Figure 7 ( A ) Immunoblots for FTMT , frataxin , mitoferrin , HSP60 , FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control ( C57BL / 6J ) mice and STAM mice treated with DFP ( 0 , 0 . 0375 , or 0 . 075 mg / g body weight ) ( n = 6 ) . The expression of FTMT , frataxin , mitoferrin , FTH1 , and TfR were normalized to HSP60 or β - actin , respectively . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . ( B ) The mean number of mitophagosome - like structures / 100 μm2 from four randomly selected areas in the livers of DFP ( 0 . 075 mg / g body weight ) - administered mice were compared between control and FTMT siRNA - treated groups for STAM mice ( n = 6 ) and DMBA + HFD mice ( n = 6 ) . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . NC : negative control siRNA , * : P < 0 . 05 . ( C ) Frozen liver sections of DFP ( 0 . 075 mg / g body weight ) - administered mice ( STAM mice and DMBA + HFD ) with or without FTMT knockdown were stained with dihydroethidium . Red fluorescence indicates the ROS production . Scale bar : 100 μm . siNC : negative control siRNA . ( D , E ) STAM mice ( D ) were fed a high - fat diet without DFP treatment or orally administered with 0 . 075 mg / g body weight of DFP from the age of 4 weeks , injected with FTMT siRNA ( 10 μl / g body weight ) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice , and followed until 16 weeks of age ( n = 6 ) . DMBA + HFD mice ( E ) were fed a high - fat diet , treated with DFP and siRNA in the same manner with STAM mice , and followed until 30 weeks of age ( n = 6 ) . The number and maximum size of liver tumors were compared among the three groups ( control without DFP , negative control siRNA , and FTMT siRNA with DFP ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . Representative liver images are shown in the upper panel . Scale bar : 1 cm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Immunoblots for FTMT, frataxin, mitoferrin, HSP60, FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control (C57BL/6J) mice and STAM mice treated with DFP (0, 0.0375, or 0.075 mg/g body weight) (n=6). The expression of FTMT, frataxin, mitoferrin, FTH1, and TfR were normalized to HSP60 or β-actin, respectively. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. (B) The mean number of mitophagosome-like structures/100 μm2 from four randomly selected areas in the livers of DFP (0.075 mg/g body weight)-administered mice were compared between control and FTMT siRNA-treated groups for STAM mice (n=6) and DMBA + HFD mice (n=6). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. NC: negative control siRNA, *: P<0.05. (C) Frozen liver sections of DFP (0.075 mg/g body weight)-administered mice (STAM mice and DMBA + HFD) with or without FTMT knockdown were stained with dihydroethidium. Red fluorescence indicates the ROS production. Scale bar: 100 μm. siNC: negative control siRNA.(D, E) STAM mice (D) were fed a high-fat diet without DFP treatment or orally administered with 0.075 mg/g body weight of DFP from the age of 4 weeks, injected with FTMT siRNA (10 μl/g body weight) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice, and followed until 16 weeks of age (n=6). DMBA + HFD mice (E) were fed a high-fat diet, treated with DFP and siRNA in the same manner with STAM mice, and followed until 30 weeks of age (n=6). The number and maximum size of liver tumors were compared among the three groups (control without DFP, negative control siRNA, and FTMT siRNA with DFP). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. Representative liver images are shown in the upper panel. Scale bar: 1 cm.


Figure 1A . Coomassie Blue stained SDS - PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C - terminally FLAG - tagged Elp1 . B . Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation ( Lane 6 of Fig 1A ) . Circled particles correspond to intact Elongator . White scale bar corresponds to 50 nm . C . Representative 2D class averages of the full Elongator complex from a total of 17 , 074 particles . All class averages shown in this figure have side width of 500 Å . D . Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure . E . Six evenly - sized densities are arranged in one Elongator lobe . A projection of the Elp456 hexamer X - ray structure ( PDB code : 4A8J ) is shown for comparison . F . Analysis of Elp2 localization using a C - terminal MBP tag . Class averages of untagged Elongator is shown for comparison . White arrowheads point towards additional densities in the tagged complex . G . Elp3 localization analysis using a C - terminal MBP tag , denoted by the white arrowhead . H . Mapping of Elp1 by incubating purified Elongator containing an Elp1 C - terminal FLAG tag with α - FLAG antibody . The additional antibody density is denoted by the white arrowhead . I . Three dimensional reconstruction of the intact Elongator complex at 25Å . J . Schematic of the approximate subunit locations based on 2D EM analysis . K . Inset showing the cavity formed through the centre of the putative Elp456 density . Black scale bars in this figure correspond to 50 Å .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Coomassie Blue stained SDS-PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C-terminally FLAG-tagged Elp1.B. Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation (Lane 6 of Fig 1A). Circled particles correspond to intact Elongator. White scale bar corresponds to 50 nm.C. Representative 2D class averages of the full Elongator complex from a total of 17,074 particles. All class averages shown in this figure have side width of 500 Å.D. Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure.E. Six evenly-sized densities are arranged in one Elongator lobe. A projection of the Elp456 hexamer X-ray structure (PDB code: 4A8J) is shown for comparison.F. Analysis of Elp2 localization using a C-terminal MBP tag. Class averages of untagged Elongator is shown for comparison. White arrowheads point towards additional densities in the tagged complex.G. Elp3 localization analysis using a C-terminal MBP tag, denoted by the white arrowhead.H. Mapping of Elp1 by incubating purified Elongator containing an Elp1 C-terminal FLAG tag with α-FLAG antibody. The additional antibody density is denoted by the white arrowhead.I. Three dimensional reconstruction of the intact Elongator complex at 25Å.J. Schematic of the approximate subunit locations based on 2D EM analysis.K. Inset showing the cavity formed through the centre of the putative Elp456 density. Black scale bars in this figure correspond to 50 Å.


Figure 2A . Number of peptides detected of each Elongator subunit purified through Elp4 - FLAG pulldown from yeast lacking ELP1 , ELP2 , or ELP3 as a fraction of the number of Elp4 peptides . Individual data points are represented as circles , while the averages are represented as bars . B . Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate ( DSS ) and analyzed by mass spectrometry . Schematic representation of the major crosslinking connectivity of Elp123 is shown . C . Major crosslinking connectivity of the Elp456 complex .

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Figure 2A. Number of peptides detected of each Elongator subunit purified through Elp4-FLAG pulldown from yeast lacking ELP1, ELP2, or ELP3 as a fraction of the number of Elp4 peptides. Individual data points are represented as circles, while the averages are represented as bars.B. Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate (DSS) and analyzed by mass spectrometry. Schematic representation of the major crosslinking connectivity of Elp123 is shown.C. Major crosslinking connectivity of the Elp456 complex.


Figure 3B . Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast . The strains were then plated onto - Ura media containing caffeine . The pRS416 vector is used for these experiments . C . Growth curve analysis of loop mutants in - Ura media with and without K . lactis toxin zymocin . Shaded regions correspond to the SEM at each time point . N = 3 . D . Lysates of Elp4 - FLAG - containing yeast expressing the Elp2 loop mutants were incubated with α - FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α - HA and α - FLAG

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Figure 3B. Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto -Ura media containing caffeine. The pRS416 vector is used for these experiments.C. Growth curve analysis of loop mutants in -Ura media with and without K. lactis toxin zymocin. Shaded regions correspond to the SEM at each time point. N=3.D. Lysates of Elp4-FLAG-containing yeast expressing the Elp2 loop mutants were incubated with α-FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α-HA and α-FLAG antibody


Figure 4A . Fitting of Elp123 structures into the 3D reconstruction of Elongator . The homology structure of the Elp1 N - terminal double WD40 - domains , the homology structure of Elp3 based on the X - ray structure of D . mccartyi , the X - ray structure of Elp1 C - terminal dimer ( 5CQS ) , and the X - ray structure of Elp2 ( 4XFV ) . Scale bar corresponds to 50 Å , and applies to panels A - C . B . Fitting of the Elp456 heterohexameric ATPase ring ( 4A8J ) into the six - membered ring from the larger Elongator lobe . C . Size comparison of a representative tRNA molecule ( 1VTQ ) and the cavity observed through the middle of the Elp456 hexamer . D . Alternate views of the Elongator model showing the subunit crosslinking patterns .

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Figure 4A. Fitting of Elp123 structures into the 3D reconstruction of Elongator. The homology structure of the Elp1 N-terminal double WD40-domains, the homology structure of Elp3 based on the X-ray structure of D. mccartyi, the X-ray structure of Elp1 C-terminal dimer (5CQS), and the X-ray structure of Elp2 (4XFV). Scale bar corresponds to 50 Å, and applies to panels A-C.B. Fitting of the Elp456 heterohexameric ATPase ring (4A8J) into the six-membered ring from the larger Elongator lobe.C. Size comparison of a representative tRNA molecule (1VTQ) and the cavity observed through the middle of the Elp456 hexamer.D. Alternate views of the Elongator model showing the subunit crosslinking patterns.


Figure 5A . CXMS and EM data , combined with the X - ray structures of Elp1 C - terminus ( 5CQS ) , Elp2 ( 4XFV ) , Elp456 ( 4A8J ) , and homology models of the Elp1 tandem WD - 40 domains and Elp3 , were used to estimate the relative positions of each subunit to generate a multi - scale model of Elongator subunit organization . B . Side view of the Elongator architectural model highlighting the cavity ( red outline ) spanning through the centre of the Elp456 hexamer . C . Comparison of the Elp123 conformations of the intact Elongator complex ( gold ) and the Elp123 subcomplex ( cyan ) . The Elp456 - unloaded lobe of Elongator and one lobe of Elp123 were aligned .

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Figure 5A. CXMS and EM data, combined with the X-ray structures of Elp1 C-terminus (5CQS), Elp2 (4XFV), Elp456 (4A8J), and homology models of the Elp1 tandem WD-40 domains and Elp3, were used to estimate the relative positions of each subunit to generate a multi-scale model of Elongator subunit organization.B. Side view of the Elongator architectural model highlighting the cavity (red outline) spanning through the centre of the Elp456 hexamer.C. Comparison of the Elp123 conformations of the intact Elongator complex (gold) and the Elp123 subcomplex (cyan). The Elp456-unloaded lobe of Elongator and one lobe of Elp123 were aligned.


Figure 6A . Silver - stained SDS - PAGE of Elongator glycerol gradient fractions purified using low salt ( 75 mM NaCl ) buffer . The fraction containing the most Elp4 , Elp5 , and Elp6 is designated by an asterisk ( * ) and the corresponding fraction from a glutaraldehyde - containing glycerol gradient is used for EM analysis . B . Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations ( 150 mM and 75 mM NaCl , respectively ) . Densities resembling a second copy of Elp456 is indicated by white triangles . D . Coomassie - stained SDS - PAGE of rElp456 glycerol gradient fractions . E . Coomassie - stained SDS - PAGE of the Elongator mixed with rElp456 glycerol gradient fractions . The fraction containing the most intact complexes is indicated with an asterisk ( * ) , and the corresponding fraction from a glutaraldehyde - containing glycerol is used for EM analysis . F . Comparison of class averages of natively purified Elongator with and without rElp456 incubation . Densities resembling a second copy of Elp456 are indicated by white triangles .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Silver-stained SDS-PAGE of Elongator glycerol gradient fractions purified using low salt (75 mM NaCl) buffer. The fraction containing the most Elp4, Elp5, and Elp6 is designated by an asterisk (*) and the corresponding fraction from a glutaraldehyde-containing glycerol gradient is used for EM analysis.B. Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations (150 mM and 75 mM NaCl, respectively). Densities resembling a second copy of Elp456 is indicated by white triangles.D. Coomassie-stained SDS-PAGE of rElp456 glycerol gradient fractions.E. Coomassie-stained SDS-PAGE of the Elongator mixed with rElp456 glycerol gradient fractions. The fraction containing the most intact complexes is indicated with an asterisk (*), and the corresponding fraction from a glutaraldehyde-containing glycerol is used for EM analysis.F. Comparison of class averages of natively purified Elongator with and without rElp456 incubation. Densities resembling a second copy of Elp456 are indicated by white triangles.


Figure 1 ( B ) Plaque formation was monitored by automated scanning Levin - Reisman et al . , 2010 ( ) on a lawn of infected PY79 ( WT ) ( 10 - 6 PFU / ml ) cells spread over an MB agar plate . Shown are images captured at the indicated time points ( hrs ) . Scale bar 2 mm . Corresponds to Movie EV2 . ( C - D ) The dynamic of SPP1 ( C ) and Phi29 ( D ) plaque formation following PY79 ( WT ) infection was monitored as described in ( B ) . Shown is the diameter of individual plaques for each phage ( n ≥ 12 ) , with the average highlighted in red .

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Figure 1(B) Plaque formation was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected PY79 (WT) (10-6 PFU/ml) cells spread over an MB agar plate. Shown are images captured at the indicated time points (hrs). Scale bar 2 mm. Corresponds to Movie EV2. (C-D) The dynamic of SPP1 (C) and Phi29 (D) plaque formation following PY79 (WT) infection was monitored as described in (B). Shown is the diameter of individual plaques for each phage (n ≥ 12), with the average highlighted in red.


Figure 2 ( A ) The indicated strains were infected with SPP1 ( 10 - 6 PFU / ml ) , spread over MB plates , and plaque diameter was monitored after 20 hrs of incubation . Shown is average plaque diameter and SD for each strain ( n ≥ 54 ) . ( D ) PY79 ( WT ) and ET19 ( ∆ sigX ) cells were infected with SPP1 at either high ( phages : bacteria 1 : 1 ) or low ( 1 : 20 ) MOI , and OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , and the average values and SD of 8 technical repeats . ( E ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) and ET191 ( PrrnE - gfp , ∆ sigX ) ( ∆ sigX , cyan ) cells were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy . Shown are overlay images from mCherry ( purple ) and GFP ( cyan ) signals of the bacterial lawn captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 µm . Corresponds to Movie EV4 . ( F ) ET27 ( PsigX - gfp ) cells were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are fluorescence from GFP ( upper panels ) and corresponding phase contrast images ( lower panels ) , captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) The indicated strains were infected with SPP1 (10-6 PFU/ml), spread over MB plates, and plaque diameter was monitored after 20 hrs of incubation. Shown is average plaque diameter and SD for each strain (n ≥ 54).(D) PY79 (WT) and ET19 (∆sigX) cells were infected with SPP1 at either high (phages:bacteria 1:1) or low (1:20) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, and the average values and SD of 8 technical repeats.(E) BDR2637 (Pveg-mCherry) (WT, purple) and ET191 (PrrnE-gfp, ∆sigX) (∆sigX, cyan) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry (purple) and GFP (cyan) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm. Corresponds to Movie EV4.(F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


Figure 3 ( C ) ET26 ( PsigX - sigX - gfp ) cells were visualized by fluorescence microscopy . Shown are signal from SigX - GFP ( cyan ) ( left panel ) , and an overlay image of phase contrast ( grey ) and signal from SigX - GFP ( cyan ) ( right panel ) . Scale bar 1 μm . ( D ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) and ET261 ( ∆ yueB , PsigX - sigX - gfp ) ( cyan ) cells were mixed , infected with SPP1 at 5 : 1 ( phages : bacteria ) MOI , placed on an agarose pad and followed by time lapse fluorescence microscopy . Shown are overlay images from mCherry ( purple ) , SigX - GFP ( cyan ) , and phase contrast ( grey ) , captured at the indicated time points post infection . Scale bar 1 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(C) ET26 (PsigX-sigX-gfp) cells were visualized by fluorescence microscopy. Shown are signal from SigX-GFP (cyan) (left panel), and an overlay image of phase contrast (grey) and signal from SigX-GFP (cyan) (right panel). Scale bar 1 μm.(D) BDR2637 (Pveg-mCherry) (WT, purple) and ET261 (∆yueB, PsigX-sigX-gfp) (cyan) cells were mixed, infected with SPP1 at 5:1 (phages:bacteria) MOI, placed on an agarose pad and followed by time lapse fluorescence microscopy. Shown are overlay images from mCherry (purple), SigX-GFP (cyan), and phase contrast (grey), captured at the indicated time points post infection. Scale bar 1 μm.


Figure 4 ( B ) ET29 ( Pveg - mCherry , PIPTG - sigX ) ( purple ) cells were grown in the presence of IPTG and mixed with PY79 ( WT ) cells . The mixture was infected with SPP1 - lysin - yfp 5 : 1 ( phages : bacteria ) MOI , placed on an IPTG - containing agarose pad , and followed by time lapse fluorescence microscopy . Shown are overlay images of phase contrast ( grey ) , signal from mCherry labeled cells ( purple ) , and signal from Lysin - SPP1 - YFP ( cyan ) , captured at the indicated time points post infection ( upper panels ) . Corresponding signal from Lysin - SPP1 - YFP ( cyan ) is shown separately ( lower panels ) . Arrows highlight the delayed infection of ET29 cells . Scale bar 1 μm . ( E ) OF83 ( Ppen - lacIΔ11 - cfp ) ( WT ) and ET40 ( Ppen - lacIΔ11 - cfp , PIPTG - sigX ) cells , grown in the presence or absence of IPTG , were infected with SPP1 - delX110lacO64 at 5 : 1 ( phages : bacteria ) MOI . The formation of LacI - CFP foci on phage DNA was monitored 10 min post infection . Non - infected OF83 cells were used for comparison . Shown are overlay images of phase contrast ( grey ) and signal from LacI - CFP ( cyan ) . Scale bar 1 µm . ( F ) Quantification of the experiment described in ( E ) . Shown is the percentage of LacI - CFP foci 10 min post infection of OF83 and ET40 cells by SPP1 , with average values and SD ( n ≥ 850 cells for each population ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) ET29 (Pveg-mCherry, PIPTG-sigX) (purple) cells were grown in the presence of IPTG and mixed with PY79 (WT) cells. The mixture was infected with SPP1-lysin-yfp 5:1 (phages:bacteria) MOI, placed on an IPTG-containing agarose pad, and followed by time lapse fluorescence microscopy. Shown are overlay images of phase contrast (grey), signal from mCherry labeled cells (purple), and signal from Lysin-SPP1-YFP (cyan), captured at the indicated time points post infection (upper panels). Corresponding signal from Lysin-SPP1-YFP (cyan) is shown separately (lower panels). Arrows highlight the delayed infection of ET29 cells. Scale bar 1 μm.(E) OF83 (Ppen-lacIΔ11-cfp) (WT) and ET40 (Ppen-lacIΔ11-cfp, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1-delX110lacO64 at 5:1 (phages:bacteria) MOI. The formation of LacI-CFP foci on phage DNA was monitored 10 min post infection. Non-infected OF83 cells were used for comparison. Shown are overlay images of phase contrast (grey) and signal from LacI-CFP (cyan). Scale bar 1 µm. (F) Quantification of the experiment described in (E). Shown is the percentage of LacI-CFP foci 10 min post infection of OF83 and ET40 cells by SPP1, with average values and SD (n ≥ 850 cells for each population).


Figure 5 ( A ) ET28 ( PIPTG - sigX ) and ET42 ( ∆ dltA , PIPTG - sigX ) cells , grown in the presence or absence of IPTG , were infected with SPP1 at 1 : 20 ( phages : bacteria ) MOI , and OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , with the average values and SD of 8 technical repeats . ( D ) PY79 ( WT ) and ET41 ( ΔdltA ) cells were infected with SPP1 ( 10 - 6 PFU / ml ) , spred over an MB agar plate , and plaque diameter was monitored after 20 hrs of incubation . Shown is plaque diameter distributions for each strain ( n ≥ 40 ) . ( F ) AR16 ( PrrnE - gfp ) ( WT , cyan ) and ET411 ( Pveg - mCherry , ∆ dltA ) ( ∆ dltA , purple ) cells were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are overlay images from GFP ( cyan ) and mCherry ( purple ) signals of the bacterial lawn captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 150 µm . ( G ) ET43 ( dltA - yfp ) cells were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are fluoresce from DltA - YFP signal ( upper panels ) and corresponding phase contrast images ( lower panels ) , captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) ET28 (PIPTG-sigX) and ET42 (∆dltA, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1 at 1:20 (phages:bacteria) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, with the average values and SD of 8 technical repeats.(D) PY79 (WT) and ET41 (ΔdltA) cells were infected with SPP1 (10-6 PFU/ml), spred over an MB agar plate, and plaque diameter was monitored after 20 hrs of incubation. Shown is plaque diameter distributions for each strain (n ≥ 40).(F) AR16 (PrrnE-gfp) (WT, cyan) and ET411 (Pveg-mCherry, ∆dltA) (∆dltA, purple) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from GFP (cyan) and mCherry (purple) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 150 µm.(G) ET43 (dltA-yfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluoresce from DltA-YFP signal (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


Figure 1A Normal human fibroblasts and HGPS fibroblasts were serum - starved for 48 h and stained for progerin ( red ) , acetylated tubulin ( green ) and DNA ( blue ) . The representative images show that the cell with high progerin ( inset 2 ) , but not low progerin ( inset 1 ) , displays defective cilia formation . Scale bars , 20 μm or 2 μm ( in the magnified images ) . B The progerin fluorescence intensity of the cells was measured by the Zeiss ZEN2 software ( n ≥ 148 ) . The dashed line indicates the background level of progerin . A . U . , arbitrary units . The cells were grown in the growth medium and stained for progerin ( red ) and F - actin with phalloidin ( green ) . The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3 . 5 x 106 ( A . U . ) , respectively . The cell spreading area was measured according to the phalloidin staining ( n ≥ 30 ) . Scale bars , 50 m . D The cells were serum - starved for 32 h to allow cilia formation and then treated with ( + ) or without ( - ) 250 nM cytochalasin D ( Cyto D ) for another 16 h before fixation . The cells were stained for progerin ( red ) , acetylated tubulin ( green ) , F - actin ( white ) , and DNA ( blue ) . The numbers of actin filaments within 60 μm2 around the cilia ( as illustrated by the circle ) from the cells under serum - starvation without Cyto D treatment were measured ( n ≥ 55 ) . Scale bars , 20 μm or 2 μm ( in the magnified insets ) . E , F The cells were subjected to immunofluorescence stain as in ( D ) . The percentage of the cells with cilia in the total counted cells ( E , n ≥ 50 ) and the cilia length ( F , n ≥ 50 ) were measured .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Normal human fibroblasts and HGPS fibroblasts were serum-starved for 48 h and stained for progerin (red), acetylated tubulin (green) and DNA (blue). The representative images show that the cell with high progerin (inset 2), but not low progerin (inset 1), displays defective cilia formation. Scale bars, 20 μm or 2 μm (in the magnified images).B The progerin fluorescence intensity of the cells was measured by the Zeiss ZEN2 software (n ≥ 148). The dashed line indicates the background level of progerin. A.U., arbitrary units.The cells were grown in the growth medium and stained for progerin (red) and F-actin with phalloidin (green). The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3.5 x 106 (A.U.), respectively. The cell spreading area was measured according to the phalloidin staining (n ≥ 30). Scale bars, 50 m.D The cells were serum-starved for 32 h to allow cilia formation and then treated with (+) or without (-) 250 nM cytochalasin D (Cyto D) for another 16 h before fixation. The cells were stained for progerin (red), acetylated tubulin (green), F-actin (white), and DNA (blue). The numbers of actin filaments within 60 μm2 around the cilia (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (n ≥ 55). Scale bars, 20 μm or 2 μm (in the magnified insets).E,F The cells were subjected to immunofluorescence stain as in (D). The percentage of the cells with cilia in the total counted cells (E, n ≥ 50) and the cilia length (F, n ≥ 50) were measured.


Figure 2A The skeletal ( tabialis anterior ) muscles from control ( Lmna + / + and Lmna + / - ) and Lmna - / - mice were stained for acetylated tubulin ( green ) and nucleus ( blue ) . The representative images with low and high magnification are shown . The insets are enlarged images for cilia . B The kidneys from control and Lmna - / - mice were stained for acetylated tubulin ( green ) and AQ1 ( a marker for the proximal tubules , red ) . The insets show the cilia of proximal renal tubule cells . C The uterus from control and Lmna - / - mice was stained for acetylated tubulin ( green ) and keratin 8 ( red ) . The insets show the cilia at the stroma of the uterus . D The ovary from control and Lmna - / - mice were stained for acetylated tubulin ( green ) and keratin 8 ( red ) . The insets show the cilia of the granulosa cells in the follicles of the ovary . E , F The cilia length ( E , n = 150 - 180 ) and cilia positive cells ( F , n ≥ 1197 ) in each organ from 4 - week - old Lmna - / - mice ( n = 3 ) and their littermate controls ( n = 3 ) were measured .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A The skeletal (tabialis anterior) muscles from control (Lmna+/+ and Lmna+/-) and Lmna-/- mice were stained for acetylated tubulin (green) and nucleus (blue). The representative images with low and high magnification are shown. The insets are enlarged images for cilia.B The kidneys from control and Lmna-/- mice were stained for acetylated tubulin (green) and AQ1 (a marker for the proximal tubules, red). The insets show the cilia of proximal renal tubule cells.C The uterus from control and Lmna-/- mice was stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia at the stroma of the uterus.D The ovary from control and Lmna-/- mice were stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia of the granulosa cells in the follicles of the ovary.E, F The cilia length (E, n=150-180) and cilia positive cells (F, n ≥ 1197) in each organ from 4-week-old Lmna-/- mice (n=3) and their littermate controls (n=3) were measured.


Figure 3A RPE cells were infected with lentiviruses encoding shRNAs specific to lamin A / C ( shLamin A / C ) or luciferase ( shLuc ) . Exogenous lamin A was re - expressed in the lamin A / C - depleted cells ( shLamin A / C - Lamin A ) . An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated . B The cells were serum - starved for 48 h and stained for acetylated tubulin ( green ) , γ - tubulin ( red ) and DNA ( blue ) . The depletion of lamin A / C ( shLamin A / C ) led to impaired ciliogenesis , as manifested by no cilia ( inset 1 ) or short cilia ( inset 2 ) . Scale bars , 20 μm or 2 μm ( insets ) . C The percentage of the cells with cilia in the total counted cells ( n ≥ 626 ) was measured . D The length of cilia was measured ( n ≥ 87 in each group ) . E Cells were stained for acetylated tubulin ( green ) and γ - tubulin ( red ) after various intervals of serum starvation . The percentage of cilia - positive cells at each time point ( n ≥ 372 ) was measured .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A RPE cells were infected with lentiviruses encoding shRNAs specific to lamin A/C (shLamin A/C) or luciferase (shLuc). Exogenous lamin A was re-expressed in the lamin A/C-depleted cells (shLamin A/C-Lamin A). An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated.B The cells were serum-starved for 48 h and stained for acetylated tubulin (green), γ-tubulin (red) and DNA (blue). The depletion of lamin A/C (shLamin A/C) led to impaired ciliogenesis, as manifested by no cilia (inset 1) or short cilia (inset 2). Scale bars, 20 μm or 2 μm (insets).C The percentage of the cells with cilia in the total counted cells (n ≥ 626) was measured.D The length of cilia was measured (n ≥ 87 in each group).E Cells were stained for acetylated tubulin (green) and γ-tubulin (red) after various intervals of serum starvation. The percentage of cilia-positive cells at each time point (n ≥ 372) was measured.


Figure 4A - C The RPE cells as described in Fig 3 were stained for paxillin ( as a marker for focal adhesion ; green ) , F - actin ( red ) and DNA ( blue ) . The representative images are shown ( A ) . Scale bars , 20 μm or 5 μm ( insets ) . The cell spreading area of the cells ( B , n ≥ 108 ) and the number of focal adhesions per cell ( C , n ≥ 30 ) were measured . D - G The cells were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D ( 250 nM ) for another 16 h . The cells were stained for acetylated tubulin ( white ) , pericentrin ( green ) , F - actin ( red ) and DNA ( blue ) . The representative images are shown ( D ) . The left insets show the merged signals of acetylated tubulin and pericentrin . Scale bars , 20 μm or 2 μm ( magnified images ) . The numbers of actin filaments within 60 μm2 around the basal body ( as illustrated by the circle ) from the cells under serum - starvation without Cyto D treatment were measured ( E , n ≥ 132 ) . The percentage of the cells with cilia in the total counted cells ( F , n ≥ 357 ) and the length of cilia ( G , n ≥ 126 ) were measured .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-C The RPE cells as described in Fig 3 were stained for paxillin (as a marker for focal adhesion; green), F-actin (red) and DNA (blue). The representative images are shown (A). Scale bars, 20 μm or 5 μm (insets). The cell spreading area of the cells (B, n ≥ 108) and the number of focal adhesions per cell (C, n ≥ 30) were measured.D-G The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D (250 nM) for another 16 h. The cells were stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (D). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of actin filaments within 60 μm2 around the basal body (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (E, n ≥ 132). The percentage of the cells with cilia in the total counted cells (F, n ≥ 357) and the length of cilia (G, n ≥ 126) were measured.


Figure 5A An equal amount of whole cell lysates from RPE cells or those expressing shLamin A / C or luciferase ( shLuc ) was analyzed by the immunoblotting with antibodies as indicated . RPE cells were infected with lentiviruses expressing shRNAs to nesprin 2 ( shNesprine 2 , clone # 1 and # 2 ) or luciferase ( shLuc ) as a control . An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated ( B ) . The cells were serum - starved for 48 h and then stained for acetylated tubulin ( white ) , pericentrin ( green ) , F - actin ( red ) and DNA ( blue ) . The representative images are shown ( C ) The left insets show the merged signals of acetylated tubulin and pericentrin . Scale bars , 20 μm or 2 μm ( magnified images ) . The numbers of F - actin stress fibers within 60 μm2 around the basal bodies ( as illustrated by the circle ) were measured ( D , n ≥ 53 ) . E , F RPE cells expressing shNesprine 2 or shLuc were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D for another 16 h . The percentage of the cells with cilia in the total counted cells ( E , n ≥ 197 ) and the length of cilia ( F , n ≥ 52 ) were measured .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A An equal amount of whole cell lysates from RPE cells or those expressing shLamin A/C or luciferase (shLuc) was analyzed by the immunoblotting with antibodies as indicated.RPE cells were infected with lentiviruses expressing shRNAs to nesprin 2 (shNesprine 2, clone #1 and #2) or luciferase (shLuc) as a control. An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated (B).The cells were serum-starved for 48 h and then stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (C) The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of F-actin stress fibers within 60 μm2 around the basal bodies (as illustrated by the circle) were measured (D, n ≥ 53).E,F RPE cells expressing shNesprine 2 or shLuc were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The percentage of the cells with cilia in the total counted cells (E, n ≥ 197) and the length of cilia (F, n ≥ 52) were measured.


Figure 6A , B RPE cells expressing shRNAs specific to lamin A / C or nesprin 2 ( shNesprin 2 , clones # 1 and # 2 ) were stained for G - actin with DNase I ( green ) . Representative images are shown ( A ) . Scale bars , 20 μm . The proportion of nuclear G - actin fluorescence intensity to the whole cell was measured ( B , n ≥ 67 ) . C An equal amount of whole cell lysates from RPE cells ( control ) and those expressing shRNAs to importin 9 ( shImportin 9 , clones # 1 and # 2 ) was analyzed by immunoblotting with antibodies as indicated . D - F The cells expressing shRNAs to importin 9 were stained for G - actin ( green ) and F - actin ( red ) . The representative images are shown ( D ) . Scale bars , 20 μm or 10 μm ( insets ) . The proportion of nuclear G - actin intensity to the whole cell ( E , n > 152 ) and the cell spreading area ( F , n ≥ 139 ) were measured . G - I RPE cells and those expressing shRNAs to importin 9 were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D for another 16 h and then stained for acetylated tubulin ( white ) , pericentrin ( green ) and F - actin ( red ) . Representative images are shown ( G ) . The left insets show the merged signals of acetylated tubulin and pericentrin . Scale bars , 20 μm or 2 μm ( magnified images ) . The percentage of the cells with cilia ( H , n ≥ 385 ) and the length of cilia ( I , n ≥ 111 ) were measured .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A,B RPE cells expressing shRNAs specific to lamin A/C or nesprin 2 (shNesprin 2, clones #1 and #2) were stained for G-actin with DNase I (green). Representative images are shown (A). Scale bars, 20 μm. The proportion of nuclear G-actin fluorescence intensity to the whole cell was measured (B, n ≥ 67).C An equal amount of whole cell lysates from RPE cells (control) and those expressing shRNAs to importin 9 (shImportin 9, clones #1 and #2) was analyzed by immunoblotting with antibodies as indicated.D-F The cells expressing shRNAs to importin 9 were stained for G-actin (green) and F-actin (red). The representative images are shown (D). Scale bars, 20 μm or 10 μm (insets). The proportion of nuclear G-actin intensity to the whole cell (E, n > 152) and the cell spreading area (F, n ≥ 139) were measured.G-I RPE cells and those expressing shRNAs to importin 9 were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h and then stained for acetylated tubulin (white), pericentrin (green) and F-actin (red). Representative images are shown (G). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The percentage of the cells with cilia (H, n ≥ 385) and the length of cilia (I, n ≥ 111) were measured.


Figure 7A RPE cells expressing shRNAs specific to luciferase ( shLuc . ) , lamin A / C , or nesprin 2 were grown in the growth medium ( + serum ) or serum - starved for 1 h ( - serum ) and then stained for Arp2 ( red ) , CEP164 ( a marker for the mother centriole , green ) and acetylated tubulin ( white ) . The representative images are shown . Note that Arp2 was found to localize exclusively at the mother centriole ( upper panels ) or at the both mother and daughter centrioles ( lower panels ) . Scale bars , 0 . 5 μm . The percentage of cells with Arp2 localized exclusively at the mother centriole in the total counted cells was measured ( n = 131 - 240 cells in each group ) . B The cells were serum - starved for 48 h and then stained for acetylated tubulin ( green ) , Arp2 ( red ) , and γ - tubulin ( white ) . The representative images are shown . Scale bars , 1 μm ( upper panels ) and 0 . 5 μm ( lower panels ) . Note that the cell with Arp2 at the both mother and daughter centrioles fails to form cilium . C The cells were serum - starved for 48 h and then stained for myosin Va ( red ) , acetylated tubulin ( green ) and γ - tubulin ( white ) . Scale bars , 0 . 5 μm . The cartoon illustrates that myosin Va - associated vesicles ( CV + PCV ) specifically accumulate at the mother centriole in most control cells ( as in the " one centriole " image ) . CV , ciliary vesicle . PCV , periciliary vesicle . The percentage of cells with myosin Va exclusively at the mother centriole in the total counted cells was measured ( n = 160 - 200 cells in each group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A RPE cells expressing shRNAs specific to luciferase (shLuc.), lamin A/C, or nesprin 2 were grown in the growth medium (+ serum) or serum-starved for 1 h (- serum) and then stained for Arp2 (red), CEP164 (a marker for the mother centriole, green) and acetylated tubulin (white). The representative images are shown. Note that Arp2 was found to localize exclusively at the mother centriole (upper panels) or at the both mother and daughter centrioles (lower panels). Scale bars, 0.5 μm. The percentage of cells with Arp2 localized exclusively at the mother centriole in the total counted cells was measured (n=131-240 cells in each group).B The cells were serum-starved for 48 h and then stained for acetylated tubulin (green), Arp2 (red), and γ-tubulin (white). The representative images are shown. Scale bars, 1 μm (upper panels) and 0.5 μm (lower panels). Note that the cell with Arp2 at the both mother and daughter centrioles fails to form cilium.C The cells were serum-starved for 48 h and then stained for myosin Va (red), acetylated tubulin (green) and γ-tubulin (white). Scale bars, 0.5 μm. The cartoon illustrates that myosin Va-associated vesicles (CV+PCV) specifically accumulate at the mother centriole in most control cells (as in the "one centriole" image). CV, ciliary vesicle. PCV, periciliary vesicle. The percentage of cells with myosin Va exclusively at the mother centriole in the total counted cells was measured (n=160-200 cells in each group).


Figure 8A RPE cells expressing shRNAs specific to luciferase ( shLuc ) or lamin A / C ( shLamin A / C ) were serum - starved for 48 h and then stained for acetylated tubulin ( Ac - tubulin , green ) and CEP164 ( red ) . The representative images show that the fluorescence intensity of CEP164 at the mother centriole of cilia - negative ( left column ) and cilia - positive ( right column ) basal bodies are similar . The graphs shown are the quantification of CEP164 at cilia - negative basal bodies and cilia - positive basal bodies . B The cells were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D for another 16 h . The cells were stained for acetylated tubulin ( Ac - tubulin , green ) and TTBK2 ( red ) . The graphs shown are the quantifications of TTBK2 intensity at cilia - negative basal bodies and cilia - positive basal bodies . C The cells as in ( B ) were stained for acetylated tubulin ( Ac - tubulin , green ) and CP110 ( red ) . The representative images show that CP110 is not removed from the mother centriole at some of cilia - negative basal bodies ( left column ) , whereas it is removed from the mother centriole at cilia - positive basal bodies ( right column ) . The graphs shown are the quantifications of CP110 - removal at cilia - negative basal bodies and cilia - positive basal bodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A RPE cells expressing shRNAs specific to luciferase (shLuc) or lamin A/C (shLamin A/C) were serum-starved for 48 h and then stained for acetylated tubulin (Ac-tubulin, green) and CEP164 (red). The representative images show that the fluorescence intensity of CEP164 at the mother centriole of cilia-negative (left column) and cilia-positive (right column) basal bodies are similar. The graphs shown are the quantification of CEP164 at cilia-negative basal bodies and cilia-positive basal bodies.B The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The cells were stained for acetylated tubulin (Ac-tubulin, green) and TTBK2 (red). The graphs shown are the quantifications of TTBK2 intensity at cilia-negative basal bodies and cilia-positive basal bodies.C The cells as in (B) were stained for acetylated tubulin (Ac-tubulin, green) and CP110 (red). The representative images show that CP110 is not removed from the mother centriole at some of cilia-negative basal bodies (left column), whereas it is removed from the mother centriole at cilia-positive basal bodies (right column). The graphs shown are the quantifications of CP110-removal at cilia-negative basal bodies and cilia-positive basal bodies.


Figure 1B t - SNE plots showing integrated analysis of Prrx1 - Cre - ( 8 - week - old mice , n = 3 males ) and Lepr - Cre - traced ( 8 - week - old mice , n = 4 males ) cells . Cells were colored by clusters ( left ) or samples ( right ) . Top 20 PCs were chosen for the clustering . D Violin plots showing the expression of feature genes for each cluster . E In situ hybridization of Col3a1 on femur sections . Col3a1 was expressed in the perichondral ( a ) and periosteal ( b ) regions , but not in the bone marrow . B : Bone ; BM : Bone marrow ; Peri : Periosteum ; Endo : Endosteum . Scale bars were 100 µm . Arrowheads indicated the periosteum .

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Figure 1B t-SNE plots showing integrated analysis of Prrx1-Cre- (8-week-old mice, n=3 males) and Lepr-Cre-traced (8-week-old mice, n=4 males) cells. Cells were colored by clusters (left) or samples (right). Top 20 PCs were chosen for the clustering.D Violin plots showing the expression of feature genes for each cluster.E In situ hybridization of Col3a1 on femur sections. Col3a1 was expressed in the perichondral (a) and periosteal (b) regions, but not in the bone marrow. B: Bone; BM: Bone marrow; Peri: Periosteum; Endo: Endosteum. Scale bars were 100 µm. Arrowheads indicated the periosteum.


Figure 2E Violin plots showing the expression of feature genes for each cluster . F Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Arrowheads indicated tdTomato + periosteal cells . Two representative periosteal regions were magnified ( a and b ) . GP : Growth plate . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm . G Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice 7 days after fracture . Aggrecan ( green ) , tdTomato ( red ) , DAPI ( blue ) . Arrowheads indicated tdTomato + periosteal cells . Two representative regions were magnified ( a and b ) . Scale bars were 100 µm .

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Figure 2E Violin plots showing the expression of feature genes for each cluster.F Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Arrowheads indicated tdTomato+ periosteal cells. Two representative periosteal regions were magnified (a and b). GP: Growth plate. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm. G Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice 7 days after fracture. Aggrecan (green), tdTomato (red), DAPI (blue). Arrowheads indicated tdTomato+ periosteal cells. Two representative regions were magnified (a and b). Scale bars were 100 µm.


Figure 3B Dot plots showing the gene expression patterns of selected genes in each cluster . D Flow cytometry analysis of BrdU incorporation in uncultured Lepr - Cre + Notch3 + / - BMSCs ( n = 4 independent experiments ) . Eight - week - old Lepr - Cre ; tdTomato mice were given a single intraperitoneal injection of BrdU ( 100 mg / kg body mass ) and maintained on 0 . 5 mg / ml of BrdU in the drinking water for 14 days . E Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Notch3 ( green ) , tdTomato ( red ) , CD31 ( gray ) , DAPI ( blue ) . Sinusoid ( a ) , arteriole ( b ) , artery ( c ) and avascular bone marrow ( BM , d ) regions were shown . Arrowheads indicated Notch3 + tdTomato + adventitial cells closely associated with ECs . Arrows indicated Notch3 - tdTomato + reticular cells in the bone marrow that were not associated with ECs . Scale bars were 10 µm . F Localization quantification of Lepr - Cre + Notch3 + cells relative to bone marrow ECs ( n = 5 biological replicates from three independent experiments ) . Cells within 5 µm diameter of ECs were considered adjacent . GH Volcano plot showing DEGs in the bulk RNA - seq analysis ( n = 3 biological replicates ) . Green dots represent down - regulated genes . Red dots represent up - regulated genes . O Heatmap showing the average expression levels ( column - wide Z score ) of secreted factors with clustering .

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Figure 3B Dot plots showing the gene expression patterns of selected genes in each cluster.D Flow cytometry analysis of BrdU incorporation in uncultured Lepr-Cre+Notch3+/- BMSCs (n=4 independent experiments). Eight-week-old Lepr-Cre; tdTomato mice were given a single intraperitoneal injection of BrdU (100 mg/kg body mass) and maintained on 0.5 mg/ml of BrdU in the drinking water for 14 days.E Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Sinusoid (a), arteriole (b), artery (c) and avascular bone marrow (BM, d) regions were shown. Arrowheads indicated Notch3+ tdTomato+ adventitial cells closely associated with ECs. Arrows indicated Notch3- tdTomato+ reticular cells in the bone marrow that were not associated with ECs. Scale bars were 10 µm. F Localization quantification of Lepr-Cre+Notch3+ cells relative to bone marrow ECs (n=5 biological replicates from three independent experiments). Cells within 5 µm diameter of ECs were considered adjacent. GH Volcano plot showing DEGs in the bulk RNA-seq analysis (n=3 biological replicates). Green dots represent down-regulated genes. Red dots represent up-regulated genes.O Heatmap showing the average expression levels (column-wide Z score) of secreted factors with clustering.


Figure 4C Heatmap showing pseudotime - dependent gene expression . Bars on the top were colored as in ( A ) . Representative genes were highlighted on both sides . F Representative images of adipogenic , osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr - Cre + BMSCs . Scale bars were 100 µm . G qPCR analysis of the expression levels of Pparg , Bglap and Acan in control or knockdown group ( n = 3 independent experiments ) . The statistical significance of differences was analyzed by one - way ANOVAs with Tukey ' s multiple comparison tests . Data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 4C Heatmap showing pseudotime-dependent gene expression. Bars on the top were colored as in (A). Representative genes were highlighted on both sides.F Representative images of adipogenic, osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr-Cre+ BMSCs. Scale bars were 100 µm.G qPCR analysis of the expression levels of Pparg, Bglap and Acan in control or knockdown group (n=3 independent experiments). The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. Data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


Figure 5B Violin plots showing the expression of feature genes for each cluster . F Immunostaining of Sca - 1 on femur sections of 8 - week - old Lepr - Cre ; tdTomato mice . Arrows indicated Sca - 1 + tdTomato + cells inside cortical bones of the femur diaphysis . Arrowheads indicated Sca - 1 + tdTomato + cells in the femur periosteum . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm . G Representative flow cytometry plots of long bone periosteum in 8 - week - old Lepr - Cre ; tdTomato mice . The percentage of DAPI - CD45 - Ter119 - Tie2 - tdTomato + Sca - 1 + / - cells were shown . I , J The average number of CFU - F colonies formed ( I ) and the average cell number per colony ( J ) were shown ( n = 4 independent experiments ) . The statistical significance of differences was assessed using one - way ANOVAs with Tukey ' s multiple comparison tests . All data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Violin plots showing the expression of feature genes for each cluster.F Immunostaining of Sca-1 on femur sections of 8-week-old Lepr-Cre; tdTomato mice. Arrows indicated Sca-1+ tdTomato+ cells inside cortical bones of the femur diaphysis. Arrowheads indicated Sca-1+ tdTomato+ cells in the femur periosteum. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm.G Representative flow cytometry plots of long bone periosteum in 8-week-old Lepr-Cre; tdTomato mice. The percentage of DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+/- cells were shown.I, J The average number of CFU-F colonies formed (I) and the average cell number per colony (J) were shown (n=4 independent experiments). The statistical significance of differences was assessed using one-way ANOVAs with Tukey's multiple comparison tests. All data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


Figure 6A , B Representative oil red staining after 7 days of adipogenic differentiation in Lepr - Cre + subsets ( A ) and qPCR analysis of Adipoq and Pparg expression ( B ) ( n = 3 independent experiments ) . Scale bars were 100 µm . C , D In vitro chondrogenic differentiation of sorted periosteal cells . Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection ( C ) . Scale bars were 100 µm . Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression ( D ) ( n = 3 independent experiments ) . E , F In vitro osteogenic differentiation of sorted periosteal cells . Representative alizarin red staining after osteogenic differentiation for 14 days . Scale bars were 200 µm . Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression ( F ) ( n = 3 independent experiments ) .

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Figure 6A, B Representative oil red staining after 7 days of adipogenic differentiation in Lepr-Cre+ subsets (A) and qPCR analysis of Adipoq and Pparg expression (B) (n=3 independent experiments). Scale bars were 100 µm.C, D In vitro chondrogenic differentiation of sorted periosteal cells. Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection (C). Scale bars were 100 µm. Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression (D) (n=3 independent experiments).E, F In vitro osteogenic differentiation of sorted periosteal cells. Representative alizarin red staining after osteogenic differentiation for 14 days. Scale bars were 200 µm. Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression (F) (n=3 independent experiments).


Figure 1 ( H ) Immunoblot representing levels mTOR - dependent targets with defined 5 ' TOP motifs 3 hours and 2 days after TAC . n = 3 biological replicates per condition . ( J ) Immunoblots and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR inhibition with Torin 1 . * p ≤ 0 . 05 vs sham surgery , # p ≤ 0 . 05 vs TAC 2d surgery . t - test , n = 5 biological replicates per group . Data information : Error bars indicate means ± SEM ( K ) RT - qPCR for indicated mRNAs 2 days after TAC for indicated groups , n = 5 biological replicates per group . Data information : Error bars indicate means ±

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Figure 1(H) Immunoblot representing levels mTOR-dependent targets with defined 5'TOP motifs 3 hours and 2 days after TAC. n=3 biological replicates per condition.(J) Immunoblots and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR inhibition with Torin 1.* p≤ 0.05 vs sham surgery, # p≤ 0.05 vs TAC 2d surgery. t-test, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM(K) RT-qPCR for indicated mRNAs 2 days after TAC for indicated groups, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM


Figure 2 ( C ) Protein expression of Cand2 in NRCMs treated with PE for 3 and 24 h , and 24h of Torin 1 treatment . mTORC1 induction was monitored by downstream targets , p4EBP1 and pS6K proteins . rpS5 - example of mTOR - dependent mRNA used as a positive control . Csq - calsequestrin used as a houskeeping protein . ( F , G ) Ribo - seq and RNA - seq expression data of Cand2 in sham and TAC operated animals ( CPM - count per million ) . One - way ANOVA , * * - P ≤ 0 . 01 , n = 2 - 4 biological replicates per group . Data information : Error bars indicate means ± SEM ( J ) Schematic representation of reporter consisting of human Cand2 5 ' UTR and downstream Renilla luciferase coding sequence . A potential TOP - like motif is highlighted in a grey box . Black arrows indicate transcription start sites in the adult heart according to the Database of Transcriptional Start Sites ( Release 10 . 1 ) . The effect of Cand2 5 ' UTR on translation measured by luciferase activity in normal conditions and after mTORC1 block with Torin 1 . eEF2 - positive control of reporter with defined 5 ' TOP motif and non - TOP 5 ' UTR of β - actin used as a negative control . Total protein content in lysates used for luciferase assay . t - test , n = 4 - 5 biological replicates per group , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

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Figure 2(C) Protein expression of Cand2 in NRCMs treated with PE for 3 and 24 h, and 24h of Torin 1 treatment. mTORC1 induction was monitored by downstream targets, p4EBP1 and pS6K proteins. rpS5 - example of mTOR-dependent mRNA used as a positive control. Csq - calsequestrin used as a houskeeping protein.(F, G) Ribo-seq and RNA-seq expression data of Cand2 in sham and TAC operated animals (CPM - count per million). One-way ANOVA, ** - P ≤ 0.01, n=2-4 biological replicates per group. Data information: Error bars indicate means ± SEM(J) Schematic representation of reporter consisting of human Cand2 5'UTR and downstream Renilla luciferase coding sequence. A potential TOP-like motif is highlighted in a grey box. Black arrows indicate transcription start sites in the adult heart according to the Database of Transcriptional Start Sites (Release 10.1). The effect of Cand2 5'UTR on translation measured by luciferase activity in normal conditions and after mTORC1 block with Torin 1. eEF2 - positive control of reporter with defined 5'TOP motif and non-TOP 5'UTR of β-actin used as a negative control. Total protein content in lysates used for luciferase assay. t-test, n=4-5 biological replicates per group, ** - P ≤ 0.01, *** - P ≤ 0.001.


Figure 3 ( A ) Representative immunoblot of Cand2 protein levels in mouse organs . ( C ) Immunofluorescent staining of paraffin sections from hearts from WT and Cand2 KO mice . CAND2 ( stained in red ) , ACTIN ( stained in green ) and nuclei ( stained with DAPI , blue ) in . Scale bar 20μm ( F ) Immunoblot of subcellular fractionation of left ventricle ( LV ) . Gapdh and Histone 3D ( H3D ) used as cytoplasmic and nuclear markers , respectively . WCL - whole cell lysate , cyt - cytoplasmic fraction , nuc - nuclear fraction .

O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative immunoblot of Cand2 protein levels in mouse organs.(C) Immunofluorescent staining of paraffin sections from hearts from WT and Cand2 KO mice. CAND2 (stained in red), ACTIN (stained in green) and nuclei (stained with DAPI, blue) in. Scale bar 20μm(F) Immunoblot of subcellular fractionation of left ventricle (LV). Gapdh and Histone 3D (H3D) used as cytoplasmic and nuclear markers, respectively. WCL - whole cell lysate, cyt - cytoplasmic fraction, nuc - nuclear fraction.


Figure 4 ( E ) NRCMs size measurement after Cand2 OE and 24h PE stimulation . Analyzed by One - way ANOVA . n > 150 cells from n = 3 independent experiments . Violin plot : the middle horizontal lane - median , upper lane - 3rd quartile , lower lane - 1st quartile . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001 ( F ) Nppa mRNA levels in vitro after Cand2 OE and PE treatment measured by RT - qPCR . Analysed by One - way ANOVA . n = 5 - 9 biological replicates per conditions . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001 ( J ) Nppa and Nppb mRNA levels 4 weeks after TAC in WT and Cand2 KO mice measured by RT - qPCR . Analyzed by One - way ANOVA . n = 6 - 13 mice per group . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(E) NRCMs size measurement after Cand2 OE and 24h PE stimulation. Analyzed by One-way ANOVA. n>150 cells from n=3 independent experiments. Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001(F) Nppa mRNA levels in vitro after Cand2 OE and PE treatment measured by RT-qPCR. Analysed by One-way ANOVA. n=5-9 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001(J) Nppa and Nppb mRNA levels 4 weeks after TAC in WT and Cand2 KO mice measured by RT-qPCR. Analyzed by One-way ANOVA. n=6-13 mice per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


Figure 5 ( B ) Immunoblots and quantification of Grk5 protein levels in vitro after Cand2 KD with siRNA and after Cand2 OE with AAV6 . t - test , n = 4 - 5 biological replicates per group . Data information : Error bars indicate means ± SEM . * - P ≤ 0 . 05 , * * * - P ≤ 0 . 001 . ( F ) Representative immunoblot of Grk5 protein levels and quantification in cytoplasmic and nuclear fractions from left ventricle lysates of WT and Cand2 KO mice after TAC surgery . Lamin B - nuclear marker , Gapdh - cytoplasmic marker . Analyzed by One - way ANOVA , n = 3 biological replicates . Data information : Error bars indicate means ± SEM . * - P ≤ 0 . 05 , * * * - P ≤ 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Immunoblots and quantification of Grk5 protein levels in vitro after Cand2 KD with siRNA and after Cand2 OE with AAV6. t-test, n=4-5 biological replicates per group. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.(F) Representative immunoblot of Grk5 protein levels and quantification in cytoplasmic and nuclear fractions from left ventricle lysates of WT and Cand2 KO mice after TAC surgery. Lamin B - nuclear marker, Gapdh - cytoplasmic marker. Analyzed by One-way ANOVA, n=3 biological replicates. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.


Figure 6 ( B ) mRNA levels of MEF2 - dependent genes Nr4a1 and Xirp2 in NRCMs with Cand2 KD for indicated groups . PE treatment for 24h ( 50uM ) . Analyzed by One - way ANOVA , n = 6 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001 ( E ) Grk5 protein and mRNA levels in NRCMs as well as MEF2 - luciferase activity after 24h of Torin 1 treatment . Analyzed by t - test , n = 4 - 6 biological replicates per conditions . Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein ( S235 / 236rpS6 ) . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001 ( I ) Grk5 , Nppa , and MEF2 targets Nr4a1 and Xirp2 mRNA levels in NRCMs after Raptor KD . Analyzed by t - test , n = 3 biological replicates per conditions . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs with Cand2 KD for indicated groups. PE treatment for 24h (50uM). Analyzed by One-way ANOVA, n=6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001(E) Grk5 protein and mRNA levels in NRCMs as well as MEF2-luciferase activity after 24h of Torin 1 treatment. Analyzed by t-test, n=4-6 biological replicates per conditions. Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein (S235/236rpS6). Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001(I) Grk5, Nppa, and MEF2 targets Nr4a1 and Xirp2 mRNA levels in NRCMs after Raptor KD. Analyzed by t-test, n=3 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


Figure 7 ( B ) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2 . NRCMs were treated with MLN4924 ( 100 nM ) to block Cul1 neddylation . Quantification of neddylated and unnedylated Cul1 protein levels after MLN4924 treatment for 24h . t - test , n = 4 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001 ( D ) Grk5 expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition ( MLN ) analysed by immunoblot and RT - qPCR . Analyzed by One - way ANOVA , n = 4 - 5 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001 ( H ) mRNA levels of MEF2 - dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups . Analyzed by One - way ANOVA , n = 6 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2. NRCMs were treated with MLN4924 (100 nM) to block Cul1 neddylation. Quantification of neddylated and unnedylated Cul1 protein levels after MLN4924 treatment for 24h. t-test, n=4 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001(D) Grk5 expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition (MLN) analysed by immunoblot and RT-qPCR. Analyzed by One-way ANOVA, n=4-5 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001(H) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups. Analyzed by One-way ANOVA, n= 6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


Figure 8 ( A ) Quantification of indicated mRNA levels 2 days after TAC measured by RT - qPCR . Analyzed by One - way ANOVA . n = 5 - 7 biological replicates per group . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 . ( C ) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 2 days after sham and TAC analyzed by immunoblotting as well as quantification of mRNA levels analyzed by RT - qPCR . Analyzed by One - way ANOVA . n = 3 - 5 biological replicates per group . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 . ( D ) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in Cand2 KO mice compared to WT mice . Analyzed by t - test . n = 5 - 8 mice per group . Cul1 neddylation was inhibited in C2C12 by MLN4924 ( - / + MLN ) for neddylated Cul1 band detection . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Quantification of indicated mRNA levels 2 days after TAC measured by RT-qPCR. Analyzed by One-way ANOVA. n=5-7 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.(C) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 2 days after sham and TAC analyzed by immunoblotting as well as quantification of mRNA levels analyzed by RT-qPCR. Analyzed by One-way ANOVA. n=3-5 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.(D) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in Cand2 KO mice compared to WT mice. Analyzed by t-test. n=5-8 mice per group. Cul1 neddylation was inhibited in C2C12 by MLN4924 (-/+ MLN) for neddylated Cul1 band detection. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.


Figure 1Macrophage - conditioned medium induces MCF10A colony formation in soft agar as compared to control medium . Colonies ≥ 50 μm were counted at 5 weeks . Error bars represent mean ± SEM from 10 independent experiments ( n = 3 per condition ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei ( clear , partially filled , almost filled and filled ) . Spheroids stained for DNA ( Hoechst 33342 in blue ) and F - actin ( Phalloidin in red ) . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Macrophage - conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control . Error bars represent mean ± SEM from 11 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Representative images of Ki67 positive nuclei of MCF10A cells . Spheroids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and Ki67 ( green ) . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage - conditioned media . Error bars represent mean ± SEM from 3 independent experiments ( 20 - 30 non - empty spheroids per condition ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . NSC23766 ( Rac1 inhibitor ; 50 μM ) reduces the filling of the spheroid lumen with cell nuclei upon macrophage - conditioned medium stimulation compared to control . Filling of the spheroid lumen with cell nuclei categorized into 4 groups ( clear , partially filled , almost filled and filled ) . Error bars represent mean ± SEM from 3 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled and filled MCF10A spheroids were combined together ( non - empty spheroids ) for statistical analysis . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Invasive protrusions of spheroids into matrigel / collagen mix marked with a white arrow . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . 3D structures of spheroids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and laminin V ( green ) . Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions . Invasive protrusions are marked with a white arrow . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Macrophage - conditioned medium induces invasive protrusions in MCF10A spheroids compared to control . Error bars represent mean ± SEM from 11 independent experiments ( n = 2 per condition ; at least 15 spheroids each from at least 2 fields of view ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Macrophage-conditioned medium induces MCF10A colony formation in soft agar as compared to control medium. Colonies ≥ 50 μm were counted at 5 weeks. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei (clear, partially filled, almost filled and filled). Spheroids stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red).16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; 50 spheroids each). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of Ki67 positive nuclei of MCF10A cells. Spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and Ki67 (green).16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage-conditioned media. Error bars represent mean ± SEM from 3 independent experiments (20-30 non-empty spheroids per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. NSC23766 (Rac1 inhibitor; 50 μM) reduces the filling of the spheroid lumen with cell nuclei upon macrophage-conditioned medium stimulation compared to control. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled MCF10A spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Invasive protrusions of spheroids into matrigel/collagen mix marked with a white arrow.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. 3D structures of spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and laminin V (green). Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions. Invasive protrusions are marked with a white arrow.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces invasive protrusions in MCF10A spheroids compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.