleated filaments, over time, starting at an average of 5 and 25 minutes after branch nucleation, in the presence (blue) or absence (gray) of VCA. Lines are exponential fits, yielding detachment rates of (12±7)x10-4 s-1 and (7±3)x10-4 s-1 without VCA for 5- and 25-minute-old filaments, respectively (p=0.2, logrank test); and detachment rates of (2.5±1.1)x10-2 s-1 and (3.1±1.7)x10-2 s-1 with 1 µM VCA for 5- and 25-minute-old filaments, respectively (p=0.4, logrank test).
Figure 7A. The fraction of remaining branches, versus time, exposed to 0.16 µM G-actin (black) supplemented with 50 nM cortactin (red). The branch junctions were exposed to an average force of 1.4 pN.B. The fraction of remaining SPIN90-Arp2/3-nucleated filaments, versus time, exposed to 0.15 µM G-actin (black) supplemented with 10 nM cortactin (red).C. The fraction of remaining SPIN90-Arp2/3-nucleated filaments, versus time, exposed to 0.15 µM G-actin, with 0.5 µM VCA (black) supplemented with 10 nM cortactin (red).
Figure 1(G) t-SNE (top) and violin plots (bottom) showing the expression of selected signature genes in different cell clusters.(J) Representative images of immunofluorescence of CCSC marker gene (LGR5) and non-CCSC cluster signature genes in the xenografts harvested at different time points. Cluster T1 (KRT19), Cluster T2 (HSPA1B), Cluster T3 (Ki67), Cluster T4 (AGR2), Cluster T5 (ODC1), and Cluster T6 (ANXA1) were immunostained. Scale bar, 60 μm.(K) Quantification of (J) showing the dynamic change of cell clusters during the progression of CD133+LGR5+ CCSC-formed xenografts. Error bars represent SEM of three xenografts. Three sections of each xenograft and 3 fields of each section were quantified.
Figure 2(A) Representative images of CD133 and LGR5 immunofluorescence in CCSC sphere cells bearing a Notch-GFP reporter. Scale bar, 60 μm. (B) Representative images of CD133 and LGR5 immunofluorescence in dividing CCSCs carrying a Notch-GFP reporter. Scale bar, 10 μm. (C) Representative time-lapse images of a Notch-GFP reporter during asymmetric division of CCSCs. Scale bar, 30 μm.(G) t-SNE (up) and Violin plot (bottom) showing the expression of representative marker genes in each cluster.
Figure 3(A) Representative immunofluorescence images showing different cell clusters in CCSC spheres. Single CCSCs with high-Notch-activity (Notch-GFP) were seeded to grow into spheres and immunofluorescence was performed to identify cell clusters using representative marker genes. Scale bar, 60 μm. (B) Pair-cell assay showing the generation of the five cell clusters by CCSC asymmetric division. Scale bar, 60 μm.(C) Edu incorporation assay showing the proliferative potential of cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells in CCSC spheres. Scale bar, 60 μm. (D) Pair-cell assay showing that cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells immediately enter the second division. Scale bar, 100 μm.(E and F) Ki67 staining showing the proliferative potential of cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells in xenografts (E) and clinical CRC tissues (F). Scale bar, 60 μm. Quantification of the immunostaining for (F) shown on the right. Error bars represent SEM of five fields in each section. P-value was calculated based on two-tailed Student's t-test. ns (p>0.05), no significant.
Figure 4(A and B) RT-qPCR showing the enrichment of cluster C3 subpopulation (CFAP54+) in CCSC1 (A) and CCSC2 (B) spheres treated with the chemotherapeutic agent 5FU or L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on two-tailed Student's t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ns, no significant.(C) Representative RNA-FISH images and quantification showing the level of cluster C3 subpopulation CFAP54+ cell (red) levels in L-OHP treated xenografts. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. *, p<0.05.(E) Fluorescent images and quantification showing AP20187-mediated ablation of cluster C4 (PLAUR+) cells from CCSC spheres carrying PLAUR-iCaspase9-mCherry construct. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. ***, p<0.001.(I) Representative H&E images and quantification (number of mice with invasive tumors) showing the invasive potential of cluster C4 cells (PLAUR+). CCSC sphere cells carrying PLAUR-iCaspase9-mCherry construct were orthotopically injected into the mice. AP20187 was applied to ablate cluster C4 (PLAUR+, red) cells. Red arrows indicate invasive tumor. Scale bars, left, 100 μm; right, 300 μm.(K) Left: Representative H&E images of liver tissue sections and quantification (number of mice with liver metastasis) showing the metastatic potential of cluster C4 cells (PLAUR+). AP20187 was applied to ablate cluster C4. Yellow arrows indicate liver metastasis. Scale bars, left, 2 mm; right, 50 μm. Right: Quantification of liver metastasis in mice with intrasplenic injection. Error bars denote mean ± SEM of three mice per group. P-value was calculated based on Student's t-test. ***, p<0.001.
Figure 5(C) RT-qPCR analysis of the signature genes of each cluster in CCSC sphere cells with control (EV) or ATF6 knockdown (shATF6-1 and shATF6-2). Error bars represent SD of three biological replicates. P-value was calculated based on Student's t-test. *, p<0.05; **, P<0.01. Diagram without asterisk indicates no statistical difference.(D) RT-qPCR analysis of signature genes of each cluster in control (EV) or ATF6 knockdown (shATF6-1 and shATF6-2) under the treatment with L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on Student's t-test. ***, p<0.001. Diagram without asterisk indicates no statistical difference.(F) Representative RNA-FISH of cluster C3 signature gene, CFAP54 (up) and immunohistochemistry of transcription factor ATF6 (bottom) showing the influence of ATF6 knockdown (shATF6-1 and shATF6-2) on cluster C3 (CFAP54+, red) population in xenografts. The mice were treated with vehicle or L-OHP after 14 days after CCSC sphere cells were implanted. Scale bars, upper, 40 μm; lower, 80 μm.(H) RT-qPCR analysis of signature genes of each cell clusters in CCSC sphere cells with control (EV) or FOXQ1 knockdown (shFOXQ1-1 and shFOXQ1-2). Error bars denote the SD between triplicates. P-value was calculated based on Student's t test. *, p<0.05; **, p<0.01; ***, p<0.001.(I) Representative H&E and immunofluorescence images showing the influence of FOXQ1 knockdown (shFOXQ1-1 and shFOXQ1-2) on cluster C4 (PLAUR+, red) population and tumor invasion. CCSC sphere cells carrying control (EV) vector or FOXQ1 shRNAs were orthotopically injected into mice (n=5). Scale bars, left, 300 μm; right, 100 μm. Arrows indicate the location of tumor invasion.
Figure 6(G) Representative immunohistochemistry images for the expression of PLAUR (cluster C4 maker gene) in primary CRC tumors and liver metastasis from CRC patients. Scale bar, 200 μm.(H and I) Representative immunohistochemistry images for ATF6 expression (H) and RNA-FISH for CFAP54 expression (I) in paired CRC tissues collected from CRC patients before chemotherapy or after chemotherapy. Scale bars, left, 200 μm; right, 40 μm. Data represent the mean ± SEM. Three to five technical replicates were analyzed. P-value was calculated based on two-tailed Student's t test. *, p<0.05; **, p<0.01; ***, p<0.001.(J and K) Kaplan-Meier survival curves for CRC patients (n=326) with different proportions of cluster C4 (J) and cluster C3 (K). P value was calculated based on log-rank test.
Figure 1C Time-lapse photographs of typical patterns obtained for bacteria alone and bacteria with phage, with corresponding model simulations. Scale bar represents 1 cm.F Visualization of infected region. E. coli FM15 was inoculated at the center of a semi-solid agar plate and the reporter phage M13-sfGFP was inoculated 1-cm away from the center. Fluorescence images were captured after overnight incubation using FITC channel and an exposure time of 200 ms. Scale bar represents 1 cm. G Relative fluorescence intensity at the center and edge positions of the infection zone as shown in (F). The relative intensity was obtained by dividing the detected values with the maximum value of green fluorescence intensity on the plate. Data represent mean values ± s.d. for three values of the center and six values of the edge from three biological replicates. Two-tailed t-test was used to compare two groups. ***, p = 0.0005.
Figure 2B Microscopic images of E. coli FM15 cells carrying plasmids with gfp gene downstream of the psp promoter with or without wild-type M13 phage infection. Scale bar: 1 μm.D Photographs of a quarter of the semi-solid agar plate of a SPACE experiment using high-copy accessory plasmid to improve within-cell activity of T7 RNAP on a synthetic promoter 1D8. An E. coli FM15 strain carrying no MP was used as control. Scale bar: 1 cm.E Activity in cells of T7 RNAP and its mutants from the SPACE experiment in (D). Two clones of the ancestor and five clones of the mutants obtained from the evolution towards 1D8 recognition were measured by in vivo transcriptional assay Two-tailed t-test was used to compare the two groups. ***, p=0.0001. Data represent mean values ± s.d..
Figure 3B Microscopic images of E. coli FM15 cells with or without phage infection. Scale bar represents 1 μm. C The experimental result shows raw photographs of a representative two-phage competition 20 h after initial inoculation with 105:1 mixture of M13w: M13s at 1 cm away from the center. The simulation result is the outcome of competition between two phages PW and PS with relative production rates of 40 and 100, respectively. Scale bar represents 1 cm.D Phage titer ratio of M13s to M13w along the central line and the edge of the fan-shaped infection zone in the experimental result in (C). Samples were collected from different positions as shown in the schematic and phage titer was quantified by qPCR Data represent mean values ± s.d. for three independent assays. Two-tailed t-test was used to compare two groups. *, p = 0.028.
Figure 4C Fold change in the relative expression activity of the evolved RNAP mutants as compared with the wild-type T7 RNAP, for the 10 selected promoter sequences corresponding to the images in (B). Results were obtained by in vitro transcriptional assay Data represent mean values ± s.d. for three biologically independent assays.D Expression activity of 10 RNAP mutants on T7 promoter and 10 promoter variants measured by in vivo transcriptional assay. Mutants are arranged in the same order as their original target promoters listed on the left side of the heatmap. Data are normalized so that the activity of wild-type RNAP on the T7 promoter is 100; the mean for three biologically independent replicates is shown
Figure 1(B) Proteasome inhibitor treatment (10 μM MG132 for 1 to 8 h) promotes accumulation of a ~110KD fragment (asterisk) in HEK293A cells.(C) The ~110KD fragment (asterisk) is related to AMOT. Wild-type (WT) and AMOT knockout (KO) HEK293A cells were treated with 10 μM proteasome inhibitor bortezomib (BTZ) for 6 h and subjected to Western blotting analysis using the indicated antibodies.(F and G) Serum and LPA treatments induce AMOT degradation and promote accumulation of a ~110 kDa protein (asterisk) in HEK293A cells. Cells were serum-starved overnight and then treated with 10% FBS or 1 μM LPA for the indicated time. The phosphorylation levels of YAP proteins were detected by Phos-tag gel and pYAP immunoblotting. Protein quantification is shown below. Data represent mean ± SEM from three independent experiments. L.E. indicates long exposure.
Figure 2C) Nelfinavir (NFV) and Lopinavir (LPV) block AMOT cleavage. HEK293A cells were pre-treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h. Cell lysates were then subjected to Western blotting.(F) Deletion of DDI2 blocks AMOT cleavage. Wild-type (WT) and DDI2 KO cells were serum-starved overnight and then treated with 1 μM LPA for 2 h.(G) Both DDI1 and DDI2 mediate AMOT cleavage. Ectopically expressed FLAG-tagged DDI1 or DDI2 rescues AMOT cleavage in DDI2 KO HEK293A cells.K) The aspartic catalytic-site mutant DDI2 exhibits a dominant negative effect through inactivation of wild-type DDI2 in HEK293A cells. Wild-type HEK293A cells were transfected with the indicated plasmids and treated with 1 μM LPA (K) before harvest.
Figure 3(A) Ubiquitination is required for AMOT cleavage. HEK293A cells were treated with 100 nM E1 inhibitor MLN7243 overnight followed by 10 μM BTZ for 6 h. Cell lysates were then subjected to Western blotting.(B and C) The TNKS1/2-RNF146 axis is crucial for AMOT cleavage. Wild-type (WT), TNKS1/2 double KO (dKO) and RNF146 KO HEK293A cells were treated with the proteasome inhibitor MG132 for 6 h before harvest. Cell lysates were subjected to Western blotting analysis using the indicated antibodies.(H and I) AMOT cleavage states in different cell lines with NF2-wild-type or NF2-deficiency.(K) Serum, LPA, and S1P stimulate PARylation and ubiquitination of AMOT. Serum-starved cells were treated with 10% FBS, 1 μM LPA, or 1 μM S1P for 4 h and subjected to an immunoprecipitation (IP) assay.
Figure 4(C) Nelfinavir and Lopinavir block AMOT cleavage in HUVECs. HUVECs were pre-treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h. Cell lysates were subjected to Western blotting. Protein quantification is shown below. (D) Aspartic protease DDI2 is responsible for AMOT cleavage in HUVECs. HUVECs were transfected with two different small interfering RNA (siRNA) and 48 h post-transfection, the cells were treated with 10 μM BTZ for 6 h. Cell lysates were subjected to Western blotting. Protein quantification is shown below.(F RNF146 are indispensable for AMOT cleavage in HUVECs. siRNAs were transfected into HUVECs, the cells were treated with 10 μM BTZ for 6 h before harvest. Protein quantifications are shown below.(H) Representative immunofluorescence images of HUVEC spheroids 18 h after VEGF stimulation. Above: inverted contrast images. Areas within the red dash box indicate region of interest (ROI) and are shown below at higher magnification. Plasma membranes were stained with CellMask (red), actin filaments with Phalloidin (green), and nucleus with DAPI (blue). Scale bar 100 μm. HUVECs infected with empty vector virus were used as control.(I and J) Quantification of the number of sprouts per spheroid and the cumulative sprout length of HUVECs. Vector (n=20), AMOT-WT (n=18), AMOT-ΔTBD (n=14), AMOT-Δcut (n=15), AMOTp80 (n=16), AMOT-NT (n=17) and AMOT-CT (n=21).
Figure 5(A and B) Knockdown of AMOT induces vascular development defects in zebrafish embryos. Lateral views of ISV and DLAV formation in Tg(flk:GFP) control and amot MO-injected zebrafish embryos at 30 h post-fertilization (hpf) are shown. Histogram depicts the quantification of normal and angiogenesis-defective embryos at 30 hpf (B). n: number of embryos analyzed. Dorsolateral anastomose vessels (DLAV), intersegmental vessels (ISV), dorsal aorta (DA) and posterior cardinal vein (PCV) are shown. Asterisks: defective ISV; dotted lines: dorsal edge of the embryo. Scale bar: 50 μm.(E and F) Non-cleavable AMOT mutant fails to support angiogenesis. Embryos were injected with amot MO together with mRNA encoding wild-type (WT) or mutant (ΔTBD, NT, CT, and Δcut) AMOT. Scale bar: 50 μm.(G and H) AMOT-CT but not full-length AMOT could rescue impaired angiogenesis in ddi2-deficient embryos. Embryos were injected with ddi2 MO together with mRNA encoding wild-type AMOT (AMOT-WT) or AMOT-CT. Scale bar: 50 μm. (I and J) Nelfinavir (NFV) and XAV939 repress ISV development. Embryos were treated with 5 μM NFV, 10 μM XAV939 at 6 hpf for 24 h. Number of ISV defects was counted at 30 hpf. Scale bar: 50 μm.
Figure 6(G) High magnification confocal images of filopodia extension at the leading edge of the retinal vascular in control and ΔcutiEC-TG mice. Scale bar: 10 μm. (H) Quantification of the number and average length of filopodia in control and ΔcutiEC-TG P6 retinal sprouts. Each data point represents the average of three measurements from one retina, control (n=7) and ΔcutiEC-TG (n=7).(L) Δcut KI mouse retinal angiogenesis shows sprouting defects in the sprouting front border. Immunofluorescent images of P7 whole-mount retinas stained for IB4 and ERG. White dash rectangles indicate regions of interest (ROI) are shown below. Arrow heads: blunt-ended tip ECs. Scale bar: 100 μm. (M) Quantification of the retinal vasculature of control and Δcut KI mice at P7. Each data point represents the average of two or three measurements from one retina, control (n=8) and Δcut KI (n=8).(N) High magnification confocal images of filopodia extension at the leading edge of the retinal vascular in control and Δcut KI mice. Tip cells are labeled with IB4. Scale bar: 10 μm. (O) Quantification of the number and average length of filopodia in control and Δcut KI P7 retinal sprouts. Each data point represents the average of three measurements from one retina, control (n=8) and Δcut KI (n=8).
Figure 7(D) Ddi2iEC-KO mice show delayed retinal angiogenesis. Immunofluorescent images of whole-mount retinas stained for IB4 and ERG of control, Ddi2iEC-KO and Ddi2iEC-KO; CTiEC-TG mice at P6. White dash rectangles indicate regions of interest (ROI) are shown below at higher magnification. Scale bar: 100 μm.(F) High magnification confocal images of filopodia extension at the leading edge of the P6 retina. Tip cells are labeled with IB4. Scale bar: 10 μm. (G) Quantification of the number and average length of filopodia in control (n=8), Ddi2iEC-KO (n=9) and Ddi2iEC-KO; CTiEC-TG (n=8) P6 retinal sprouts. Each data point represents the average of three measurements from one retina.(H and I) Representative images and comparisons of Ki67 positive endothelial cells in control (n=8), Ddi2iEC-KO (n=8) and Ddi2iEC-KO; CTiEC-TG (n=7) P6 retinas. Each data point represents the average of three measurements from one retina. Scale bar: 50 μm.
Figure 8(D) Nf2iEC-KO retinas show endothelial cell accumulation in the sprouting front and sparse vascular plexus in the region close to optic stalk. CT overexpression can partially rescue the phenotype. Immunofluorescent images of whole-mount P6 retinas stained for IB4 and ERG are shown. White rectangles indicate regions of interest (ROI). Scale bar: 100 μm.(F and G) High magnification confocal images and quantification of filopodia extension at the leading edge of the P6 retinas. Tip cells are labeled with IB4. Scale bar: 10 μm. Control (n=10), Nf2iEC-KO (n=10), Nf2iEC-KO; CTiEC-TG (n=10) and Nf2iEC-KO; ΔcutiEC-TG (n=8).(H and I) Representative images and comparisons of Ki67 positive endothelial cells in control (n=8), Nf2iEC-KO (n=8), Nf2iEC-KO; CTiEC-TG (n=8) and Nf2iEC-KO; ΔcutiEC-TG (n=7) P6 retinas. Each data point represents the average of three measurements from one retina. Scale bar: 50 μm.
Figure 9(B) CT overexpression promotes revascularization in the OIR model. Upper, IB4 staining of the whole retina of P15 control and CTiEC-TG mice after OIR. Lower, higher magnification images of sprouting vessels from veins. Scale bars are shown in the figures.(D) Blocking AMOT cleavage inhibits revascularization in the OIR model. Upper, IB4 staining of the whole retina of P17 WT and Δcut KI mice after OIR. Lower, higher magnification images of sprouting vessels from veins. Scale bars are shown in the figures.
Figure 1A. Levels of ROW protein in rowRNAi lines were decreased in both male and female fly heads. Western blot for the two rowRNAi lines compared to controls was performed using rat polyclonal αROW and rat αTUBULIN (TUB). Genotype description: rowRNAi-1(Act-GAL4/+; UAS-rowRNAi-1/+), rowRNAi-2(Act-GAL4/+; UAS-rowRNAi-2/+), rowRNAi-1 Control (CyO/+; UAS-rowRNAi-1/+), rowRNAi-2 Control (CyO/+; UAS- rowRNAi-2) and Act-GAL4 Control (Act-GAL4 /+).B. Decrease in the viability of rowRNAi lines. To examine the viability of rowRNAi flies, we counted the offspring generated from the cross between the heterozygous Act-GAL4/CyO driver line with the homozygotes rowRNAi, or WT (w1118) flies as control. Values are the percentages from the total progeny ± standard error of the mean (SEM).C. Decrease in pupal eclosion of rowRNAi line. Pupae were collected from the cross between the heterozygous Act-GAL4/CyO driver line with the homozygotes rowRNAi-1 or WT (w1118) flies as control. Values are the percentages for each genotype of pupal eclosion ± SEM.D. Survival curve for rowRNAi flies showing reduced life span compared to controls. n=3 (biological replicates). Significance was tested using the Kaplan-Meier estimate of survival and log-rank test.E. Crossing of females/males rowRNAi flies with WT males/females (w1118) flies resulted in a strong reduction in offspring number. Values are the number of offspring from crosses between WT (w1118) flies and flies with different genotypes.
Figure 3A. Western blot for flies expressing endogenous FLAG-tagged ROW (ROW-FLAG), with αFlag tag antibody to validate the tagging. αTubulin (TUB) antibody was used as a reference. Het, Heterozygotes; Hom, Homozygotes for tagged ROW.D. BEAF-32 coimmunoprecipitate with ROW. Lysates from S2 cells not transfected (-) or transfected (+) with ROW-FLAG tagged plasmid were subjected to immunoprecipitation with αFlag tag beads. The immunoprecipitates and input (5%) were analyzed by western blot with αFlag tag and αBEAF-32 antibodies.
Figure 5A. Central enrichment of AT-rich sequence in ROW binding regions. The logo shows the most significant enriched sequence (based on MEME-ChIP analysis). The plot shows the motif's probability relative to the ROW ChIP-seq peaks (calculated with centriMo).B. On the top are the results of DNA affinity pull-down followed by western blot with ROW antibody. None is a pull-down with no probe, AT-rich is a pull-down with a biotin-labeled AT-rich dsDNA probe, and control is a pull-down with a probe composed of 49% A/T bases. On the bottom is the stain-free gel as a loading control.D. ChIP-qPCR results using FLAG-tag antibody for S2 cells transfected with WT or AT-hook mutant ROW-FLAG tagged plasmids (n = 3, technical and biological replicates). As a control, cells not transfected with ROW-FLAG tagged plasmid were used. Input percentages for 3 biological replicates are shown for three promoters containing AT-rich sequences (Hcf, Phax, and Hcs).E. Co-IP results using αFlag antibody in S2 cells transfected with WT or AT-hook mutant ROW-FLAG tagged plasmids or with a plasmid containing only the ZNF domains of ROW (with FLAG-tag). As a control, cells not transfected with ROW-FLAG tagged plasmid were used. Western blot using αFLAG, αWOC, αBEAF-32, αHP1c, and αTUB (loading control) antibodies is shown for input and IP samples.