EMBO/SourceData huggingface dataset:
Figure 2(A) TIRF images of fixed EL4-MyD88-GFP cells off grids and stained with antibodies against K63-Ub, M1-Ub, pIKK and pp65. Cells were activated on IL-1 functionalized SLBs for 30 min before fixation. Scale bar, 5 µm. (B) A magnified view of the large patch-like Myddosome clusters from the highlighted region of interest in panel A (yellow box on merge images). Scale bar, 1 µm.(C) Structured illumination microscopy images of Myddosome clusters stained with anti-pIKK. Top row right, insets show detail of Myddosome staining with anti-pIKK. Inset taken from regions of interest overlaid the merge image (yellow boxes 1 and 2). Bottom row, x-z view slice taken from yellow line overlaid on the merge image (top row). Myddosome and pIKK staining localize the cell-SLB interface. Blue dashed line defines the nucleus volume determined from the DAPI stain. Scale bar in main image and Z projection, 1 µm; scale bar inset, 0.5 µm.
Figure 3(A and B) Top, TIRF images of fixed EL4-MyD88-GFP cells incubated with IL-1 functionalized SLBs for 30 minutes, and stained with antibodies against pp65 (A) or pIKK (B). Scale bar, 5 µm. Region of interest (red box, merge image) shows an example of MyD88-GFP puncta that colocalizes with pp65 (A) or pIKK (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated pp65 (A) or pIKK (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms). Blue shaded regions on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88-GFP channel and associated pp65 (A) and pIKK (B) channel (pp65/pIKK images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88-GFP image (numbered red arrows). Scale bar, 1 µm.(E and F) 2D histogram of MyD88-GFP puncta intensity and associated pp65 (E) or pIKK (F) staining intensity on 2.5 µm and 1 µm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms).(G and H) Quantification of mean pp65 (G) or pIKK (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 µm grids. The normalized mean intensity for clusters, single Myddosomes MyD88 puncta on 2.5 and 1 µm grids are the following: for pp65 0.318 ± 0.044, 0.163 ± 0.009, 0.059 ± 0.005 and 0.057 ± 0.008; for pIKK 0.393 ± 0.051, 0.130 ± 0.004, 0.037 ± 0.006 and 0.035 ± 0.007 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. p values are *<0.05, ***<0.001, ****<0.0001. Bars represent mean ± SEM (n = 3 - 4 biological replicates for pp65, with 10273, 14009 and 2657 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates; n = 4 - 5 biological replicates for pIKK, with 2375, 35496 and 59593 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates). Statistical significance is determined using unpaired two-tailed Student's t-test.
Figure 4(A and B) Top, TIRF images of fixed EL4-MyD88-GFP cells incubated with IL-1 functionalized SLBs for 30 minutes, and stained with anti-K63-Ub (A) or anti-M1-Ub (B). Scale bar, 5 µm. Region of interest (red box, merge image) shows an example of MyD88-GFP puncta that is colocalized with K63-Ub (A) or M1-Ub (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (A) or M1-Ub (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms). Blue shaded region on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88-GFP channel and associated K63-Ub (A) and M1-Ub (B) channel (K63/M1-Ub images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88-GFP image (numbered red arrows). Scale bar, 1 µm.(E and F) 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (E) or M1-Ub (F) staining intensity on 2.5 µm and 1 µm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient of linear fit labeled on 2D histograms).(G and H) Quantification of mean K63-Ub (G) or M1-Ub (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 µm grids. The normalized mean intensity for clustered, single Myddosomes, and MyD88 puncta on 2.5 and 1 µm grids are the following: for K63-Ub 0.444 ± 0.030, 0.257 ± 0.025, 0.113 ± 0.015 and 0.104 ± 0.008; for M1-Ub 0.520 ± 0.020, 0.183 ± 0.008, 0.174 ± 0.022 and 0.153 ± 0.016 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. p values are **<0.01, ***<0.001. Bars represent mean ± SEM (n = 3 - 4 biological replicates for K63, with 14571, 27494 and 24026 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates; n = 3 - 4 biological replicates for M1, with 3114, 6091 and 1844 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates). Statistical significance is determined using unpaired two-tailed Student's t-test.
Figure 5(A and B) Top: TIRF images of MyD88-GFP and mScarlet-TRAF6 (A) or mScarlet-HOIL1 (B). Region of interest (yellow box, merge image) shows an example of a MyD88-GFP puncta colocalized with mScarlet-TRAF6 (A) or mScalet-HOIL1 (B). Bottom: Time-series TIRF images from the region of interest (left) and fluorescence intensity times series (right) of MyD88 and TRAF6 (A) or HOIL1 (B).(C and D) Quantifications of percentage of MyD88-GFP puncta that colocalized with TRAF6 (C) or HOIL1 (D) grouped for all puncta, puncta containing ≤1x, >1x and ≥2x Myddosome complexes. The percentages for TRAF6 (C) in these groups are 15.4 ± 2.4%, 6.1 ± 1.8%, 36.2 ± 5.2% and 58.4 ± 5.9%, respectively (mean ± SEM). The percentages for HOIL1 (D) in these groups are 8.6 ± 0.5%, 0.4 ± 0.2%, 14.9 ± 1.9% and 25.9 ± 5.0%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 6 biological replicates for TRAF6, with 191 cells measured in total across all replicates,
Figure 6(A and B) TIRF images of EL4 cells expressing MyD88-GFP and mScarlet-TRAF6 stimulated on IL-1 functionalized SLBs at a ligand density of 1 IL-1/µm2 off grids (A) or on 1 µm grids (B). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 µm. (C) Quantification of percentage of MyD88-GFP puncta that colocalized with TRAF6 off grids and on 1 µm grids at a ligand density of 1 IL-1/µm2, and the percentages are 16.2 ± 2.5% and 6.1 ± 1.5%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 4 experimental replicates off grids, with a total of 24315 MyD88 puncta from 91 cells; n = 3 biological replicates on 1 µm grids, with a total of 23161 MyD88 puncta from 70 cells). Statistical significance is determined using unpaired two-tailed Student's t-test.
Figure 7(A-B) Histogram showing the lifetime of TRAF6 and HOIL1 recruitment to single Myddsomes, and clusters containing 2-4 and 5-7 Myddosome complexes, off (blue) and on (red) 1 µm grids.
Figure 1A A heatmap showing the differential miRNAs expression as determined by miRNA-seq in progerin-expressing cells relative to control cells, P< 0.05. Lowly expressed miRNAs were shown in blue, highly expressed ones in red.B Volcano Plot of top 10 miRNAs upregulated and 2 miRNAs downregulated in HGPS cells compared to control.C The level of top 10 upregulated miRNAs in HGPS cells was assessed by qPCR.D The level of novel-miR-27, miR-3656 and miR-59 in HGPS patient cells (HGAFDFN003 p21 and HGAFDFN167 p19) versus control (CRL-1474 cells p28) was assessed by qPCR.HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days, cells were subjected to SA-β-gal staining. HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days, The percentage of the Ki67 positive cells was calculated by immunofluorescence (F).HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days The level of cyclin A2 and lamin B1 was detected by western blot (G).Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The percentage of SA-β-gal positive cells was calculated. Scale bars: 100 μm (H).Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The percentage of the Ki67 positive cells was calculated by immunofluorescence (I).Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The level of cyclin A2 and lamin B1 was detected by western blot (J).K The level of miR-59 was detected in 88- and 92-year-old cells versus 7-year-old cells was assessed by qPCR.L 7-year-old cells and 88-year-old cells were transfected with anti-miR-ctrl or anti-miR-59, the level of cyclin A2 and lamin B1 was detected.
Figure 2HGAFDFN003 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days, the mRNA level of miR-59-targeting potential genes was assessed by qPCR (B).HGAFDFN003 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days, The protein expression of HMGA1 and HMGA2 was detected by western blot (C).D, After transfection with anti-miR-ctrl or anti-miR-59 in progerin-expressing CRL-1474 cells, the protein expression (D) of HMGAs were detected.After transfection with anti-miR-ctrl or anti-miR-59 in progerin-expressing CRL-1474 cells, mRNA level (E) of HMGAs were detected.Western blot analysis the expression of HMGAs (F) in miR-ctrl or miR-59 mimics-transfected CRL-1474 cells.qPCR analysis the mRNA levels (G) in miR-ctrl or miR-59 mimics-transfected CRL-1474 cells.Compared HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL-1474 p26), the protein expression (H) of HMGAs were assessed.Compared HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL-1474 p26), mRNA level (I) of HMGAs were assessed.The protein (J) level of HMGAs were detected in 7, 88 and 92-year-old cells.mRNA (K) level of HMGAs were detected in 7, 88 and 92-year-old cells.Transfected anti-miR-ctrl or anti-miR-59 in 88-year-old cells, the level of HMGAs was detected by Western blot (L)Transfected anti-miR-ctrl or anti-miR-59 in 88-year-old cells, the level of HMGAs was detected by qPCR (M).N, O The binding sites of miR-59 on HMGAs 3′ UTR were analyzed by TargetScan and miRanda. Luciferase experiments with the wild-type and the mutated 3′ UTR of HMGA1 (N) / HMGA2 (O).
Figure 3In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), the level of cyclin A2, lamin B1 was detected by western blot (A).In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), SA-β-gal staining were performed, the percentage of SA-β-gal positive cells was calculated.In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), Expression of Ki67 was analyzed by immunofluorescence. Scale bars: 50 μm (C).Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. SA-β-gal staining was performed. The percentage of SA-β-gal positive cells was calculated. Scale bars: 100 μm (D).Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. The level of cyclin A2, lamin B1 was detected by western blot (E).Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti-miR-59-transfected HGPS cells (HGAFDFN003), cells were subjected to SA-β-gal staining. Scale bars: 100 μm (F). Cells were subjected to immunofluorescence using anti-Ki67(red) antibody (left). The percentage of the Ki67 positive cells was calculated. Scale bars: 50 μm (G).Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti-miR-59-transfected HGPS cells (HGAFDFN003) The level of cyclin A2, lamin B1, HMGA1 and HMGA2 was detected by western blot (H).
Figure 4A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA1 antibody in H1299 cells. Pulled-down protein complexes were analyzed. Benzonase (B) was added in cell lysate. B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA2 antibody in H1299 cells. Pulled-down protein complexes were analyzed.D Interaction of endogenous CDK7 with HMGAs, XPB and TFIIH4 was analyzed by Co-IP assay using anti-CDK7 antibody in H1299 cells. Pulled-down protein complexes were analyzed.HEK293T cells were transfected with Flag-HMGA1 (E) HMGA1 was IP with anti-Flag antibody and anti-CDK7 antibody.HEK293T cells were transfected with CDK7 (F). HMGA1 or CDK7 was IP with anti-Flag antibody and anti-CDK7 antibody.G HEK293T cell lysates were incubated with GST or GST-HMGA1 sepharose beads. Pulled-down protein complexes were analyzed.I HEK293T cells were transfected with Flag-HMGA1 deletion mutants. IP of Flag was performed. Pulled-down protein complexes were analyzed.J HEK293T cell lysates were incubated with GST or GST-HMGA1 deletion mutants sepharose beads. Recombinant proteins from pull-down assays visualized by silver staining and western blot.K, L Progerin-expressing H1299 cells were transfected with His-control or His-HMGA1. Immunoprecipitation of RNA Pol II (K) or CDK7 (L) with anti-RNAPII antibody or anti-CDK7 antibody was performed, respectively. Pulled-down protein complexes were analyzed.RNAPII, the RNAPII Ser2P and RNAPII Ser5P in HGAFDFN003 cells (M) compared to CRL-1474 cells were detected by western blot.RNAPII, the RNAPII Ser2P and RNAPII Ser5P in 88, 92-year-old cells (N) compared to CRL-1474 cells were detected by western blot.O Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. Indicated proteins and modifications were detected by western blot.P Infection with siCtrl or siCDK7 in Flag-HMGA1-transfected HGPS cells (HGAFDFN003), cyclin A1, RNAPII, the RNAPII Ser2P and RNAPII Ser5P was detected.Q Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti-miR-59-transfected HGAFDFN003 cells. Indicated proteins and modifications were detected by western blot.
Figure 5The mRNA level of cell cycle genes in HGPS patient cells (HGAFDFN003 and HGAFDFN167) (D) was assessed by qPCR.The mRNA level of cell cycle genes in HGPS and 88- and 92-year-old cells (E) was assessed by qPCR.F HGAFDFN003 cells were infected with His-HMGA1 and the mRNA level of cell cycle genes was assessed by qPCR.G Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti-miR-59-transfected HGAFDFN003 cells. The mRNA level of cell cycle genes was assessed by qPCR.
Figure 6A miR-59 was detected in heart, liver, lung, kidney, skin, muscle, and brain of LmnaG609G/G609G mice by qPCR at 12 weeks of life (n=5 for each group).B, C The mRNA level of Hmga1 (B) and Hmga2 (C) was detected in heart, liver, lung, kidney, skin, muscle and brain of LmnaG609G/G609G mice by qPCR at 12 weeks of life (n=5 for each group).D Transfected anti-miR-Ctrl or anti-miR-59 in progerin-expressing NIH3T3 cells. Western blot analysis of HMGAs.G, H Luciferase experiments with the wild-type and the mutated 3′ UTR of Hmga1 and Hmga2.
Figure 7A The in vivo therapy scheme. Eight-day-old mice were injected intraperitoneally with AAV9-mCherry-anti-miR-59 (5x1011 viral particles). The mCherry signals were detected 30 days post-injection (DPI) in LmnaG609G/G609G mice versus PBS-injected control (dorsal).B Expression of the mCherry reporter in different organs at 30DPI in LmnaG609G/G609G mice versus PBS-injected control. Hrt, Mscl, Ver, Kid, Liv: heart, Muscle, vertebrae, kidney, liver. AAV9-mCherry-anti-miR-59 was injected into 8-day-old mice and detected at different points.C, D The gross morphology of the wild-type and LmnaG609G/G609G mice which injected with anti-ctrl or anti-miR-59 for 14 weeks.E Progression of body weight of male mice transduced with anti-Ctrl versus anti-miR-59 was detected from 4 weeks of life. The sample size (n) indicates the initial number of mice at week 4, initial n=10 WT; n=9 anti-Ctrl-transduced mice; n=7 anti-miR-59-transduced mice. Shown are mean values ± standard deviations, two-sided t-test was used when comparing anti-Ctrl-transduced mice and anti-miR-59-transduced mice from week 6-16.F Body weight comparison at 7- and 11-week-old male mice (n=9 anti-Ctrl-transduced mice; n=7 anti-miR-59-transduced mice). Data are presented as the mean ± SD (*P<0.05, **P<0.01). P-values were calculated by unpaired Student's t-test.G Progression of body weight of female mice transduced with anti-Ctrl versus anti-miR-59 was detected from four weeks of life. The sample size (n) indicates the initial number of mice at week 4, initial n=5 WT; n=6 anti-Ctrl-transduced mice; n=6 anti-miR-59-transduced mice. Shown are mean values ± standard deviations, two-sided t-test was used when comparing anti-Ctrl-transduced mice and anti-miR-59-transduced mice from week 6-16.H Body weight comparison at 7- and 11-week-old female mice (n=6 for each group). Data are presented as the mean ± SD (*P<0.05). P-values were calculated by unpaired Student's t-test.I Kaplan-Meier survival plot of the wild-type and LmnaG609G/G609G mice which injected with anti-Ctrl or anti-miR-59 (n=12 WT; n=13 anti-Ctrl-transduced mice; n=13 anti-miR-59-transduced mice, two-sided log-rank test).
Figure 8A Running ability of male mice transduced with anti-Ctrl versus anti-miR-59 at 12 weeks of life (n=5 for each group).Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle (B) The boxes and arrows denoted the aortic adventitia.Masson staining showed the adventitial fibrosis (blue areas) and the adventitial width of aorta (C). The boxes and arrows denoted the aortic adventitia.D H&E staining in skin showed the epidermis and fat layer of skin. Scale bars, 150 μm. The boxes and arrows denoted epidermis and vertical bars indicated fat layer.E The level of miR-59 was detected in heart, liver, lung, kidney, skin, and muscle of LmnaG609G/G609G mice (n=5 for each group). F, G The mRNA level of Hmga1 (F) and Hmga2 (G) was detected in heart, liver, lung, kidney, skin, and muscle of LmnaG609G/G609G mice (n=5 for each group). H Hmgas immunohistochemistry in skin of WT mice and LmnaG609G/G609G mice. Scale bars, 50 μm. D
Figure 1B Plot of the whole-cell current (Vhold -65 mV) in control conditions (ctrl) and after application of NASPM (NSP, p<0.0001, n=18).
Figure 2B Current-voltage relationship of the tonic Ba2+-sensitive (100 µM) outward GIRK current demonstrating reversal near expected EK and inward rectification (n=6 biological replicates).
Figure 3A With GDPβS-containing internal solution, noradrenaline-induced GluD1R current (INA) was diminished by >20 minutes post-dialysis, shown in a representative trace.C Plot of the magnitude of tonic GluD1R current measured after dialysis with GTP + LiCl-, GDPβS-Li3-, and GTP-containing internal solution, displaying no significant difference between the groups (p=0.46, n=9, 13, 8 biological replicates respectively).
Figure 4B-C Plot of the amplitude of the α1-AR-EPSC in control conditions (ctrl) and in glycine (1 mM, p=0.43, n=10 biological replicates, B), or D-serine (1 mM, p=0.13, n=9 biological replicates, C).E Glycine had no significant effect on the amplitude of the α1-AR-EPSC (arrow) or whole-cell current, shown in a representative trace.
Figure 5A Increasing extracellular calcium to 4.8 mM reduced the amplitude of the α1-AR-EPSC, shown in a time-course plot (n=18 and 26 biological replicates for 2.4 and 4.8 mM respectively).F Plot of the magnitude of tonic GluD1R current measured by application of NASPM in control conditions (1.2 mM), 2.4 mM and 4.8 mM calcium. The magnitude of tonic GluD1R current in 2.4 and 4.8 mM calcium was reduced relative to control conditions (1.2 mM: n=18; 2.4 mM: p=0.03, n=17; 4.8 mM: p=0.005, n=22 biological replicates).J Elevating extracellular calcium to 4.8 mM reduces the change in whole-cell current to NASPM and accelerates recovery of the α1-AR-EPSC from NASPM-block in control conditions (1.2 mM extracellular calcium), shown in a representative whole-cell voltage-clamp recording.
Figure 6A Representative traces of whole-cell current clamp recordings of membrane potential and AP firing evoked by current injection (1.5 s) demonstrating hyperpolarization by NASPM. Dashed line is at -80 mV.C Plot of the membrane potential (Vm) versus injected current relative to rheobase (rb) in control and NASPM, demonstrating that NASPM produced a hyperpolarization at subthreshold potentials (n=10 biological replicates).
Figure 1B: Single-cell growth of drug-treated cells as in A as a function of time since MreB-motion stop according to panel A. Left and middle: Relative increase (left) and single-cell rates (middle) of volume, mass, surface, and length. Gray asterisks indicate single-cell mass at lysis. Right: Relative changes of dry-mass density, surface-to-mass ratio, surface-to-volume ratio, and width.D: Complex nutrient shift of non-dividing cells (S382) growing on agarose pad (MM+0.02% glucose) in the presence of vancomycin (100 μg/mL). Same quantities as in B, as a function of time since MreB-motion stop. Cells were transiently starved for glucose (through addition of 0.5% (w/v) αMG and 0.25% (w/v) 2DG), followed by addition of excess glucose (1%). Different from A-C, all compounds were added to the agarose pad as droplets and reached cells by diffusion.
Figure 2A: Single-cell growth of non-dividing b42 cells (ΔmepS/pBAD30-mepS) grown on LB agarose pads as a function of time after addition of arabinose (0.2%) in form of a droplet. Left: Relative increase of volume, mass, surface, and length. Middle: Elongation rate and surface-growth rate. Right: Relative changes of surface-to-mass ratio and width.
Figure 3B-C: Cell bending: B: Time-lapses of bending filamentous cells (S290) in donut microchambers (3% agarose; RDM+glu) and grown without (top) or with (bottom) D-cycloserine (1 mM) as a function of time since D-cycloserine treatment, starting >10 min after D-cycloserine treatment (the time of MreB-motion stop D-cycloserine-treated cells were osmotically shocked (NaCl) after 33 min of treatment, which releases a fraction of cells C: Change of cell centerline curvature as a function of cell length between 10 min after start of D-cycloserine treatment and release of cells through osmotic shockD-E: Cell straightening. D: Time-lapses of straightening filamentous cells (S290) previously bent in donuts: control (top) and D-cycloserine treated cells (bottom) as a function of time since simultaneous release from donuts and beginning of drug treatment. E: Change of centerline curvature as a function of relative length from 10 min after D-cycloserine treatment (time of MreB-motion stop).
Figure 4C: Relative change of cell width of non-dividing cells (S257) grown in LB during combined D-cycloserine treatment (5 mM) and a hypo-osmotic ramp as in A (DCS + Hypo ramp), as compared to cells exposed to the hypo-osmotic ramp only (Hypo ramp) or treated with D-cycloserine only (DCS). D-cycloserine was contained in both agarose pads, and treatment started 15 min prior to the shock.