G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells ( p ≤ 0 . 001 ) . All groups were compared against each other with a one - way ANOVA analysis , followed by Turkey test . M phase cells have the lowest NICD . Each point in the box - plot represents the normalized intensity of a single cell . Data from three different experiments ( n = 3 ) was randomised collated and plotted .

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G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells (p≤0.001). All groups were compared against each other with a one-way ANOVA analysis, followed by Turkey test. M phase cells have the lowest NICD. Each point in the box-plot represents the normalized intensity of a single cell. Data from three different experiments (n=3) was randomised collated and plotted.


Purvalanol B treatment reduced the NICD - FBXW7 interaction . 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0 . 1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin has been used as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 treated samples . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * * p ≤ 0 . 001 .

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Purvalanol B treatment reduced the NICD-FBXW7 interaction. 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0.1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin has been used as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.


E10 . 5 mouse tails were bissected down the midline . One half ( + ) was cultured for 4 hrs in the presence of Purvalanol B ( 1 µM ) . The contralateral half ( - ) was cultured for 4h in the presence of DMSO . Control or treated explants were pooled and NICD levels were detected by western blot . β - Actin was used as loading control .

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E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of Purvalanol B (1 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin was used as loading control.


E10 . 5 mouse tails were bissected down the midline . One half ( + ) was cultured for 4 hrs in the presence of RO - 3306 ( 10 µM ) . The contralateral half ( - ) was cultured for 4h in the presence of DMSO . Control or treated explants were pooled and NICD levels were detected by western blot . β - Actin has been used as loading control .

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E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of RO-3306 (10 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin has been used as loading control.


Bissected E10 . 5 mouse PSM explants were cultured in the absence ( - ) or presence ( + ) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression . Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the " - " explant . n = 18 . Scale bar is 100 µm .

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Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the "-" explant. n=18. Scale bar is 100 µm.


Bissected E10 . 5 mouse PSM explants were cultured in the absence ( - ) or presence ( + ) of 10 µM of RO - 3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression . RO - 3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the " - " explant . n = 10 . Scale bar is 100 µm .

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Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 10 µM of RO-3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. RO-3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the "-" explant. n=10. Scale bar is 100 µm.


The mean log2 fold change in successive somite length at position + 1 ( last formed somite ) is plotted for different treatment conditions . Error bars denote standard error of the mean . Paired t - test analysis was performed at 5 % significance level , with p = 0 . 0437 for RO - 3006 and p = 0 . 0524 for PurB , compared to DMSO . n = 14 . The mean log2 fold change in successive somite length at position + 2 ( penultimate formed somite ) is plotted for different treatment conditions . Error bars denote standard error of the mean . Paired t - test analysis was performed at 5 % significance level , with p = 0 . 6409 for RO - 3006 and p = 0 . 4065 for PurB , compared to DMSO . n = 14 .

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The mean log2 fold change in successive somite length at position +1 (last formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.0437 for RO-3006 and p= 0.0524 for PurB, compared to DMSO. n=14.   The mean log2 fold change in successive somite length at position +2 (penultimate formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.6409 for RO-3006 and p= 0.4065 for PurB, compared to DMSO. n=14.


A Speed of propidium iodide ( PI ) penetration into fibroblasts during 60 min of fixation with either 4 % PFA or 3 % glyoxal . N = 3 independent experiments . Glyoxal fixation enables PI to penetrate far more rapidly into the cells .

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A Speed of propidium iodide (PI) penetration into fibroblasts during 60 min of fixation with either 4 % PFA or 3 % glyoxal. N = 3 independent experiments. Glyoxal fixation enables PI to penetrate far more rapidly into the cells.


B Speed of FM 1 - 43 penetration in similar experiments . The arrowhead points to one example of ongoing endocytosis during PFA fixation . N = 3 - 4 independent experiments . The general pattern of FM 1 - 43 entry was similar to that of propidium iodide . Only the first 10 minutes are shown , to enable an optimal observation of the kinetics of the first stages of FM 1 - 43 entry . The results parallel those obtained with PI : faster penetration during glyoxal fixation . Scale bar = 40 µm , * p < 0 . 05 , * * p < 0 .

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B Speed of FM 1-43 penetration in similar experiments. The arrowhead points to one example of ongoing endocytosis during PFA fixation. N = 3-4 independent experiments. The general pattern of FM 1-43 entry was similar to that of propidium iodide. Only the first 10 minutes are shown, to enable an optimal observation of the kinetics of the first stages of FM 1-43 entry. The results parallel those obtained with PI: faster penetration during glyoxal fixation. Scale bar = 40 µm, * p < 0.05, ** p < 0.01


Figure 2 A comparison of morphological changes taking place during fixation with PFA or glyoxal . The changes were visualized by DIC images taken at 5 minute intervals during fixation . The graph shows the correlation of each image to the first frame . N = 50 ( PFA ) and 54 ( glyoxal ) cellular regions analyzed , from 3 independent experiments . The higher correlation value indicates that glyoxal preserves the initial cell morphology with higher accuracy than PFA . Scale bar = 20 µm , * p < 0 . 05 , * * p < 0 . 01

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Figure 2 A comparison of morphological changes taking place during fixation with PFA or glyoxal. The changes were visualized by DIC images taken at 5 minute intervals during fixation. The graph shows the correlation of each image to the first frame. N = 50 (PFA) and 54 (glyoxal) cellular regions analyzed, from 3 independent experiments. The higher correlation value indicates that glyoxal preserves the initial cell morphology with higher accuracy than PFA. Scale bar = 20 µm, * p < 0.05, ** p < 0.01


A SDS - PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives . The graph shows the summed intensity of the bands in each lane . Fixed proteins either no longer run into the gel or form only smears . To compare the efficiency of fixation , the bands that survive fixation were summed and were expressed as % of an unfixed control . The intensity of PFA - fixed samples was significantly higher than that of glyoxal - fixed samples ( N = 5 independent experiments ) . Glut = 0 . 2 % glutaraldehyde .

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A SDS-PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives. The graph shows the summed intensity of the bands in each lane. Fixed proteins either no longer run into the gel or form only smears. To compare the efficiency of fixation, the bands that survive fixation were summed and were expressed as % of an unfixed control. The intensity of PFA-fixed samples was significantly higher than that of glyoxal-fixed samples (N = 5 independent experiments). Glut = 0.2% glutaraldehyde.


B Staining of nucleic acids after fixation . The propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 ( N = 6 - 8 ) . To test whether the fixed nucleic acids were still available for specific detection , we performed FISH for GAPDH in cultured neurons , using a standard protocol provided by the company Affymetrix . The fluorescence signal of the samples fixed with glyoxal ( pH 4 ) was significantly higher than for PFA - fixed samples ( N = 5 - 6 ) . Scale bar = 10 µm , * p < 0 . 05 , * * p < 0 .

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B Staining of nucleic acids after fixation. The propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 (N = 6-8). To test whether the fixed nucleic acids were still available for specific detection, we performed FISH for GAPDH in cultured neurons, using a standard protocol provided by the company Affymetrix. The fluorescence signal of the samples fixed with glyoxal (pH 4) was significantly higher than for PFA-fixed samples (N = 5-6). Scale bar = 10 µm, * p < 0.05, ** p < 0.01


Figure 4 STED imaging of primary hippocampal neurons fixed with either PFA or glyoxal . Strong differences in labeling patterns can be observed . The images are brighter and less " spotty " for the glyoxal - fixed samples . Structures such as filaments or organelles are more easily detected . Quantification of the fluorescence signal ( fold over background ) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to the PFA fixed samples . N = 35 - 132 objects . Scale bar = 6 µm for β - actin and α - tubulin , and 3 µm for the other proteins . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 4 STED imaging of primary hippocampal neurons fixed with either PFA or glyoxal. Strong differences in labeling patterns can be observed. The images are brighter and less "spotty" for the glyoxal-fixed samples. Structures such as filaments or organelles are more easily detected. Quantification of the fluorescence signal (fold over background) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to the PFA fixed samples. N = 35 - 132 objects. Scale bar = 6 µm for β-actin and α-tubulin, and 3 µm for the other proteins. ** p < 0.01, *** p < 0.001


Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal . HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA , glyoxal pH 4 or glyoxal pH 5 . Fluorescence intensities ( fold over background ) were compared and are shown in the graph . The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings . N = 73 - 156 cells per condition analyzed . Scale bar = 10 µm . * * * p < 0 . 001

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Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal. HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA, glyoxal pH 4 or glyoxal pH 5. Fluorescence intensities (fold over background) were compared and are shown in the graph. The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings. N = 73 - 156 cells per condition analyzed. Scale bar = 10 µm. *** p < 0.001


Figure 6 Comparison of immunostained AtT20 cells after fixation with either PFA or glyoxal . AtT20 cells stained for the SNARE proteins syntaxin1 and SNAP25 and the autophagy marker LC3B , were compared with regard to the fluorescence intensity ( fold over background ) of the stainings . Quantification of the intensity shows that glyoxal fixation allows for significantly brighter stainings of the membrane SNARE proteins . LC3B staining is brighter in PFA fixed cells . N = 9 - 20 cells per condition . Scale bar = 5 µm . * p < 0 . 05 , * * * p < 0 . 001

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Figure 6 Comparison of immunostained AtT20 cells after fixation with either PFA or glyoxal. AtT20 cells stained for the SNARE proteins syntaxin1 and SNAP25 and the autophagy marker LC3B, were compared with regard to the fluorescence intensity (fold over background) of the stainings. Quantification of the intensity shows that glyoxal fixation allows for significantly brighter stainings of the membrane SNARE proteins. LC3B staining is brighter in PFA fixed cells. N = 9 - 20 cells per condition. Scale bar = 5 µm. * p < 0.05, *** p < 0.001


A Primary hippocampal neurons were stained for Actin and Ankyrin G . A comparison between PFA - and glyoxal - fixed samples shows that actin staining with phalloidin works as least as well in both , showing the prominent actin rings . Ankyrin G staining is brighter in PFA - fixed cells .

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A Primary hippocampal neurons were stained for Actin and Ankyrin G. A comparison between PFA- and glyoxal-fixed samples shows that actin staining with phalloidin works as least as well in both, showing the prominent actin rings. Ankyrin G staining is brighter in PFA-fixed cells.


B Primary hippocampal neurons were stained for pan - Nav and Kv7 . 2 . Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons . Staining of K - channels shows a slightly more regular pattern in glyoxal fixed neurons .

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B Primary hippocampal neurons were stained for pan-Nav and Kv7.2. Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons. Staining of K-channels shows a slightly more regular pattern in glyoxal fixed neurons.


C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin . While the spectrin staining seems to be equally well in both fixation conditions , neurofilament staining is brighter in glyoxal fixed cells .

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C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin. While the spectrin staining seems to be equally well in both fixation conditions, neurofilament staining is brighter in glyoxal fixed cells.


D Growth cones of hippocampal neurons were stained for actin and βIII - tubulin after either glyoxal or PFA + glutaraldehyde fixation . The latter is a standard fixation used for the co - labeling of tubulin and actin , and is a stronger fixation than normal PFA fixation , which is incompatible with many organelle immunostainings ( unlike glyoxal fixation ) . The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal , whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures . Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde . Scale bars = 1 µm .

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D Growth cones of hippocampal neurons were stained for actin and βIII-tubulin after either glyoxal or PFA + glutaraldehyde fixation. The latter is a standard fixation used for the co-labeling of tubulin and actin, and is a stronger fixation than normal PFA fixation, which is incompatible with many organelle immunostainings (unlike glyoxal fixation). The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal, whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures. Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde. Scale bars = 1 µm.


Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal . Sepia fin was fixed with the respective fixative and stained for the neuropeptide FMRFamide . A clear change in morphology can be observed between samples fixed with PFA and samples fixed with glyoxal . The former appear broken and swollen , while the glyoxal - fixed ones appear complete . The effect is presumably due to the different speed of penetration into tissue and / or fixation . Scale bar = 5 µm .

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Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal. Sepia fin was fixed with the respective fixative and stained for the neuropeptide FMRFamide. A clear change in morphology can be observed between samples fixed with PFA and samples fixed with glyoxal. The former appear broken and swollen, while the glyoxal-fixed ones appear complete. The effect is presumably due to the different speed of penetration into tissue and/or fixation. Scale bar = 5 µm.


Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal . Freshly isolated murine ventricular myocytes were either fixed with 4 % PFA or 3 % glyoxal and immunostained for caveolin - 3 or ryanodine receptor type 2 . Quantification of the fluorescence intensity of the stainings show significantly brighter stainings for glyoxal fixed myocytes . The graph shows mean values and error bars represent standard deviations . N = 10 ( RyR2 ) and 12 ( Cav3 ) myocytes per condition . Scale bar = 2 µm . * * * p < 0 . 001

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Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal. Freshly isolated murine ventricular myocytes were either fixed with 4% PFA or 3% glyoxal and immunostained for caveolin-3 or ryanodine receptor type 2. Quantification of the fluorescence intensity of the stainings show significantly brighter stainings for glyoxal fixed myocytes. The graph shows mean values and error bars represent standard deviations. N = 10 (RyR2) and 12 (Cav3) myocytes per condition. Scale bar = 2 µm. *** p < 0.001


Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal . Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins . The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins ( CtBP2 , Calretinin and Homer1 ) fixed with glyoxal . None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation . Representative images show maximum intensity projections from z - stacks of inner hair cell ribbon synapses . N = 5 independent stainings from two animals per condition ( PSD95 , CtBP2 , Otoferlin , Calretinin ) and 10 - 15 images per condition ( CaV1 . 3 and Homer1 ) . Scale bar = 2 µm . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal. Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins. The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins (CtBP2, Calretinin and Homer1) fixed with glyoxal. None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation. Representative images show maximum intensity projections from z-stacks of inner hair cell ribbon synapses. N = 5 independent stainings from two animals per condition (PSD95, CtBP2, Otoferlin, Calretinin) and 10 - 15 images per condition (CaV1.3 and Homer1). Scale bar = 2 µm. ** p < 0.01, *** p < 0.001


Figure 11 Comparison of stained vimentin and α - synuclein in human neuroglioma cells after fixation with either PFA or glyoxal . Cells were fixed with PFA or glyoxal for 10 minutes , and were stained for endogenous vimentin , or for expressed alpha - synuclein . A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha - synuclein , but that PFA was superior for endogenous vimentin ( leftmost graph ) . The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal ; the latter allowed more mOrange2 fluorescence to be detected ( rightmost graph ) . N = 29 - 81 cell regions per condition . * p < 0 . 05 , * * * p < 0 . 001 Scale bar : 10 µm .

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Figure 11 Comparison of stained vimentin and α-synuclein in human neuroglioma cells after fixation with either PFA or glyoxal. Cells were fixed with PFA or glyoxal for 10 minutes, and were stained for endogenous vimentin, or for expressed alpha-synuclein. A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha-synuclein, but that PFA was superior for endogenous vimentin (leftmost graph). The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal; the latter allowed more mOrange2 fluorescence to be detected (rightmost graph). N = 29 - 81 cell regions per condition. * p < 0.05, *** p < 0.001 Scale bar: 10 µm.


Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal . Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B , COA6 , NDUFA9 and TIM23 . Quantification of the staining intensity shows a significant increase of fluorescence ( signal over background ) for 2 markers ( ATP5B and Mitotracker ) after fixation with glyoxal , whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA , albeit the differences are small . N = 18 - 128 cells per condition . Scale bar = 5 µm . * * * p < 0 . 001

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Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal. Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B, COA6, NDUFA9 and TIM23. Quantification of the staining intensity shows a significant increase of fluorescence (signal over background) for 2 markers (ATP5B and Mitotracker) after fixation with glyoxal, whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA, albeit the differences are small. N = 18 - 128 cells per condition. Scale bar = 5 µm. *** p < 0.001


Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal . The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal . Quantification is shown as percentage of signal derived from PFA fixed cells . 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal . Only 4 proteins show significantly reduced staining intensity . For LC3B the intensity of less than 5 cells was quantified , therefore single data points were plotted in addition to the bars . N = 3 - 44 cells per condition . Scale bar = 10 µm . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal. The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal. Quantification is shown as percentage of signal derived from PFA fixed cells. 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal. Only 4 proteins show significantly reduced staining intensity. For LC3B the intensity of less than 5 cells was quantified, therefore single data points were plotted in addition to the bars. N = 3 - 44 cells per condition. Scale bar = 10 µm. ** p < 0.01, *** p < 0.001


Figure 14 Comparison of immunostained U2OS cells and primary hippocampal neurons after either PFA or glyoxal fixation . Immunostaining of the Na / K ATPase in primary hippocampal neurons shows a different distribution of the protein between PFA fixed and nucleus fixed samples . While in PFA - fixed neurons the antibody falsely stains the glyoxal as well as the cytoplasm ( 100 % of the 82 cells we analyzed ) , in nucleus - fixed neurons the U2OS is devoid of signal , and the membrane appears to be correctly labeled ( arrowheads ; 100 % of the 60 cells we analyzed ) . mitochondria cells were immunostained for KDEL ( Tom20 ) and ER ( EGFP - KDEL ) . The fixation and / or staining of mitochondria seems to be comparable in glyoxal and in PFA fixed cells . The staining of the ER shows an improved signal to noise ratio . The signal appears de - localized from the ER for multiple PFA - stained cells ( 25 % of 36 analyzed cells , see arrows ) , while this is rare for the glyoxal - stained cells ( 3 . 4 % of 58 analyzed cells ) . Scale bar = 10 µm .

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Figure 14 Comparison of immunostained U2OS cells and primary hippocampal neurons after either PFA or glyoxal fixation. Immunostaining of the Na/K ATPase in primary hippocampal neurons shows a different distribution of the protein between PFA fixed and nucleus fixed samples. While in PFA-fixed neurons the antibody falsely stains the glyoxal as well as the cytoplasm (100% of the 82 cells we analyzed), in nucleus-fixed neurons the U2OS is devoid of signal, and the membrane appears to be correctly labeled (arrowheads; 100% of the 60 cells we analyzed). mitochondria cells were immunostained for KDEL (Tom20) and ER (EGFP-KDEL). The fixation and/or staining of mitochondria seems to be comparable in glyoxal and in PFA fixed cells. The staining of the ER shows an improved signal to noise ratio. The signal appears de-localized from the ER for multiple PFA-stained cells (25% of 36 analyzed cells, see arrows), while this is rare for the glyoxal-stained cells (3.4% of 58 analyzed cells). Scale bar = 10 µm.


A - B Confocal images showing staining of Olfactory Marker Protein ( OMP ) and ß3 - tubulin along the dorsal aspect of the mouse olfactory epithelium . While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata , their dendrites , and axons , the axon bundles ( green arrow ) located above the olfactory epithelium exemplify the clear signal - to - noise ratio benefits of glyoxal fixation versus that of the PFA - fixative . Immunostaining with the ß3 - tubulin antibody stains the dendrites and axons in both PFA and glyoxal - fixed tissue , but strong staining of the cilia can only be observed in the glyoxal - fixed sections ( B ) .

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A - B Confocal images showing staining of Olfactory Marker Protein (OMP) and ß3-tubulin along the dorsal aspect of the mouse olfactory epithelium. While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata, their dendrites, and axons, the axon bundles (green arrow) located above the olfactory epithelium exemplify the clear signal-to-noise ratio benefits of glyoxal fixation versus that of the PFA-fixative. Immunostaining with the ß3-tubulin antibody stains the dendrites and axons in both PFA and glyoxal-fixed tissue, but strong staining of the cilia can only be observed in the glyoxal-fixed sections (B).


C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb . Identities of the axons are in part defined by the neuropilin - 1 ( Nrp1 ) and neuropilin - 2 ( Nrp2 ) expression levels , visualized here with antibodies raised against the two proteins . While complementary expression of the two molecules can be seen in the PFA - fixed sections ( C ) , the glyoxal - fixed sections ( D ) exhibit profoundly improved signal - to - noise ratios for Nrp - 1 ( red arrows ) , and in the case of Nrp - 2 , also the segmentation of the axon bundle into varying levels of Nrp - 2 ( green arrows ) .

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C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb. Identities of the axons are in part defined by the neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) expression levels, visualized here with antibodies raised against the two proteins. While complementary expression of the two molecules can be seen in the PFA-fixed sections (C), the glyoxal-fixed sections (D) exhibit profoundly improved signal-to-noise ratios for Nrp-1 (red arrows), and in the case of Nrp-2, also the segmentation of the axon bundle into varying levels of Nrp-2 (green arrows).


E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons . The axons of olfactory sensory neurons can be readily visualized with PFA staining ( green ) in the superficial olfactory nerve layer and terminating in glomeruli located below ( green arrows ) . While sections fixed with either PFA or glyoxal display adequate staining levels , the signal distribution of the Immunostaining - fixed tissue appears more irregular ( E ) , seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal - fixed sections ( F ) . The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral / tufted cells as well as PFA of periglomerular neurons . Immunostaining with Vesicular Glutamate Transporter 2 ( VGLUT2 ) allows visualization of these structures and is easily seen in the glyoxal - fixed section ( F ) , while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA - fixed panel ( red arrows in E ) . Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal , albeit weaker versus Glyoxal ( inset ) . Staining with ß3 - tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal - fixed tissue , but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli ( blue arrows ) and in the external plexiform layer located below the glomeruli .

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E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons. The axons of olfactory sensory neurons can be readily visualized with PFA staining (green) in the superficial olfactory nerve layer and terminating in glomeruli located below (green arrows). While sections fixed with either PFA or glyoxal display adequate staining levels, the signal distribution of the Immunostaining-fixed tissue appears more irregular (E), seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal-fixed sections (F). The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral/tufted cells as well as PFA of periglomerular neurons. Immunostaining with Vesicular Glutamate Transporter 2 (VGLUT2) allows visualization of these structures and is easily seen in the glyoxal-fixed section (F), while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA-fixed panel (red arrows in E). Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal, albeit weaker versus Glyoxal (inset). Staining with ß3-tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal-fixed tissue, but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli (blue arrows) and in the external plexiform layer located below the glomeruli.


Morphological changes of vacuolar membranes and peroxisomal GFP - SKL during microautophagic degradation of peroxisomes after glucose adaptation . Double fluorescence of methanol - grown cells of P . pastoris cells at various times after the switch to glucose medium . ( a ) 0 h , ( b ) 30 min , ( c ) 2 h .

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Morphological changes of vacuolar membranes and peroxisomal GFP-SKL during microautophagic degradation of peroxisomes after glucose adaptation. Double fluorescence of methanol-grown cells of P. pastoris cells at various times after the switch to glucose medium. (a) 0 h, (b) 30 min, (c) 2 h.


Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes . The three panels in each figure are as follows ( a - e , left ) FM4 - 64 ; ( center ) GFP - SKL ; ( right ) superimposed FM4 - 64 and GFP - SKL signals . ( a , f ) stage 0 ; ( b , g ) early stage 1 ; ( c , h ) late stage 1 ; ( d , i ) stage 2 ; ( e ) stage 3 . ( d , arrowhead ) During the capture of these fluorescence images , this peroxisome - containing vesicle had just pinched off from the outer vacuolar membrane . Therefore , the FM4 - 64 - stained vesicle membrane and GFP - SKL could not be superimposed in this picture .

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Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes. The three panels in each figure are as follows (a-e, left) FM4-64; (center) GFP-SKL; (right) superimposed FM4-64 and GFP-SKL signals. (a, f) stage 0; (b, g) early stage 1; (c, h) late stage 1; (d, i) stage 2; (e) stage 3. (d, arrowhead) During the capture of these fluorescence images, this peroxisome-containing vesicle had just pinched off from the outer vacuolar membrane. Therefore, the FM4-64-stained vesicle membrane and GFP-SKL could not be superimposed in this picture.


Kinetics of morphological changes in vacuoles and peroxisomalGFP - SKL during microautophagic degradation of P . pastoris in glucose adaptation . At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point . Cells were counted in three randomly selected fields , and standard deviations are shown as vertical bars .

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Kinetics of morphological changes in vacuoles and peroxisomalGFP-SKL during microautophagic degradation of P. pastoris in glucose adaptation. At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point. Cells were counted in three randomly selected fields, and standard deviations are shown as vertical bars.


Macroautophagic degradation of peroxisomes after transfer of methanol - grown P . pastoris cells to ethanol medium . ( a - e ) Electron micrographs of intermediates seen during macroautophagy . ( a ) After 1 h of ethanol adaptation , peroxisomes ( P ) were often surrounded by autophagosomal membranes , higher magnifications of which are in d and e . ( b ) After 1 h of ethanol adaptation , the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane . ( c ) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole . Magnified pictures of samples obtained at the ( d ) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes , and at the ( e ) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes . ( f ) Kinetics of morphological changes in vacuoles and peroxisomal GFP - SKL . Other conditions are described in Materials and Methods , and are the same as in Fig . 4 . ( g ) Fluorescence images taken after 2 h of ethanol adaptation of wild - type P . pastoris cells .

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Macroautophagic degradation of peroxisomes after transfer of methanol-grown P. pastoris cells to ethanol medium. (a-e) Electron micrographs of intermediates seen during macroautophagy. (a) After 1 h of ethanol adaptation, peroxisomes (P) were often surrounded by autophagosomal membranes, higher magnifications of which are in d and e. (b) After 1 h of ethanol adaptation, the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane. (c) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole. Magnified pictures of samples obtained at the (d) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes, and at the (e) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes. (f) Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL. Other conditions are described in Materials and Methods, and are the same as in Fig. 4. (g) Fluorescence images taken after 2 h of ethanol adaptation of wild-type P. pastoris cells.


Effect of inhibitors on transport of peroxisomal GFP - SKL to , and degradation in , vacuoles during ( a ) glucose adaptation and ( b ) ethanol adaptation . Wortmannin and PMSF were dissolved in DMSO , and added to the glucose medium . ( Filled circle ) Control ( 2 % DMSO ) ; ( open circle ) 1 mM wortmannin ( with 2 % DMSO ) ; ( filled square ) 3 mM PMSF ( with 2 % DMSO ) ; ( filled triangle ) 10 mg / ml cycloheximide .

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Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation. Wortmannin and PMSF were dissolved in DMSO, and added to the glucose medium. (Filled circle) Control (2% DMSO); (open circle) 1 mM wortmannin (with 2% DMSO); (filled square) 3 mM PMSF (with 2% DMSO); (filled triangle) 10 mg/ml cycloheximide.


Microautophagic degradation of peroxisomes is not observed in a vacuolar protease - negative strain ( STK7 ) of P . pastoris . Kinetics of morphological changes in vacuoles and peroxisomal GFP - SKL during ( a ) glucose adaptation and ( b ) ethanol adaptation . ( c ) Fluorescence images taken after 2 h of glucose adaptation . Arrows show GFP - SKL in vacuolar matrix . Some cells showed diffusion of GFP - SKL into the vacuolar matrix .

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Microautophagic degradation of peroxisomes is not observed in a vacuolar protease-negative strain (STK7) of P. pastoris. Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL during (a) glucose adaptation and (b) ethanol adaptation. (c) Fluorescence images taken after 2 h of glucose adaptation. Arrows show GFP-SKL in vacuolar matrix. Some cells showed diffusion of GFP-SKL into the vacuolar matrix.


( a ) Visualization of alcohol oxidase activity in colonies after ( a ) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild - type ( strain PPY12 ) and pag mutant cells ( strains STK1 - STK6 , Materials and Methods ) of P . pastoris . ( b ) Western - blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time . Antibodies used were AOX , alcohol oxidase ; F1 , mitochondrial F1β subunit ; G6PDH , glucose - 6 - phosphate dehydrogenase . Growth curve of the wild - type strain PPY12 and the pag4 mutant ( strain STK4 ) after shift from methanol to glucose .

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(a) Visualization of alcohol oxidase activity in colonies after (a) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild-type (strain PPY12) and pag mutant cells (strains STK1-STK6, Materials and Methods) of P. pastoris. (b) Western-blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time. Antibodies used were AOX, alcohol oxidase; F1, mitochondrial F1β subunit; G6PDH, glucose-6-phosphate dehydrogenase. Growth curve of the wild-type strain PPY12 and the pag4 mutant (strain STK4) after shift from methanol to glucose.


Fluorescence images of ( a ) pag2 ( strain STK2 ) and ( b ) pag3 ( strain STK3 ) mutants of P . pastoris after a 3 h shift to glucose . ( b ) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant .

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Fluorescence images of (a) pag2 (strain STK2) and (b) pag3 (strain STK3) mutants of P. pastoris after a 3 h shift to glucose. (b) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant.


Fluorescence images of the pag4 ( strain STK4 ) mutant of P . pastoris after ( a ) 0 , ( b ) 3 , or ( c ) 6 h of glucose adaptation . The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation .

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Fluorescence images of the pag4 (strain STK4) mutant of P. pastoris after (a) 0, (b) 3, or (c) 6 h of glucose adaptation. The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation.


Microautophagy of peroxisomes in a pex1 - ts mutant ( strain SJH242 ) of P . pastoris expressing GFP - SKL from the constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) promoter at the restrictive temperature for peroxisome assembly ( 34 ° C ) . ( a ) Kinetics of peroxisome degradation at 34 ° C . Strain SJH242 was induced on methanol at 23 ° C , and glucose adaptation was performed at 34 ° C . ( b ) Changes in vacuole morphology were seen even in the absence of normal peroxisomes . At 0 h , SJH242 cells were grown at 34 ° C when GFP - SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies . After 2 h , ∼ 20 - 30 % vacuoles showed complicated membrane structures representative of stage 2 cells .

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Microautophagy of peroxisomes in a pex1-ts mutant (strain SJH242) of P. pastoris expressing GFP-SKL from the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter at the restrictive temperature for peroxisome assembly (34°C). (a) Kinetics of peroxisome degradation at 34°C. Strain SJH242 was induced on methanol at 23°C, and glucose adaptation was performed at 34°C. (b) Changes in vacuole morphology were seen even in the absence of normal peroxisomes. At 0 h, SJH242 cells were grown at 34°C when GFP-SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies. After 2 h, ∼20-30% vacuoles showed complicated membrane structures representative of stage 2 cells.


A Deletion of siaA , siaB , siaC , or siaD regulates biofilm formation . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down )

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A Deletion of siaA, siaB, siaC, or siaD regulates biofilm formation. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down)


B The intracellular levels of c - di - GMPin PAO1 , ΔsiaC , ΔsiaB and the corresponding complemented strains were detected by LC - MS / MS

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B The intracellular levels of c-di-GMPin PAO1, ΔsiaC, ΔsiaB and the corresponding complemented strains were detected by LC-MS/MS


E Bacterial two - hybrid assay reveals an interaction between SiaC and SiaD . The recombinant strains harboring different vectors were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . NA represents the empty vector pBT or pTRG . The strain expressing LGF2 and Gal11P was used as positive controls

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E Bacterial two-hybrid assay reveals an interaction between SiaC and SiaD. The recombinant strains harboring different vectors were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P was used as positive controls


F Pull down assays confirmed the interaction between SiaC and SiaD . Cell lysates of P . aeruginosa containing pMM67EH - siaD - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads

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F Pull down assays confirmed the interaction between SiaC and SiaD. Cell lysates of P. aeruginosa containing pMM67EH-siaD-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads


G SPR measurements of SiaC ( left ) or SiaCP ( right ) binding at varying concentrations to SiaD . SiaC specifically interacted with SiaD with a KD of 24 . 7nM , whereas SiaCP failed to interact with SiaB . Shown are measured binding responses ( black ) and curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant

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G SPR measurements of SiaC (left) or SiaCP (right) binding at varying concentrations to SiaD. SiaC specifically interacted with SiaD with a KD of 24.7nM, whereas SiaCP failed to interact with SiaB. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant


H Production of c - di - GMP by either SiaD or SiaD with SiaC at the indicated time - point was determined by HPLC

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H Production of c-di-GMP by either SiaD or SiaD with SiaC at the indicated time-point was determined by HPLC


A Bacterial two - hybrid assay reveals an interaction between SiaB and SiaC . The recombinant strains harboring different proteins were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . NA represents the empty vector pBT or pTRG . The strain expressing LGF2 and Gal11P were used as positive controls .

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A Bacterial two-hybrid assay reveals an interaction between SiaB and SiaC. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P were used as positive controls.


B Pull - down assays confirmed the interaction between SiaB and SiaC . Cell lysates of P . aeruginosa containing pMM67EH - siaC - Flag were incubated with GST or GST - SiaB individually , and protein complexes were captured by glutathione beads .

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B Pull-down assays confirmed the interaction between SiaB and SiaC. Cell lysates of P. aeruginosa containing pMM67EH-siaC-Flag were incubated with GST or GST-SiaB individually, and protein complexes were captured by glutathione beads.


C SPR measurements of SiaC binding at varying concentrations to SiaB . SiaC specifically interacted with SiaB with a KD of 40 . 1 nM . Shown are measured binding responses ( black ) and curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant .

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C SPR measurements of SiaC binding at varying concentrations to SiaB. SiaC specifically interacted with SiaB with a KD of 40.1 nM. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant.


D SiaB - mediated regulation of biofilm formation is dependent on SiaC . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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D SiaB-mediated regulation of biofilm formation is dependent on SiaC. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


E Bacterial two - hybrid assayreveals an interaction between SiaC and SiaA PP2C - like domain . NA represents the empty vector pBT or pTRG . The recombinant strains harboring different proteins were separately streaked on nonselective and dual - selective media ( 3 - amino - 1 , 2 , 4 - triazole + streptomycin ) . The strain expressing LGF2 and Gal11P were used as positive controls .

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E Bacterial two-hybrid assayreveals an interaction between SiaC and SiaA PP2C-like domain. NA represents the empty vector pBT or pTRG. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). The strain expressing LGF2 and Gal11P were used as positive controls.


F Pull - down assays confirmed the interaction between SiaC and SiaA . Cell lysates of P . aeruginosa containing pMM67EH - siaA - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads .

F Pull-down assays confirmed the interaction between SiaC and SiaA. Cell lysates of P. aeruginosa containing pMM67EH-siaA-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.


G Pull - down assays confirmed the interaction between SiaC and SiaA PP2C - like domain . Cell lysates of P . aeruginosa containing pMMB67EH - siaA386 - Flag were incubated with GST or GST - SiaC individually , and protein complexes were captured by glutathione beads .

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G Pull-down assays confirmed the interaction between SiaC and SiaA PP2C-like domain. Cell lysates of P. aeruginosa containing pMMB67EH-siaA386-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.


H The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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H The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate . Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides .

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B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate. Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides.


C Determination of modification sites by collision induced dissociation ( CID ) analysis . The MS / MS spectrum of modified L63LYLNTSSIK72 is shown . The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation , suggesting phosphorylation of Thr68 .

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C Determination of modification sites by collision induced dissociation (CID) analysis. The MS/MS spectrum of modified L63LYLNTSSIK72 is shown. The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation, suggesting phosphorylation of Thr68.


D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro . SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP . Phosphorylated products were separated by SDS - PAGE .

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D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro. SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP. Phosphorylated products were separated by SDS-PAGE.


E Over - expression of SiaCT68A restored the ability of ΔsiaA to form biofilm . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm . The biofilm formation of the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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E Over-expression of SiaCT68A restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


G Production of c - di - GMP at the indicated time - point by SiaD with SiaC or SiaCT68A was determined by HPLC . To evaluate the effect of SiaB - mediated phosphorylation on SiaD DGC activity , SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation .

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G Production of c-di-GMP at the indicated time-point by SiaD with SiaC or SiaCT68A was determined by HPLC. To evaluate the effect of SiaB-mediated phosphorylation on SiaD DGC activity, SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation.


C SiaCE33A partially restored the biofilm formation of ΔsiaA . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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C SiaCE33A partially restored the biofilm formation of ΔsiaA. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


D Triple mutation of E61A - Q64A - R67A is important for the function of SiaB . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) .

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D Triple mutation of E61A-Q64A-R67A is important for the function of SiaB. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


E Leu110 and Phe174 residues are important for SiaB function . The biofilm formation by the indicated strains was displayed with crystal violet staining ( up ) and quantified with optical density measurement ( down ) . Data represent the means and SDs of three biological replicates . * * , P < 0 . 01 based on one - way ANOVA test .

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E Leu110 and Phe174 residues are important for SiaB function. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). Data represent the means and SDs of three biological replicates. **, P<0.01 based on one-way ANOVA test.


D , E Images of FRET efficiency in mock - treated ( - ant . A , D ) and Antimycin A - treated ( + ant . A . , E ) ubi - AT1 . 03NL wing discs at time point 2 h . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . F Line graphs showing the FRET efficiency in individual mock - treated ( - ant . A , n = 26 ) and ant . A - treated ( + ant . A . , n = 19 ) ubi - AT1 . 03NL wing discs before ( 0 h ) and after 2 h of treatment . Paired t - test , ns = not significant , * * * p ≤ 0 . 001 .

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D, E Images of FRET efficiency in mock-treated (- ant.A, D) and Antimycin A-treated (+ ant. A., E) ubi-AT1.03NL wing discs at time point 2 h. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars= 50 µm. F Line graphs showing the FRET efficiency in individual mock-treated (- ant. A, n=26) and ant. A-treated (+ ant. A., n=19) ubi-AT1.03NL wing discs before (0 h) and after 2 h of treatment. Paired t-test, ns= not significant, ***p≤0.001.


G - J Time - controlled knock - down of metabolic enzymes in the dorsal compartment of ubi - AT1 . 03NL wing discs using the apGalts driver . Images of FRET efficiency in control ( G ) , apGalts > PfkRNAi ( H ) , apGalts > GapdhRNAi ( I ) , and apGalts > Glo1RNAi ( J ) wing discs after RNAi induction for 48 h ( G ) , 120 h ( H , J ) , and 93 hours ( I ) . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control ( n = 27 ) , apGalts > PfkRNAi ( n = 7 ) , apGalts > GapdhRNAi ( n = 10 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Loss of Pfk , Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , ns = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001.


L Time - controlled knock - down of ecdysoneless ( ecd ) in the dorsal compartment of ubi - AT1 . 03NL wing discs using apGalts > ecdRNAi . Image of FRET efficiency in apGalts > ecdRNAi wing disc 48 h after RNAi induction . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . M Line graph showing the FRET in the dorsal and ventral compartment of apGalts > ecdRNAi ( n = 28 ) wing discs . Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , * * * p ≤ 0 . 001 .

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L Time-controlled knock-down of ecdysoneless (ecd) in the dorsal compartment of ubi-AT1.03NL wing discs using apGalts>ecdRNAi. Image of FRET efficiency in apGalts>ecdRNAi wing disc 48 h after RNAi induction. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. M Line graph showing the FRET in the dorsal and ventral compartment of apGalts>ecdRNAi (n=28) wing discs. Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ***p≤0.001.


A - D Adult wing phenotype of knock - down of Gapdh ( C765 > GapdhRNAi ) , over - expression of full - length Ecd ( C765 > ecd ) , over - expression of C - terminally truncated form of Ecd ( C765 > ecdDN ) , and knock - down of ecd ( C765 > ecdRNAi ) in the whole wing disc ( see Appendix Fig S3A for the expression pattern of C765 - Gal4 ) . Images of adult wings of control ( A ) , C765 > GapdhRNAi ( A ' ) , C765 > ecd ( B ) , C765 > ecdDN ( C ) , and C765 > ecdRNAi ( D ) male flies . ( A ' ' ) Overlay of the wings shown in A and A ' . ( C ' ) Overlay of control wing and wing shown in C and C ' . Loss of Ecd throughout the wing discs produces adults with vestigal wings ( D ) . In contrast , the effects of ecdDN over - expression or Gapdh knock - down are less severe on tissue survival , giving rise to smaller wings with altered proportions . Images consist of a series of tiles that were stitched together by the microscope software . Scale bar = 500 μm . E , F Tukey box - and - whiskers plot showing quantifications of the area ( E ) and shape ( aspect ratio : major / minor axis , F ) of control , C765 > GapdhRNAi , C765 > ecd , and C765 > ecdDN wings . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to ± 1 . 5 times the interquartile range . Sample size is indicated for each perturbation . Loss of Gapdh or over - expression of EcdDN leads to smaller wings that are increased along the anterior - posterior ( A / P ) axis compared to the proximal - distal ( P / D ) axis . Statistical analysis was performed using one - way ANOVA , followed by Dunnett ' s multiple comparisons test . * * * p ≤ 0 . 001 .

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A-D Adult wing phenotype of knock-down of Gapdh (C765>GapdhRNAi), over-expression of full-length Ecd (C765>ecd), over-expression of C-terminally truncated form of Ecd (C765>ecdDN), and knock-down of ecd (C765>ecdRNAi) in the whole wing disc (see Appendix Fig S3A for the expression pattern of C765-Gal4). Images of adult wings of control (A), C765>GapdhRNAi (A'), C765>ecd (B), C765>ecdDN (C), and C765>ecdRNAi (D) male flies. (A'') Overlay of the wings shown in A and A'. (C') Overlay of control wing and wing shown in C and C'. Loss of Ecd throughout the wing discs produces adults with vestigal wings (D). In contrast, the effects of ecdDN over-expression or Gapdh knock-down are less severe on tissue survival, giving rise to smaller wings with altered proportions. Images consist of a series of tiles that were stitched together by the microscope software. Scale bar= 500 μm. E, F Tukey box-and-whiskers plot showing quantifications of the area (E) and shape (aspect ratio: major/minor axis, F) of control, C765>GapdhRNAi, C765>ecd, and C765>ecdDN wings. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to ± 1.5 times the interquartile range. Sample size is indicated for each perturbation. Loss of Gapdh or over-expression of EcdDN leads to smaller wings that are increased along the anterior-posterior (A/P) axis compared to the proximal-distal (P/D) axis. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test. ***p≤0.001.


A - B ' Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A , A ' ) and apGalts > ecdRNAi ( B , B ' ) wing discs , stained for the Hedgehog ( Hh ) pathway components Smoothened ( Smo , A , B ) , and Ci155 ( A ' , B ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 5 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > ecdRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 001 ) wing discs . Thus , loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B' Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A, A') and apGalts>ecdRNAi (B, B') wing discs, stained for the Hedgehog (Hh) pathway components Smoothened (Smo, A, B), and Ci155 (A', B'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=5) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>ecdRNAi (Smo: p≤0.001, Ci155: p≤0.001) wing discs. Thus, loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


C - E ' Time - controlled knock - down of metabolic enzymes in the dorsal compartment of the wing disc . IF of apGalts > PfkRNAi , apGalts > GapdhRNAi , and apGalts > Glo1RNAi wing discs , stained for Smo ( C , D , E ) and Ci155 ( C ' , D ' , E ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts > PfkRNAi ( n = 5 ) , apGalts > GapdhRNAi ( n = 5 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > PfkRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 01 ) , apGalts > GapdhRNAi ( Smo , Ci155 : p ≤ 0 . 05 ) , or apGalts > Glo1RNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 05 ) wing discs . Thus , loss of Pfk , Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 120 h after RNAi induction . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E' Time-controlled knock-down of metabolic enzymes in the dorsal compartment of the wing disc. IF of apGalts>PfkRNAi, apGalts>GapdhRNAi, and apGalts>Glo1RNAi wing discs, stained for Smo (C, D, E) and Ci155 (C', D', E'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts>PfkRNAi (n=5), apGalts>GapdhRNAi (n=5), and apGalts>Glo1RNAi (n=6) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>PfkRNAi (Smo: p≤0.001, Ci155: p≤0.01), apGalts>GapdhRNAi (Smo, Ci155: p≤0.05), or apGalts>Glo1RNAi (Smo: p≤0.001, Ci155: p≤0.05) wing discs. Thus, loss of Pfk, Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 120 h after RNAi induction. Scale bars= 50 μm.


A - D ' ' ' IF of NIH3T3 cells stably expressing Smo - mEos2 treated with mock ( control , A - A ' ' ' ) , the Smo agonist ( + SAG , B - B ' ' ' ) or the glycolytic inhibitors 3 - bromopyruvate ( + 3 - BP , C - C ' ' ' ) and 2 - deoxyglucose ( + 2 - DG , D - D ' ' ' ) . DAPI staining , shown in gray , was used to visualize cell nuclei . Acetylated tubulin ( AcTub ) , used as a ciliary marker , is shown in magenta , Smo - mEos2 is shown in green . Zoomed - in images of cilia marked by white arrows in A ' ' , B ' ' , C ' ' , and D ' ' are shown in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . Scale bars = 5 μm , but 2 μm in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . E , F Box plot showing quantification of Smo - mEos2 ciliary localization in NIH3T3 cells upon 3 - BP ( E ) and 2 - DG ( F ) treatment , respectively . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to the minimum and maximum values . Sample size is indicated for each perturbation . Treatment with glycolytic inhibitors induces Smo - mEos2 localization to the primary cilium . t - test , * * * p ≤ 0 . 001 .

O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D''' IF of NIH3T3 cells stably expressing Smo-mEos2 treated with mock (control, A-A'''), the Smo agonist (+ SAG, B-B''') or the glycolytic inhibitors 3-bromopyruvate (+3-BP, C-C''') and 2-deoxyglucose (+ 2-DG, D-D'''). DAPI staining, shown in gray, was used to visualize cell nuclei. Acetylated tubulin (AcTub), used as a ciliary marker, is shown in magenta, Smo-mEos2 is shown in green. Zoomed-in images of cilia marked by white arrows in A'', B'', C'', and D'' are shown in A''', B''', C''', and D'''. Scale bars= 5 μm, but 2 μm in A''', B''', C''', and D'''. E, F Box plot showing quantification of Smo-mEos2 ciliary localization in NIH3T3 cells upon 3-BP (E) and 2-DG (F) treatment, respectively. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to the minimum and maximum values. Sample size is indicated for each perturbation. Treatment with glycolytic inhibitors induces Smo-mEos2 localization to the primary cilium . t-test, ***p≤0.001.


A - B Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A ) and apGalts > ecdRNAi ( B ) wing discs , stained for Hh . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 4 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analysis ( t - test ) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts > ecdRNAi wing discs . Thus , loss of Ecd does not affect Hh production and spreading . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A) and apGalts>ecdRNAi (B) wing discs, stained for Hh. Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=4) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analysis (t-test) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts>ecdRNAi wing discs. Thus, loss of Ecd does not affect Hh production and spreading. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


C - F Time - controlled knock - down of ecd in the dorsal compartment of dispatched mutant wing discs ( dispS037707 , apGalts > ecdRNAi , see Fig 1C for the expression pattern of apGalts ) . IF of control ( C ) , apGalts > ecdRNAi ( D ) , dispS037707 ( E ) and dispS037707 , apGalts > ecdRNAi ( F ) wing discs expressing the decapentaplegic ( dpp ) reporter gene dpp - lacZ ( dppZ ) , stained for dppZ . Next to the images are quantifications of the respective stainings ( n = 4 ( C , F ) , n = 5 ( D ) , n = 3 ( E ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts > ecdRNAi ( p ≤ 0 . 01 ) ( C vs D ) , and dispS037707 and dispS037707 , apGalts > ecdRNAi ( p ≤ 0 . 01 ) wing discs ( E vs F ) . Loss of Ecd elevates the expression of dppZ in a broader than normal stripe . However , lack of Ecd function alone is not sufficient to trigger dpp expression , which instead depends on the presence of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-F Time-controlled knock-down of ecd in the dorsal compartment of dispatched mutant wing discs (dispS037707, apGalts>ecdRNAi, see Fig 1C for the expression pattern of apGalts). IF of control (C), apGalts>ecdRNAi (D), dispS037707 (E) and dispS037707, apGalts>ecdRNAi (F) wing discs expressing the decapentaplegic (dpp) reporter gene dpp-lacZ (dppZ), stained for dppZ. Next to the images are quantifications of the respective stainings (n=4 (C, F), n=5 (D), n=3 (E)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts>ecdRNAi (p≤0.01) (C vs D), and dispS037707 and dispS037707, apGalts>ecdRNAi (p≤0.01) wing discs (E vs F). Loss of Ecd elevates the expression of dppZ in a broader than normal stripe. However, lack of Ecd function alone is not sufficient to trigger dpp expression, which instead depends on the presence of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


G - J IF of dispS037707 ( G , I ) and dispS037707 , apGalts > ecdRNAi ( H , J ) wing discs , stained for Smo ( G , H ) and Ci155 ( I , J ) . Next to the images are quantifications of the respective stainings ( n = 2 ( G , H ) , n = 3 ( I ) , n = 4 ( J ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Although statistical analyses ( t - test ) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707 , apGalts > ecdRNAi wing discs , loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-J IF of dispS037707 (G, I) and dispS037707, apGalts>ecdRNAi (H, J) wing discs, stained for Smo (G, H) and Ci155 (I, J). Next to the images are quantifications of the respective stainings (n=2 (G, H), n=3 (I), n=4 (J)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Although statistical analyses (t-test) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707, apGalts>ecdRNAi wing discs, loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing discs 48 h or 120 h after RNAi induction , respectively . V / D indicates the boundary between the ventral and dorsal compartments . Next to the maximal projections are shown the sum projections of cross - sections along the A / P axis . Loss of Ecd or Gapdh alters the plasma membrane potential . Scale bars = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs 48 h or 120 h after RNAi induction, respectively. V/D indicates the boundary between the ventral and dorsal compartments. Next to the maximal projections are shown the sum projections of cross-sections along the A/P axis. Loss of Ecd or Gapdh alters the plasma membrane potential. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs