Cell fractionation upon inhibition of DPP9 activity . HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO . Cells were subjected to fractionation into ' cytosol ' ( supernatant ) and ' mitochondria ' ( pellet ) by differential digitonin lysis and centrifugation . The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration ( lanes 2 , 5 , 8 , 11 ) irrespective of 1G244 treatment . Likewise , the cytosolic marker protein LDH localized under all conditions to the same fractions . Conversely , endogenous AK2 appeared at this digitonin concentration in the ' cytosol ' fraction but only upon 1G244 treatment ( compare lanes 2 and 5 ) . Thus , inhibition of DPP9 results in appearance of AK2 in the cytosol . Quantification shows loading - control adjusted AK2 - signals of 1G244 - treated ' cytosol ' fraction ( lane 5 ) over DMSO - treated ' cytosol ' fraction ( lane 2 ) . White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed .

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

Cell fractionation upon inhibition of DPP9 activity. HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO. Cells were subjected to fractionation into 'cytosol' (supernatant) and 'mitochondria' (pellet) by differential digitonin lysis and centrifugation. The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration (lanes 2,5,8,11) irrespective of 1G244 treatment. Likewise, the cytosolic marker protein LDH localized under all conditions to the same fractions. Conversely, endogenous AK2 appeared at this digitonin concentration in the 'cytosol' fraction but only upon 1G244 treatment (compare lanes 2 and 5). Thus, inhibition of DPP9 results in appearance of AK2 in the cytosol. Quantification shows loading-control adjusted AK2-signals of 1G244-treated 'cytosol' fraction (lane 5) over DMSO-treated 'cytosol' fraction (lane 2). White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed.


Localization of DPP9 - insensitive AK2 - HA variants . Experiment was performed as described in Figure 2C . Compared to the wild type , DPP9 - insensitive and import - competent AK2 - HA variants co - localized with cytosolic markers indicating partial cytosolic localization . The colocalization of AK2 - HA variants with Mitotracker was quantified using Fiji ( Pearson ' s correlation coefficient ) . A one - way ANOVA with post - hoc Tukey test was performed . Numbers of analysed cells are indicated in the plot . * * represents p < 0 . 01 . Data shown in Figures 2C , 5B , S2 and S5 are derived from the same experiments . The black horizontal line represents the median . Data information : Scale bars : 10 μm .

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Localization of DPP9-insensitive AK2-HA variants. Experiment was performed as described in Figure 2C. Compared to the wild type, DPP9-insensitive and import-competent AK2-HA variants co-localized with cytosolic markers indicating partial cytosolic localization. The colocalization of AK2-HA variants with Mitotracker was quantified using Fiji (Pearson's correlation coefficient). A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. ** represents p<0.01. Data shown in Figures 2C, 5B, S2 and S5 are derived from the same experiments. The black horizontal line represents the median. Data information: Scale bars: 10 μm.


Localization of AK2C40S - HA upon proteasomal inhibition . Experiment was performed as described in Figure 2C . Upon MG132 treatment , colocalization of AK2C40S - HA with the cytosolic marker increased . A one - way ANOVA with post - hoc Tukey test was performed . Numbers of analysed cells are indicated in the plot . * * * represents p < 0 . 001 . The black horizontal line represents the median . Data information : Scale bars : 10 μm .

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Localization of AK2C40S-HA upon proteasomal inhibition. Experiment was performed as described in Figure 2C. Upon MG132 treatment, colocalization of AK2C40S-HA with the cytosolic marker increased. A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. *** represents p<0.001.The black horizontal line represents the median. Data information: Scale bars: 10 μm.


AK2 activity in dependence of its substrate AMP . Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0 . 012 mM AMP . The profiles for both AK2C40S , C232S and AK2C40S , C42S , C92S , C232S are superimposable . Reported values are the mean of at least 3 independent experiments ; error bars represent ± SD . AK2 activity in dependence of its substrate ADP . Maximal activity was observed at around 1 - 2 mM ADP with an apparent KM of 0 . 12 mM and 0 . 1 mM ADP for AK2C40S , C232S and AK2C40S , C42S , C92S , C232S , respectively . The profiles for both AK2C40S , C232S and AK2C40S , C42S , C92S , C232S are thus highly similar . Reported values are the mean of at least 3 independent experiments ; error bars represent ± SD .

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AK2 activity in dependence of its substrate AMP. Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0.012 mM AMP. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are superimposable. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   AK2 activity in dependence of its substrate ADP. Maximal activity was observed at around 1-2 mM ADP with an apparent KM of 0.12 mM and 0.1 mM ADP for AK2C40S,C232S and AK2C40S,C42S,C92S,C232S, respectively. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are thus highly similar. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.


Activity assay for AK2 - HA variants isolated from HEK293 cells . Indicated AK2 - HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants . Then , the AK2 activity assay described in ( B ) was performed . While import - competent AK2 variants exhibited full activity , a cytosolic AK2 variant with the native N - terminus showed very low activity . This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9 - insensitive cytosolic AK2 variants . The enzymatic inactive K28Q variants served as negative control indicating that only AK2 - HA - dependent activity ways measured by our assay . Reported values are the mean of 2 independent experiments ; error bars represent ± SD .

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Activity assay for AK2-HA variants isolated from HEK293 cells. Indicated AK2-HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants. Then, the AK2 activity assay described in (B) was performed. While import-competent AK2 variants exhibited full activity, a cytosolic AK2 variant with the native N-terminus showed very low activity. This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9-insensitive cytosolic AK2 variants. The enzymatic inactive K28Q variants served as negative control indicating that only AK2-HA-dependent activity ways measured by our assay. Reported values are the mean of 2 independent experiments; error bars represent ±SD.


Steady state levels of NDUFB10 , NDUFS5 , NDUFA8 , COA6 , CHCHD2 , and TIMM9 upon incubation with the DPP9 inhibitor 1G244 . Cells were treated for 24h with inhibitor or DMSO as control , and were then analysed by immunoblot against the indicated proteins . Proteins except TIMM9 are stabilized upon DPP9 inhibition . Reported values are the mean of 3 - 5 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * * represents p < 0 . 01 .

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

Steady state levels of NDUFB10, NDUFS5, NDUFA8, COA6, CHCHD2, and TIMM9 upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. Proteins except TIMM9 are stabilized upon DPP9 inhibition. Reported values are the mean of 3-5 independent experiments; error bars represent ±SD. Student's t-test was performed. ** represents p<0.01.


Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244 . Cells were treated for 24h with inhibitor or DMSO as control , and were then analysed by immunoblot against the indicated proteins . NDUFB10C107S is stabilized upon DPP9 inhibition . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - test was performed for the respective control and treatment samples . * * represents p < 0 . 01 .

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

Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. NDUFB10C107S is stabilized upon DPP9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed for the respective control and treatment samples. ** represents p<0.01.


Localization of NDUFB10 variants upon 1G244 treatment . Experiment was performed as in ( C ) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins . Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition . Reported values are the mean of 2 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 .

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Localization of NDUFB10 variants upon 1G244 treatment. Experiment was performed as in (C) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins. Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition. Reported values are the mean of 2 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.


( A ) Western blot showing the protein expression of ASCL1 and / or NEUROD1 in NCI - H69 , NCI - H82 , NCI - H146 , NCI - H510A , NCI - H526 , SHP - 77 and DMS - 53 SCLC cells ( lanes 4 - 10 ) ; A549 and NCI - H460 NSCLC cells and a IMR - 90 HFL cell lines ( lanes 1 - 3 ) . Tubulin ( α - Tub ) is shown as loading control . Each blot is representative from at least three independent experiments .

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(A) Western blot showing the protein expression of ASCL1 and/or NEUROD1 in NCI-H69, NCI-H82, NCI-H146, NCI-H510A, NCI-H526, SHP-77 and DMS-53 SCLC cells (lanes 4-10); A549 and NCI-H460 NSCLC cells and a IMR-90 HFL cell lines (lanes 1-3). Tubulin (α-Tub) is shown as loading control. Each blot is representative from at least three independent experiments.


( A - E ) ChIP - seq experiments indicates the localization of ( A ) ASCL1 , ( B ) NEUROD1 and ( E ) RNAPII . ATAC - seq ( C ) and ( D ) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t = 0 ( blue ) and t = 4h ( green ) after lurbinectedin treatment ; - 2 . 0 and + 2 . 0 kb to TSS coordinates of all hg19 genes .

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(A-E) ChIP-seq experiments indicates the localization of (A) ASCL1, (B) NEUROD1 and (E) RNAPII. ATAC-seq (C) and (D) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t=0 (blue) and t=4h (green) after lurbinectedin treatment; -2.0 and +2.0 kb to TSS coordinates of all hg19 genes.


( A - D ) Chip - seq genome track of ASCL1 / NEUROD1 , RNAPII , and H3K27Ac on ASCL1 , BCL2 ; INSM1 and MYB ( hg19 ) , in untreated ( dark blue ) and in lurbinectedin treated ( green ) conditions respectively ; to be noticed the location of CGG rich motifs ( lower part of each panel ) which parallel the presence of lurbinectedin ( red ) .

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(A-D) Chip-seq genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1 and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red).


( a ) Western blot showing RNAPII across the panel of ; A549 and NCI - H460 NSCLC cells ; IMR - 90 HFL cells ; NCI - H146 , NCI - H82 , DMS - 53 , NCI - H510A , NCI - H526 and SHP - 77 SCLC cells before ( - ) and after ( + ) treatment with lurbinectedin . α - Tubulin ( α - Tub ) is shown as loading control ; lower panel , Histogram showing the average of RNAPII before ( dark blue ) and after ( light Blue ) lurbinectedin treatment in arbitrary units ( a . u . ) normalized against α - Tub . Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák ' s multiple comparisons test ( n = 3 biological replicas ) . * * * * P ≤ 0 . 0001 .

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(a) Western blot showing RNAPII across the panel of; A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526 and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin. α-Tubulin (α-Tub) is shown as loading control; lower panel, Histogram showing the average of RNAPII before (dark blue) and after (light Blue) lurbinectedin treatment in arbitrary units (a.u.) normalized against α-Tub. Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák's multiple comparisons test (n=3 biological replicas). ****P ≤0.0001.


( B Immunofluorescence of RNAPII ( red ) before ( Ctrl ) and after lurbinectedin treatment . DAPI ( blue ) was used to stain nuclei ; scale bar is 65 μm .

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(B Immunofluorescence of RNAPII (red) before (Ctrl) and after lurbinectedin treatment. DAPI (blue) was used to stain nuclei; scale bar is 65 μm.


( D ) Affinity purification of Bio - lur fractions containing elongating RNAPII Ser2 ( lane 4 ) . Input ( lanes 1 - 2 ) represents 10 % of the starting material . Respective molecular weights are shown on the right ( kDa ) .

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(D) Affinity purification of Bio-lur fractions containing elongating RNAPII Ser2 (lane 4). Input (lanes 1-2) represents 10% of the starting material. Respective molecular weights are shown on the right (kDa).


( E ) Upper panel : GST - pulldown of ubiquitinated proteins showing hypo - ( IIA ) , - hyper ( II0 ) phosphorylated RNAPII ( lanes 4 - 5 upper panel ) and Ser2 Phosphorylated RNAPII ( Ser2P ) ( lanes 4 - 5 lower panel ) after lurbinectedin treatment . Empty beads ( Mock ) ( lane 3 ) as a negative control ; Lower Panel : GST - pulldown of ubiquitinated proteins Ser2P ) ( lanes 4 - 5 ) . Representative smear of successful ubiquitinated proteins pulldown ( Lanes 4 and 5 ) as compared to input ( lanes 1 and 2 ) . Empty beads ( Mock ) ( lane 3 ) as a negative control . Each Western blot is representative of three independent experiments .

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(E) Upper panel: GST-pulldown of ubiquitinated proteins showing hypo- (IIA), -hyper (II0) phosphorylated RNAPII (lanes 4-5 upper panel) and Ser2 Phosphorylated RNAPII (Ser2P) (lanes 4-5 lower panel) after lurbinectedin treatment. Empty beads (Mock) (lane 3) as a negative control; Lower Panel: GST-pulldown of ubiquitinated proteins Ser2P) (lanes 4-5). Representative smear of successful ubiquitinated proteins pulldown (Lanes 4 and 5) as compared to input (lanes 1 and 2). Empty beads (Mock) (lane 3) as a negative control. Each Western blot is representative of three independent experiments.


( B ) In vivo epifluorescence microscopy following de novo expressed UapA - GFP in a single growing germling , at 70 , 80 , 100 , 120 , 140 , 160 and 170 min after derepression of transcription via its native uapA promoter ( for details see text and Materials and methods ) . Notice that in the growing germling tip UapA localizes in a membranous mesh and some cytosolic puncta whereas in more tip - distal parts of the germlings UapA is in PM - localized puncta ( see white arrows ) , which progressively become more abundant so that at more posterior areas the PM is homogeneously labeled .

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(B) In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a single growing germling, at 70, 80, 100, 120, 140, 160 and 170 min after derepression of transcription via its native uapA promoter (for details see text and Materials and methods). Notice that in the growing germling tip UapA localizes in a membranous mesh and some cytosolic puncta whereas in more tip-distal parts of the germlings UapA is in PM-localized puncta (see white arrows), which progressively become more abundant so that at more posterior areas the PM is homogeneously labeled.


( C ) In vivo epifluorescence microscopy following de novo expressed UapA - GFP in a germling maturing to young hypha at 180 , 240 , 300 , 360 and 400 min after derepression of transcription via its native uapA promoter . During this developmental transition , UapA apical localization is gradually diminished . As the cell tip acquires a faster growth rate , UapA is completely retracted from the hyphal apex ( see zoon - in panels on the right and white arrows ) .

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(C) In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a germling maturing to young hypha at 180, 240, 300, 360 and 400 min after derepression of transcription via its native uapA promoter. During this developmental transition, UapA apical localization is gradually diminished. As the cell tip acquires a faster growth rate, UapA is completely retracted from the hyphal apex (see zoon-in panels on the right and white arrows).


( D ) In a mature hyphal cell , UapA is no longer found in the PM of the extreme apical compartment , but in a cytoplasmic a membrane network resembling the ER . As the distance from the apex increases , UapA progressively populates the PM as cortical puncta ( see white arrows ) .

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(D) In a mature hyphal cell, UapA is no longer found in the PM of the extreme apical compartment, but in a cytoplasmic a membrane network resembling the ER. As the distance from the apex increases, UapA progressively populates the PM as cortical puncta (see white arrows).


( F ) In vivo epifluorescence microscopy following neosynthesized alcAp - UapA - GFP in a single hypha at 120 , 130 , 140 , 150 , 160 and 170 min , under overexpressing ( derepression / ethanol - induction ) conditions , as described in Materials and methods . Notice that UapA labels the perinuclear ER rings ( white arrows ) .

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(F) In vivo epifluorescence microscopy following neosynthesized alcAp-UapA-GFP in a single hypha at 120, 130, 140, 150, 160 and 170 min, under overexpressing (derepression/ethanol-induction) conditions, as described in Materials and methods. Notice that UapA labels the perinuclear ER rings (white arrows).


( G ) In vivo epifluorescence microscopy following de novo expressed alcAp - GFP - SynA in a single young hyphal cell , at 60 , 70 , 80 , 90 and 100 min after transcriptional derepression ( for strain details see Materials and methods ) . SynA is a standard polar membrane cargo that traffics to the hyphal tip via Golgi - dependent secretion . Notice the very distinct GFP fluorescent signals obtained using UapA ( non - polar membrane network and PM ) versus SynA ( Golgi - like puncta and polar depositioning at the tip ; see also alter ) .

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(G) In vivo epifluorescence microscopy following de novo expressed alcAp-GFP-SynA in a single young hyphal cell, at 60, 70, 80, 90 and 100 min after transcriptional derepression (for strain details see Materials and methods). SynA is a standard polar membrane cargo that traffics to the hyphal tip via Golgi-dependent secretion. Notice the very distinct GFP fluorescent signals obtained using UapA (non-polar membrane network and PM) versus SynA (Golgi-like puncta and polar depositioning at the tip; see also alter).


( H ) Similar experiment as in ( B ) performed in an isogenic strain where ArtA , the arrestin required for UapA endocytosis , is genetically depleted .

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(H) Similar experiment as in (B) performed in an isogenic strain where ArtA, the arrestin required for UapA endocytosis, is genetically depleted.


( A , B ) Co - localization analysis of neosynthesized alcAp - UapA - GFP with early ( SedV ) or late ( PHosbp ) markers , respectively , tagged with mCherry or mRFP . Images show single hyphal cells . For conditions of transcriptional derepression see Materials and methods . Quantification of co - localization was performed by calculating Pearson ' s Correlation Coefficient ( PCC ) . One sample t - test was performed to test the significance of differences in PCCs . Biological / technical replicates : 2 / 9 for alcAp - UapA - GFP mCherry - SedV and 2 / 8 for alcAp - UapA - GFP mRFP - PHosbp . For the definition of the two categories of replicates see Materials and methods . Results of quantification , shown on the middle , suggest that there is no significant overlapping fluorescent signal of UapA with SedV ( PCC = 0 . 25 ± 0 . 09 , P < 0 . 0001 ) or with PHOSBP ( PCC = 0 . 34 ± 0 . 06 , P < 0 . 0001 ) , as PCC values close to 0 . 2 - 0 . 3 are also commonly obtained when distinct compartments ( e . g . ER and early Golgi or early and late Golgi ) are followed with different fluorophores . Scale bars : 2 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

(A, B) Co-localization analysis of neosynthesized alcAp-UapA-GFP with early (SedV) or late (PHosbp) markers, respectively, tagged with mCherry or mRFP. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization was performed by calculating Pearson's Correlation Coefficient (PCC). One sample t-test was performed to test the significance of differences in PCCs. Biological/technical replicates:2/9 for alcAp-UapA-GFP mCherry-SedV and 2/8 for alcAp-UapA-GFP mRFP-PHosbp. For the definition of the two categories of replicates see Materials and methods. Results of quantification, shown on the middle, suggest that there is no significant overlapping fluorescent signal of UapA with SedV (PCC=0.25±0.09, P<0.0001) or with PHOSBP (PCC=0.34±0.06, P<0.0001), as PCC values close to 0.2-0.3 are also commonly obtained when distinct compartments (e.g. ER and early Golgi or early and late Golgi) are followed with different fluorophores. Scale bars: 2 μm.


( C , D ) Co - localization analysis of neosynthesized alcAp - GFP - SynA , used as a conventional cargo that traffics through the Golgi , with early ( SedV ) or late ( PHospb ) markers tagged with mCherry or mRFP , respectively . Images show single hyphal cells . For conditions of transcriptional derepression see Materials and methods . Quantification of co - localization and statistical analysis was performed as in ( A , B ) . Biological / technical replicates : 2 / 10 for each strain . Statistical analysis showed significant colocalization of SynA with the late Golgi marker ( PCC = 0 . 67 ± 0 . 04 , P < 0 . 0001 ) , but not with early Golgi marker ( PCC = 0 . 37 ± 0 . 06 , P < 0 . 0001 ) . An explanation for lack of co - localization of SynA with SedV is given in the text . Scale bars : 2 μm . The cartoon at the bottom depicts the localization of early or late markers as established here in several previous

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Co-localization analysis of neosynthesized alcAp-GFP-SynA, used as a conventional cargo that traffics through the Golgi, with early (SedV) or late (PHospb) markers tagged with mCherry or mRFP, respectively. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization and statistical analysis was performed as in (A, B). Biological/technical replicates: 2/10 for each strain. Statistical analysis showed significant colocalization of SynA with the late Golgi marker (PCC = 0.67±0.04, P<0.0001), but not with early Golgi marker (PCC=0.37±0.06, P<0.0001). An explanation for lack of co-localization of SynA with SedV is given in the text. Scale bars: 2 μm. The cartoon at the bottom depicts the localization of early or late markers as established here in several previous studies


( B ) Key endogenous genes controlling Golgi ( sedV , geaA , hypB ) or post - Golgi trafficking ( rabE , ap1σ , claH ) were genetically replaced by versions transcribed under the highly repressible thiAp promoter via targeted homologous recombination . In the absence of thiamine from the growth medium ( derepressed conditions ) the relative proteins are expressed , while upon addition of thiamine at the onset of conidiospore germination ( ab initio repression ) the expression of these proteins is tightly repressed . Proteins are detected by a standard western blot analysis using either anti - FLAG or anti - GFP antibodies for Golgi and Post - Golgi proteins . Equal loading and protein steady state levels are normalized against the amount of actin , detected with a specific antibody .

O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O B-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

(B) Key endogenous genes controlling Golgi (sedV, geaA, hypB) or post-Golgi trafficking (rabE, ap1σ, claH) were genetically replaced by versions transcribed under the highly repressible thiAp promoter via targeted homologous recombination. In the absence of thiamine from the growth medium (derepressed conditions) the relative proteins are expressed, while upon addition of thiamine at the onset of conidiospore germination (ab initio repression) the expression of these proteins is tightly repressed. Proteins are detected by a standard western blot analysis using either anti-FLAG or anti-GFP antibodies for Golgi and Post-Golgi proteins. Equal loading and protein steady state levels are normalized against the amount of actin, detected with a specific antibody.


( C ) In the absence of thiamine from the growth medium ( derepressed conditions ) the corresponding strains grow nearly as an isogenic wild - type control , although a delay in growth is observed for thiAp - sedV and less so for thiAp - ap1σ ( upper row ) . In the presence of thiamine to the growth medium most cells do not form colonies , except for thiAp - hypB which forms a compact slow growing colony ( lower row ) .

O O O O O O O B-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 B-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

(C) In the absence of thiamine from the growth medium (derepressed conditions) the corresponding strains grow nearly as an isogenic wild-type control, although a delay in growth is observed for thiAp-sedV and less so for thiAp-ap1σ (upper row). In the presence of thiamine to the growth medium most cells do not form colonies, except for thiAp-hypB which forms a compact slow growing colony (lower row).


( D ) Microscopic examination of the corresponding strains under thiamine ( repressing conditions ) shows that , in most cases , the apical region of germlings is enlarged and growth is arrested . This morphological phenotype , taken as a strong indication of blocked secretion , is more evident in thiAp - sedV and thiAp - rabE but concerns all strains , except for thiAp - hypB . Scale bar : 5 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

(D) Microscopic examination of the corresponding strains under thiamine (repressing conditions) shows that, in most cases, the apical region of germlings is enlarged and growth is arrested. This morphological phenotype, taken as a strong indication of blocked secretion, is more evident in thiAp-sedV and thiAp-rabE but concerns all strains, except for thiAp-hypB. Scale bar: 5 μm.


( E ) Subcellular localization of UapA , after 6 - 8 hours of initiation of transcription , via its native uapA promoter , while conventional sedV , geaA or hypB transcription is repressed by thiamine ab initio . Scale bars : 2 μm . Notice that UapA - GFP translocates in the PM of germlings in all cases . Results shown are confirmed by quantification ( right panel ) of UapA - GFP PM / cytosolic intensity ratios for the four strains ( for details see Materials and methods ) . Mean PM / cytosolic intensity ratios for wild - type , thiAp - sedV , thiAp - geaA and thiAp - hypB are 0 . 53 ± 0 . 02 , 0 . 52 ± 0 . 02 , 0 . 52 ± 0 . 02 and 0 . 52 ± 0 . 02 , respectively . For the statistical analysis , Tukey ' s Multiple Comparison test was performed ( One - way ANOVA ) . No statistical significance was found between the wild - type and each of the mutant strains . Biological / technical replicates : 3 / 15 for wild - type , 3 / 18 for thiAp - sedV , 3 / 15 for thiAp - geaA and 3 / 18 for thiAp - hypB .

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

(E) Subcellular localization of UapA, after 6-8 hours of initiation of transcription, via its native uapA promoter, while conventional sedV, geaA or hypB transcription is repressed by thiamine ab initio. Scale bars: 2 μm. Notice that UapA-GFP translocates in the PM of germlings in all cases. Results shown are confirmed by quantification (right panel) of UapA-GFP PM/cytosolic intensity ratios for the four strains (for details see Materials and methods). Mean PM/cytosolic intensity ratios for wild-type, thiAp-sedV, thiAp-geaA and thiAp-hypB are 0.53±0.02, 0.52±0.02, 0.52±0.02 and 0.52±0.02, respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). No statistical significance was found between the wild-type and each of the mutant strains. Biological/technical replicates: 3/15 for wild-type, 3/18 for thiAp-sedV, 3/15 for thiAp-geaA and 3/18 for thiAp-hypB.


( F ) alcAp - UapA - GFP , under derepressing conditions , translocates to the PM of a strain carrying a thermosensitive mutation in SedVts both at the permissive ( 25oC ) and restrictive temperature ( 42oC ) . A degree of increased degradation of UapA , caused by exposure to high temperature ( 42oC ) , is apparent as cytosolic puncta , which correspond to membrane aggregates and sorting to vacuoles . Scale bar : 5 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

(F) alcAp-UapA-GFP, under derepressing conditions, translocates to the PM of a strain carrying a thermosensitive mutation in SedVts both at the permissive (25oC) and restrictive temperature (42oC). A degree of increased degradation of UapA, caused by exposure to high temperature (42oC), is apparent as cytosolic puncta, which correspond to membrane aggregates and sorting to vacuoles. Scale bar: 5 μm.


( G ) Unlike UapA , de novo made apical markers , such as ChsB or SynA , lose their polar localization , when the expression of sedV , geaA or hypB is repressed . Scale bars : 2 μm . Quantification : GFP - ChsB PM / cytosolic and alcAp - GFP - SynA PM / cytosolic intensity ratios are plotted to the right upper and lower panel , respectively . For GFP - ChsB , mean PM / cytosolic intensity ratios are : 0 . 60 ± 0 . 02 ( wild - type ) , 0 . 11 ± 0 . 01 ( thiAp - sedV ) , 0 . 26 ± 0 . 02 ( thiAp - null ) and 0 . 34 ± 0 . 01 ( thiAp - hypB ) . For alcAp - GFP - SynA mean PM / cytosolic intensity ratios for wild - type , thiAp - sedV , thiAp - geaA and thiAp - hypB are 0 . 61 ± 0 . 03 , 0 . 14 ± 0 . 02 , 0 . 26 ± 0 . 03 and 0 . 39 ± 0 . 03 , respectively . The statistical analysis was performed as in ( E ) . A significant difference ( * * * * P < 0 . 0001 ) of GFP - ChsB fluorescence in the apical PM membrane was found between the wild - type and the three strains lacking the key - Golgi proteins . This is also the case for SynA , as seen in the scatter plot on the lower right panel , where the fluorescence intensity of SynA is diminished ( * * * * P < 0 . 0001 ) when sedV , geaA and hypB are repressed . Biological / technical replicates : 3 / 15 for each strain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Unlike UapA, de novo made apical markers, such as ChsB or SynA, lose their polar localization, when the expression of sedV, geaA or hypB is repressed. Scale bars: 2 μm. Quantification: GFP-ChsB PM/cytosolic and alcAp-GFP-SynA PM/cytosolic intensity ratios are plotted to the right upper and lower panel, respectively. For GFP-ChsB, mean PM/cytosolic intensity ratios are: 0.60±0.02 (wild-type), 0.11±0.01 (thiAp-sedV), 0.26±0.02 (thiAp-null) and 0.34±0.01 (thiAp-hypB). For alcAp-GFP-SynA mean PM/cytosolic intensity ratios for wild-type, thiAp-sedV, thiAp-geaA and thiAp-hypB are 0.61±0.03, 0.14±0.02, 0.26±0.03 and 0.39±0.03, respectively. The statistical analysis was performed as in (E). A significant difference (****P<0.0001) of GFP-ChsB fluorescence in the apical PM membrane was found between the wild-type and the three strains lacking the key-Golgi proteins. This is also the case for SynA, as seen in the scatter plot on the lower right panel, where the fluorescence intensity of SynA is diminished (****P<0.0001) when sedV, geaA and hypB are repressed. Biological/technical replicates: 3/15 for each strain.


( A ) Growth test showing that Sec24 expression is essential for growth as its transcriptional repression by thiamine ( + thi ) leads to absence of colony formation , despite initial germination The strains shown are isogenic except for the sec24 locus . thiAp - sec24 signifies the strain where the endogenous sec24 promoter was replaced by the thiAp promoter .

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

(A) Growth test showing that Sec24 expression is essential for growth as its transcriptional repression by thiamine (+thi) leads to absence of colony formation, despite initial germination The strains shown are isogenic except for the sec24 locus. thiAp-sec24 signifies the strain where the endogenous sec24 promoter was replaced by the thiAp promoter.


( B ) Epifluorescence microscopy analysis of the subcellular localization of UapA - GFP under conditions where sec24 transcription is ab initio derepressed ( upper panel ) or repressed by thiamine ( lower panel ) . o / n ( overnight ) means addition of thiamine from the onset of germination . Germination of conidiospores takes place until full repression of Sec24 is achieved ( 10 - 12 h ) . Notice the total lack of PM - associated signal of UapA under conditions of Sec24 repression . Scale bar : 5 μm . Quantification : In the scatter plot on the right , UapA - GFP PM / cytosolic intensity ratios are quantified when sec24 is derepressed ( - thi ) or repressed ( + thi ) . Mean PM / cytosolic intensity ratios are 0 . 55 ± 0 . 02 and 0 . 32 ± 0 . 02 respectively . To test the significance of differences , an unpaired t - test was performed , which verified the significant difference ( * * * * P < 0 . 0001 ) in the presence of thiamine . Biological / technical replicates : 2 / 15 for each condition .

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

(B) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec24 transcription is ab initio derepressed (upper panel) or repressed by thiamine (lower panel). o/n (overnight) means addition of thiamine from the onset of germination. Germination of conidiospores takes place until full repression of Sec24 is achieved (10-12 h). Notice the total lack of PM-associated signal of UapA under conditions of Sec24 repression. Scale bar: 5 μm. Quantification: In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when sec24 is derepressed (-thi) or repressed (+thi). Mean PM/cytosolic intensity ratios are 0.55±0.02 and 0.32±0.02 respectively. To test the significance of differences, an unpaired t-test was performed, which verified the significant difference (****P<0.0001) in the presence of thiamine. Biological/technical replicates: 2/15 for each condition.


( C ) Epifluorescence microscopy analysis of the subcellular localization of the apical marker chitin synthase ( GFP - ChsB ) under sec24 derepressed or repressed conditions . Notice the loss of apical depositioning and the concurrent labeling of cytoplasmic foci and membranous structures under repression conditions . Scale bar : 5 μm . Quantification : GFP - ChsB PM / cytosolic intensity ratios are plotted in cases where sec24 is derepressed ( - thi ) or repressed ( + thi ) . Mean values are 0 . 56 ± 0 . 03 and 0 . 12 ± 0 . 02 respectively . The fluorescence intensity of GFP - ChsB is statistically lower ( * * * * P < 0 . 0001 ) when the expression of sec24 is repressed ( statistical analysis as in B ) . Biological / technical replicates : 2 / 15 for each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-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

(C) Epifluorescence microscopy analysis of the subcellular localization of the apical marker chitin synthase (GFP-ChsB) under sec24 derepressed or repressed conditions. Notice the loss of apical depositioning and the concurrent labeling of cytoplasmic foci and membranous structures under repression conditions. Scale bar: 5 μm. Quantification: GFP-ChsB PM/cytosolic intensity ratios are plotted in cases where sec24 is derepressed (-thi) or repressed (+thi). Mean values are 0.56±0.03 and 0.12±0.02 respectively. The fluorescence intensity of GFP-ChsB is statistically lower (****P<0.0001) when the expression of sec24 is repressed (statistical analysis as in B). Biological/technical replicates: 2/15 for each condition.


( D ) Co - localization analysis by epifluorescence microscopy of Sec24 - mRFP and de novo made UapA - GFP ( 8h derepressed transcription ) . Quantification of co - localization was made by calculating Pearson ' s Correlation Coefficient . For the statistical analysis one sample t - test was used . Notice the significant co - localization ( PCC = 0 . 61 ± 0 . 07 , * * * * P < 0 . 0001 ) . Due to the partially functional mRFP - tagged version of Sec24 ( evident in the growth test shown on the left panel ) , UapA trafficking is significantly delayed , reflected in the appearance of prominent labeling of uniform perinuclear ER . Scale bar : 5 μm . Biological / technical replicates : 3 / 5 .

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

(D) Co-localization analysis by epifluorescence microscopy of Sec24-mRFP and de novo made UapA-GFP (8h derepressed transcription). Quantification of co-localization was made by calculating Pearson's Correlation Coefficient. For the statistical analysis one sample t-test was used. Notice the significant co-localization (PCC=0.61±0.07, ****P<0.0001). Due to the partially functional mRFP-tagged version of Sec24 (evident in the growth test shown on the left panel), UapA trafficking is significantly delayed, reflected in the appearance of prominent labeling of uniform perinuclear ER. Scale bar: 5 μm. Biological/technical replicates: 3/5.


( E ) Epifluorescence microscopy analysis of the subcellular localization of UapA - GFP under conditions where sec13 transcription is ab initio derepressed ( left panel ) or repressed by thiamine ( right panel ) . The inserts in the upper right corner in both panels reflect growth tests showing that Sec13 repression leads to arrest in growth and absence of colony formation . Scale bars : 5 μm . All images reflect practically identical results obtained in several experiments . Quantification : UapA - GFP PM / cytosolic intensity ratios are quantified when sec13 is derepressed ( - thi ) or repressed ( + thi ) , with mean values being 0 . 54 ± 0 . 02 and 0 . 31 ± 0 . 02 , respectively . There is a significant difference on UapA PM fluorescence intensity ( * * * * P < 0 . 0001 ) in the absence of the COPII outer coat protein ( statistical analysis as in B ) . Biological / technical replicates : 2 / 15 for each condition .

O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-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

(E) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec13 transcription is ab initio derepressed (left panel) or repressed by thiamine (right panel). The inserts in the upper right corner in both panels reflect growth tests showing that Sec13 repression leads to arrest in growth and absence of colony formation. Scale bars: 5 μm. All images reflect practically identical results obtained in several experiments. Quantification: UapA-GFP PM/cytosolic intensity ratios are quantified when sec13 is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.31±0.02, respectively. There is a significant difference on UapA PM fluorescence intensity (****P<0.0001) in the absence of the COPII outer coat protein (statistical analysis as in B). Biological/technical replicates: 2/15 for each condition.


( A ) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA - GFP , after 6 - 8 hours of initiation of transcription in strains where the expression of RabE , AP - 1σ or ClaH ( thiAp - rabE , thiAp - ap1σ or thiAp - claH ) has been repressed ab initio ( o / n ) by addition of thiamine . Notice that when rabE or ap1σ is repressed UapA - GFP still reaches the PM . In contrast , repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network . Scale bars : 5 μm . Quantification : UapA - GFP PM / cytosolic intensity ratios are plotted on the right . Mean ratios for wild - type , thiAp - rabE , thiAp - ap1σ and thiAp - claH are 0 . 55 ± 0 . 02 , 0 . 53 ± 0 . 03 , 0 . 54 ± 0 . 02 and 0 . 29 ± 0 . 02 respectively . For the statistical analysis , Tukey ' s Multiple Comparison test was performed ( One - way ANOVA ) . After 6 - 8h of transcriptional derepression , UapA - GFP fluorescence to the PM does not change when rabE or ap1σ are repressed . On the other hand , in the absence of ClaH , UapA does not reach the PM , as its fluorescence intensity there is statistically lower ( * * * * P < 0 . 0001 ) in comparison to that of the wild - type strain . Biological / technical replicates : 2 / 15 for each strain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA-GFP, after 6-8 hours of initiation of transcription in strains where the expression of RabE, AP-1σ or ClaH (thiAp-rabE, thiAp-ap1σ or thiAp-claH) has been repressed ab initio (o/n) by addition of thiamine. Notice that when rabE or ap1σ is repressed UapA-GFP still reaches the PM. In contrast, repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network. Scale bars: 5 μm. Quantification: UapA-GFP PM/cytosolic intensity ratios are plotted on the right. Mean ratios for wild-type, thiAp-rabE, thiAp-ap1σ and thiAp-claH are 0.55±0.02, 0.53±0.03, 0.54±0.02 and 0.29±0.02 respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). After 6-8h of transcriptional derepression, UapA-GFP fluorescence to the PM does not change when rabE or ap1σ are repressed. On the other hand, in the absence of ClaH, UapA does not reach the PM, as its fluorescence intensity there is statistically lower (****P<0.0001) in comparison to that of the wild-type strain. Biological/technical replicates: 2/15 for each strain.


( B ) In a similar experiment , the apical polarized localization of de novo made SynA , used as a control of conventional secretion , is shown to be abolished in all three strains where RabE , AP - 1σ or ClaH are repressed . Scale bars : 5 μm . mCherry - SynA PM / cytosolic intensity ratios are plotted with values being 0 . 52 ± 0 . 02 for wild - type , 0 . 14 ± 0 . 02 for thiAp - rabE , 0 . 24 ± 0 . 02 for thiAp - ap1σ and 0 . 16 ± 0 . 02 for thiAp - claH . The statistical evaluation was performed as in ( A ) . There is a significant difference ( * * * * P < 0 . 0001 ) of mCherry - SynA fluorescence in the apical PM membrane between the wild - type and the three strains lacking the post - Golgi proteins . Biological / technical replicates : 2 / 15 for each strain .

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(B) In a similar experiment, the apical polarized localization of de novo made SynA, used as a control of conventional secretion, is shown to be abolished in all three strains where RabE, AP-1σ or ClaH are repressed. Scale bars: 5 μm. mCherry-SynA PM/cytosolic intensity ratios are plotted with values being 0.52±0.02 for wild-type, 0.14±0.02 for thiAp-rabE, 0.24±0.02 for thiAp-ap1σ and 0.16±0.02 for thiAp-claH. The statistical evaluation was performed as in (A). There is a significant difference (****P<0.0001) of mCherry-SynA fluorescence in the apical PM membrane between the wild-type and the three strains lacking the post-Golgi proteins. Biological/technical replicates: 2/15 for each strain.


( C ) Co - localization analysis and relevant quantification of strains co - expressing de novo made UapA - GFP ( 120 min after initiation of transcription ) with RabE - mRFP , AP - 1 - mRFP or ClaH - mRFP . Scale bars : 2 μm . Quantification by calculating Pearson ' s Correlation Coefficient ( PCC ) shows clear non - colocalization of UapA with all the Post - Golgi markers tested , as confirmed by one sample t - test ( PCC = 0 . 31 ± 0 . 08 , 0 . 29 ± 0 . 04 or 0 . 07 ± 0 . 01 , with * * * * P < 0 . 0001 , for RabE , AP - 1 or ClaH , respectively ) . Biological / technical replicates : 3 / 7 for each strain .

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(C) Co-localization analysis and relevant quantification of strains co-expressing de novo made UapA-GFP (120 min after initiation of transcription) with RabE-mRFP, AP-1-mRFP or ClaH-mRFP. Scale bars: 2 μm. Quantification by calculating Pearson's Correlation Coefficient (PCC) shows clear non-colocalization of UapA with all the Post-Golgi markers tested, as confirmed by one sample t-test (PCC=0.31±0.08, 0.29±0.04 or 0.07±0.01, with ****P<0.0001, for RabE, AP-1 or ClaH, respectively). Biological/technical replicates: 3/7 for each strain.


( A ) Time course of treatment with the actin polymerization drug latrunculin B for 2 , 5 , 10 , 30 or 50 minutes of a strain expressing neosynthesized UapA - GFP under conditions of derepression compared to an untreated strain included as control ( 150 min ) . In all cases latrunculin B was added at 100 min of UapA derepression , so that total time of UapA - GFP expression was 102 , 105 , 110 , 130 , or 150 minutes in the different samples . Notice the abolishment of sorting of UapA to the PM after 130 or 150 min of expression when latrunculin B was present for the last 30 or 50 min , respectively . Scale bar : 5 μm .

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(A) Time course of treatment with the actin polymerization drug latrunculin B for 2, 5, 10, 30 or 50 minutes of a strain expressing neosynthesized UapA-GFP under conditions of derepression compared to an untreated strain included as control (150 min). In all cases latrunculin B was added at 100 min of UapA derepression, so that total time of UapA-GFP expression was 102, 105, 110, 130, or 150 minutes in the different samples. Notice the abolishment of sorting of UapA to the PM after 130 or 150 min of expression when latrunculin B was present for the last 30 or 50 min, respectively. Scale bar: 5 μm.


( B ) Time course of treatment of strains expressing neosynthesized alcAp - UapA - GFP and mCherry - TubA with the anti - microtubule drug benomyl for 5 , 55 , or 95 minutes . In all cases benomyl was added at 95 min of UapA derepression , so that total time of UapA - GFP expression was 100 , 150 or 190 minutes Benomyl abolished the thread - like appearance microtubules in all samples added , evident by the diffuse cytoplasmic signal of mCherry - TubA . Notice that UapA reaches normally the PM ( at 190 min of derepression ) , similarly to the untreated control ( right panel ) . Scale bar : 5 μm .

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(B) Time course of treatment of strains expressing neosynthesized alcAp-UapA-GFP and mCherry-TubA with the anti-microtubule drug benomyl for 5, 55, or 95 minutes. In all cases benomyl was added at 95 min of UapA derepression, so that total time of UapA-GFP expression was 100, 150 or 190 minutes Benomyl abolished the thread-like appearance microtubules in all samples added, evident by the diffuse cytoplasmic signal of mCherry-TubA. Notice that UapA reaches normally the PM (at 190 min of derepression), similarly to the untreated control (right panel). Scale bar: 5 μm.


( C ) Subcellular localization of neosynthesized alcAp - GFP - ChsB in the absence ( left panel ) or presence ( ≈ 40 min ) of benomyl ( middle panel ) or latrunculin B ( right panel ) , after ≈ 2h of derepression . Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases . Scale bars : 5 μm .

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(C) Subcellular localization of neosynthesized alcAp-GFP-ChsB in the absence (left panel) or presence (≈40 min) of benomyl (middle panel) or latrunculin B (right panel), after ≈2h of derepression. Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases. Scale bars: 5 μm.


( D ) Effect of latrunculin B or benomyl on COPII vesicle formation , followed through the subcellular localization Sec24 - GFP . Notice that 40 min of latrunculin B led to loss of the wild - type punctuate appearance of Sec24 cytoplasmic puncta and the appearance of a diffuse fluorescent haze in the cytoplasm . In contrast , benomyl treatment did not affect the wild - type punctuate localization of Sec24 . Scale bar : 5 μm .

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(D) Effect of latrunculin B or benomyl on COPII vesicle formation, followed through the subcellular localization Sec24-GFP. Notice that 40 min of latrunculin B led to loss of the wild-type punctuate appearance of Sec24 cytoplasmic puncta and the appearance of a diffuse fluorescent haze in the cytoplasm. In contrast, benomyl treatment did not affect the wild-type punctuate localization of Sec24. Scale bar: 5 μm.


( A ) Subcellular localization of de novo made alcAp - GFP - SsoA in the PM in a single hypha . Notice the ER - like membranous network ( white arrows ) and several cytosolic puncta labelled before SsoA reaches eventually the PM . Scale bar : 5 μm .

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(A) Subcellular localization of de novo made alcAp-GFP-SsoA in the PM in a single hypha. Notice the ER-like membranous network (white arrows) and several cytosolic puncta labelled before SsoA reaches eventually the PM. Scale bar: 5 μm.


( B ) Subcellular localization of neosynthesized UapA - GFP ( 8 hours after transcriptional depression ) while ssoA transcription is ab initio repressed . Notice the localization of UapA in the ER - membranous network , often at very close proximity with the PM . Scale bar : 5 μm . In the scatter plot on the right , UapA - GFP PM / cytosolic intensity ratios are quantified when ssoA is derepressed ( - thi ) or repressed ( + thi ) , with mean values being 0 . 54 ± 0 . 02 and 0 . 30 ± 0 . 02 respectively . There is statistically lower fluorescence intensity to the PM ( * * * * P < 0 . 0001 ) when SsoA is absent from the cell , as confirmed by an unpaired t - test . Biological / technical replicates : 2 / 15 for each condition .

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(B) Subcellular localization of neosynthesized UapA-GFP (8 hours after transcriptional depression) while ssoA transcription is ab initio repressed. Notice the localization of UapA in the ER-membranous network, often at very close proximity with the PM. Scale bar: 5 μm. In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when ssoA is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.30±0.02 respectively. There is statistically lower fluorescence intensity to the PM (****P<0.0001) when SsoA is absent from the cell, as confirmed by an unpaired t-test. Biological/technical replicates: 2/15 for each condition.


( A ) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp - AzgA - GFP and alcAp - FurA - GFP , after 6 - 8 hours transcriptional derepression in strains where the expression of Sec24 , SedV , GeaA , HypB , RabE , AP - 1σ and SsoA was repressed ab initio ( o / n ) by addition of thiamine . Images showing that sorting of AzgA or FurA to the PM does not require early ( SedV , GeaA ) , late ( HypB ) or post - Golgi ( RabE , AP - 1σ ) key proteins . On the other hand , COPII coat protein Sec24 and PM t - SNARE SsoA are essential for proper localization of the two transporters to the PM . Scale bar : 5 μm .

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(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm.


( B ) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters ( see text for details ) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated . Notice that addition of Benomyl , has no effect on translocation of AzgA and FurA to the PM , whereas Latrunculin B abolishes their PM localization , as seen also for UapA . Scale bar : 5 μm .

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(B) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters (see text for details) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated. Notice that addition of Benomyl, has no effect on translocation of AzgA and FurA to the PM, whereas Latrunculin B abolishes their PM localization, as seen also for UapA. Scale bar: 5 μm.


( A ) Muscular exophers contain organelles and large protein complexes . Arrows : white - exopher , blue - muscle cell , green - mitochondria , red - proteasome foci . MOM - mitochondrial outer membrane . ( B ) BMW actively releases significant amounts of exopher . The image shows the middle part of the worm ' s body with muscles marked with dashed lines . Arrows indicate representative exophers , and the asterisk indicates the position of the vulva .

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(A) Muscular exophers contain organelles and large protein complexes. Arrows: white - exopher, blue - muscle cell, green - mitochondria, red - proteasome foci. MOM - mitochondrial outer membrane. (B) BMW actively releases significant amounts of exopher. The image shows the middle part of the worm's body with muscles marked with dashed lines. Arrows indicate representative exophers, and the asterisk indicates the position of the vulva.


( H ) Knockdown of two autophagy genes , atg - 7 and lgg - 1 significantly reduce the number of generated exophers . n = 91 - 103 ; N = 3 .

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(H) Knockdown of two autophagy genes, atg-7 and lgg-1 significantly reduce the number of generated exophers. n = 91-103; N = 3.


( A ) The highest number of exophers is produced during the hermaphrodite reproductive period and in aging animals . Males do not produce exophers during the first days of adulthood and begin to generate a small number of exophers later in life . Starting n = 90 hermaphrodites and 150 males ; N = 3 .

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(A) The highest number of exophers is produced during the hermaphrodite reproductive period and in aging animals. Males do not produce exophers during the first days of adulthood and begin to generate a small number of exophers later in life. Starting n = 90 hermaphrodites and 150 males; N = 3.


( B ) Feminized hermaphrodites of thermosensitive a fem - 1 mutant strain do not produce exophers regardless of growth temperature . This phenotype can be partially rescued by mating fem - 1 mutants with males . n = 10 - 26 ; N = 2 .

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(B) Feminized hermaphrodites of thermosensitive a fem-1 mutant strain do not produce exophers regardless of growth temperature. This phenotype can be partially rescued by mating fem-1 mutants with males. n = 10-26; N = 2.


( D ) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal . n = 118 and 112 animals ; N = 3 .

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(D) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal. n = 118 and 112 animals; N = 3.


( E ) Males and sterile hermaphrodites ( via FUdR treatment ) show the formation of spherical structures in the BWM that resemble mature exophers . MOM - mitochondrial outer membrane .

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(E) Males and sterile hermaphrodites (via FUdR treatment) show the formation of spherical structures in the BWM that resemble mature exophers. MOM - mitochondrial outer membrane.


( B ) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production . n = 50 - 60 ; N = 2 .

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(B) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production. n = 50-60; N = 2.


( F ) Increased eggshell permeability caused by emb - 27 knockdown elevates exopher production . n = 103 and 89 ; N = 3 .

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(F) Increased eggshell permeability caused by emb-27 knockdown elevates exopher production. n = 103 and 89; N = 3.


( C , D ) Animals with overactive exopheresis have reduced exploratory behaviour presented as a reduction in straight line distance travelled ( C ) and by representative worms traces on the plate ( D ) . n = 11 - 34 ; N = 3 .

(C, D) Animals with overactive exopheresis have reduced exploratory behaviour presented as a reduction in straight line distance travelled (C) and by representative worms traces on the plate (D). n = 11-34; N = 3.


( E ) RNAi knockdown of the egg yolk precursor protein VIT - 1 increases the number of muscular exophers . n = 20 ; N = 2 .

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(E) RNAi knockdown of the egg yolk precursor protein VIT-1 increases the number of muscular exophers. n = 20; N = 2.


( F ) Embryos from hermaphrodite mothers that produce a high number of exophers contain more egg yolk precursor protein VIT - 2 . Representative images of embryos with endogenous VIT - 2 : : GFP levels from mothers with different exopheresis activity are shown above the graph . n = 15 and 14 , N = 5 and 6 . Scale bar is 10 µm .

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(F) Embryos from hermaphrodite mothers that produce a high number of exophers contain more egg yolk precursor protein VIT-2. Representative images of embryos with endogenous VIT-2::GFP levels from mothers with different exopheresis activity are shown above the graph. n = 15 and 14, N = 5 and 6. Scale bar is 10 µm.


( G ) RNAi knockdown of RME - 2 yolk receptor abolish exopher production . n = 110 - 123 ; N = 3 .

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(G) RNAi knockdown of RME-2 yolk receptor abolish exopher production. n = 110-123; N = 3.


( H ) Muscle - produced VIT - 2 is released from muscles via exophers . The image shows the midbody of worms expressing the proteasome subunit RPN - 5 tagged with RFP in BWM and VIT - 2 : : GFP endogenous expression . Arrows : white - exopher , blue - muscle cell . Dashed lines mark embryos present in the uterus . Scale bar is 10 µm .

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(H) Muscle-produced VIT-2 is released from muscles via exophers. The image shows the midbody of worms expressing the proteasome subunit RPN-5 tagged with RFP in BWM and VIT-2::GFP endogenous expression. Arrows: white - exopher, blue - muscle cell. Dashed lines mark embryos present in the uterus. Scale bar is 10 µm.


B . Representative E - cadherin ( E - cad ) and DAPI immunofluorescence images of AECs treated apically with TGFβ1 or vehicle control after 72 h in the co - culture system ( scale bars 25 µm ) .

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B. Representative E-cadherin (E-cad) and DAPI immunofluorescence images of AECs treated apically with TGFβ1 or vehicle control after 72 h in the co-culture system (scale bars 25 µm).


E . ELISA analysis shows increased αSMA levels in a TGFβ1 dose - dependent manner from MCs and IPF - MCs in the co - culture system . Note a stronger αSMA response in IPF - MCs ( mean + s . d . , n = 3 biological replicates from 6 MCs and 6 IPF - MCs donors , * * p = 0 . 0014 , * * * * p < 0 . 0001 , Nonlinear Regression followed by ANOVA / Sidak ' s ) .

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E. ELISA analysis shows increased αSMA levels in a TGFβ1 dose-dependent manner from MCs and IPF-MCs in the co-culture system. Note a stronger αSMA response in IPF-MCs (mean + s.d., n = 3 biological replicates from 6 MCs and 6 IPF-MCs donors, **p = 0.0014, ****p < 0.0001, Nonlinear Regression followed by ANOVA/Sidak's).


G , H . 72 h after substitutions in the co - culture , αSMA and E - cad levels of MCs and AECs , respectively , were measured with ELISA and normalised to the control . Note replacement of injured AECs with untreated AECs or replacement of media and MCs attenuates αSMA expression and increases E - cad levels ( mean + s . d . , ns = non - significant , * * * p < 0 . 001 , * * p < 0 . 01 , n = 3 biological replicates with 6 MCs donors , repeated measurement ANOVA for MCs or ordinary ANOVA for AECs with Dunnett ´ s posthoc test ) .

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G, H. 72 h after substitutions in the co-culture, αSMA and E-cad levels of MCs and AECs, respectively, were measured with ELISA and normalised to the control. Note replacement of injured AECs with untreated AECs or replacement of media and MCs attenuates αSMA expression and increases E-cad levels (mean + s.d., ns = non-significant, ***p < 0.001, **p < 0.01, n = 3 biological replicates with 6 MCs donors, repeated measurement ANOVA for MCs or ordinary ANOVA for AECs with Dunnett´s posthoc test).


B . Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co - culture medium upon apical TGFβ1 stimulation ( p - adj < 0 . 05 , t - test ) , with examples of profibrotic secreted proteins ( red dots indicating significantly upregulated proteins , blue dots indicating significantly downregulated proteins , and grey dots indicating no significant change in protein expression levels ) .

B. Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co-culture medium upon apical TGFβ1 stimulation (p-adj < 0.05, t-test), with examples of profibrotic secreted proteins (red dots indicating significantly upregulated proteins, blue dots indicating significantly downregulated proteins, and grey dots indicating no significant change in protein expression levels).


H . Representative H3K27me3 and EZH2 immunofluorescence images and box plots ( minimum , first quartile , median , third quartile and maximum ) showing decreased H3K27me3 levels but increased total EZH2 levels in TGFβ1 - injured AECs in co - culture with MCs ( n = 5 biological replicates with > 50 cells per experiment , scale bars 50 µm , * p < 0 . 05 , unpaired t - test ) .

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H. Representative H3K27me3 and EZH2 immunofluorescence images and box plots (minimum, first quartile, median, third quartile and maximum) showing decreased H3K27me3 levels but increased total EZH2 levels in TGFβ1-injured AECs in co-culture with MCs (n = 5 biological replicates with >50 cells per experiment, scale bars 50 µm, *p < 0.05, unpaired t-test).


G . EZH2 - deleted AECs ( sgEZH2 ) reintroducing empty vector ( EV ) , T311 wildtype ( WT ) , a phosphorylated - deficient T311A , or a phosphomimetic T311D form of EZH2 were quantified for the expression of profibrotic genes . Vehicle and TGFβ1 treated AECs ( sgNEG + vehicle / TGFβ1 ) were used as control . mRNA levels are normalised to HPRT1 expression . ( mean + s . d . , n = 4 biological replicates , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Kruskal - Wallis / Dunn ' s ) .

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G. EZH2-deleted AECs (sgEZH2) reintroducing empty vector (EV), T311 wildtype (WT), a phosphorylated-deficient T311A, or a phosphomimetic T311D form of EZH2 were quantified for the expression of profibrotic genes. Vehicle and TGFβ1 treated AECs (sgNEG + vehicle / TGFβ1) were used as control. mRNA levels are normalised to HPRT1 expression. (mean + s.d., n = 4 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis/Dunn's).


B . Nuclear fractionation followed by simple western analysis ( Peggy Sue ) of AECs exposed to 72 h of TGFβ1 in the co - culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph - EZH2 . Quantifications ( lower panels ) show mean + s . d . , n = 3 biological replicates ( ns = non - significant , * * * p < 0 . 001 , Kruskal - Wallis / Dunn ' s ) .

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B. Nuclear fractionation followed by simple western analysis (Peggy Sue) of AECs exposed to 72 h of TGFβ1 in the co-culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph-EZH2. Quantifications (lower panels) show mean + s.d., n = 3 biological replicates (ns = non-significant, ***p < 0.001, Kruskal-Wallis/Dunn's).


E . ChIP - qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5 - OZ ( mean + s . d . , n = 3 biological replicates ) . Unspecific IgG was used as negative control .

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E. ChIP-qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5-OZ (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control.


C , D . ChIP - qPCR shows increased occupancy of ( C ) POL2 - S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h , whereas no enrichment of ( D ) POL2 - S2p at the gene bodies of these genes was detected . Negative IgG control is shown in Appendix Figure S5B ( mean + s . d . , n = 3 biological replicates ) . E , F . ChIP - qPCR shows increased occupancy of ( E ) POL2 - S5p at promoters and ( F ) POL2 - S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h . Negative IgG control is shown in Appendix Figure S5C ( mean + s . d . , n = 3 biological replicates ) .

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C, D. ChIP-qPCR shows increased occupancy of (C) POL2-S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h, whereas no enrichment of (D) POL2-S2p at the gene bodies of these genes was detected. Negative IgG control is shown in Appendix Figure S5B (mean + s.d., n = 3 biological replicates). E, F. ChIP-qPCR shows increased occupancy of (E) POL2-S5p at promoters and (F) POL2-S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h. Negative IgG control is shown in Appendix Figure S5C (mean + s.d., n = 3 biological replicates).


B . Simple western analysis ( Peggy Sue ) of EZH2 immunoprecipitates shows abolition of TGFβ1 - induced profibrotic transcriptional complex of EZH2 / POL2 / actin upon the convergent treatment of TGFβ1 and Y27632 . Unspecific IgG binding was used as a negative control . Representative from 3 biological replicates is shown .

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B. Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows abolition of TGFβ1-induced profibrotic transcriptional complex of EZH2/POL2/actin upon the convergent treatment of TGFβ1 and Y27632. Unspecific IgG binding was used as a negative control. Representative from 3 biological replicates is shown.


E . qPCR analysis of profibrotic genes in MCs co - culture with AECs shows that depletion of IPO9 in TGFβ1 - injured AECs blocks the fibrotic crosstalk with MCs . Data show mRNA levels of profibrotic genes normalised to S26 ( mean + s . d . , n = 3 biological replicates with 5 MCs donors , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA / Tukey ' s ) .

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E. qPCR analysis of profibrotic genes in MCs co-culture with AECs shows that depletion of IPO9 in TGFβ1-injured AECs blocks the fibrotic crosstalk with MCs. Data show mRNA levels of profibrotic genes normalised to S26 (mean + s.d., n = 3 biological replicates with 5 MCs donors, *p < 0.05, ***p < 0.001, ANOVA/Tukey's).


A . Simple western analysis ( Peggy Sue ) and quantifications ( right panels ) of mouse lung epithelial cells shows increased ph - EZH2 ( T311 ) , ph - TAK1 , myosin activity ( ph - MLC2 ) and POL2 - K7m levels in AAV - mediated TGFβ1 over - expression . Note , increased ph - EZH2 and POL2 - K7m levels are attenuated by the EZH2 inhibitor GSK126 , whereas ph - TAK1 and ph - MLC2 levels cannot be rescued by GSK126 . Quantifications ( right panels ) show violin plots , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = non - significant , Kruskal - Wallis / Dunn ' s . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice .

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A. Simple western analysis (Peggy Sue) and quantifications (right panels) of mouse lung epithelial cells shows increased ph-EZH2 (T311), ph-TAK1, myosin activity (ph-MLC2) and POL2-K7m levels in AAV-mediated TGFβ1 over-expression. Note, increased ph-EZH2 and POL2-K7m levels are attenuated by the EZH2 inhibitor GSK126, whereas ph-TAK1 and ph-MLC2 levels cannot be rescued by GSK126. Quantifications (right panels) show violin plots, *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant, Kruskal-Wallis/Dunn's. Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.


B . Representative of 3D computed tomography ( CT ) reconstruction of the lung from control , AAV - TGFβ1 and GSK126 - treated AAV - TGFβ1 mice ( green : lung tissue , red : airways , and region - of - interest ( ROI ) : blue ) . Insets show µCT slices in the middle of the lung from respective mice . Note , GSK126 attenuates TGFβ1 - induced lung injury . Quantification ( right panel ) shows mean intensity of ROIs from the whole lung ( violin plots , * p = 0 . 0385 , * * p = 0 . 0012 , ANOVA / Holm - Sidak ' s ) . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice .

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B. Representative of 3D computed tomography (CT) reconstruction of the lung from control, AAV-TGFβ1 and GSK126-treated AAV-TGFβ1 mice (green: lung tissue, red: airways, and region-of-interest (ROI): blue). Insets show µCT slices in the middle of the lung from respective mice. Note, GSK126 attenuates TGFβ1-induced lung injury. Quantification (right panel) shows mean intensity of ROIs from the whole lung (violin plots, *p = 0.0385, **p = 0.0012, ANOVA/Holm-Sidak's). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.


D . Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph - EZH2 ( scale bars 100 µm ) , pro - SFTPC as a marker for alveolar type 2 epithelial cells ( scale bars 50 µm ) and quantifications ( right panels ) show percentage of KRT5 + pods area per 10X field and percentage of pro - SFTPC + cells per 20X field ( * p < 0 . 05 , unpaired t - test ) . Data information : All violin plots display minimum , first quartile , median , third quartile and maximum ; n = 5 control , 12 AAV - TGFβ1 and 13 GSK126 - treated AAV - TGFβ1 mice .

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D. Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph-EZH2 (scale bars 100 µm), pro-SFTPC as a marker for alveolar type 2 epithelial cells (scale bars 50 µm) and quantifications (right panels) show percentage of KRT5+ pods area per 10X field and percentage of pro-SFTPC+ cells per 20X field (*p < 0.05, unpaired t-test). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.


B MAPKKK5 - C interacts with PBL27 in yeast two - hybrid experiments . The growth of yeast colonies on plates ( - ULWH ) lacking uracil ( U ) , leucine ( L ) , tryptophan ( W ) , and histidine ( H ) with 2 mM 3 - aminotriazole ( 3 - AT ) indicates a positive interaction .

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B MAPKKK5-C interacts with PBL27 in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 2 mM 3-aminotriazole (3-AT) indicates a positive interaction.


C Chitin - induced MAPK activation was analyzed by immunoblots with α - pMAPK .

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C Chitin-induced MAPK activation was analyzed by immunoblots with α-pMAPK.


D Complementation of chitin - induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5 - FLAG . MAPK activation was analyzed by immunoblots with α - pMAPK .

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D Complementation of chitin-induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5-FLAG. MAPK activation was analyzed by immunoblots with α-pMAPK.


E Flg22 - induced MAPK activation was analyzed by immunoblots with α - pMAPK . Experiments ( B ) - ( E ) were performed three times with similar results .

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E Flg22-induced MAPK activation was analyzed by immunoblots with α-pMAPK. Experiments (B) - (E) were performed three times with similar results.


A Chitin - induced callose deposition in the mapkkk5 mutants . Seedlings were analyzed at 18 hours after treatment with 10 μM chitin . Representative pictures from the biological replicates are presented . Data are means ± SD from three independent biological replicates , where each biological replicate consists of two technical replicates . The asterisks indicate statistically significant differences from the WT controls by Student ' s t - test ( P < 0 . 05 ) .

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A Chitin-induced callose deposition in the mapkkk5 mutants. Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Representative pictures from the biological replicates are presented. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


B Leaf discs were treated with 10 μM ( GlcNAc ) 7 in a solution containing 500 μM L - 012 and 10 μg / ml horseradish peroxidase . ROS production was quantified using a luminescence microplate reader . Data are means ± SD calculated using three biological replicates , where each biological replicate consisted of two technical replicates .

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B Leaf discs were treated with 10 μM (GlcNAc)7 in a solution containing 500 μM L-012 and 10 μg/ml horseradish peroxidase. ROS production was quantified using a luminescence microplate reader. Data are means ±SD calculated using three biological replicates, where each biological replicate consisted of two technical replicates.


C mapkkk5 mutations reduce resistance to Alternaria brassiciicola . Lesions sizes were measured 6 days after inoculation . Values are mean ± S . E . , n ≥ 92 . Asterisks indicate significant difference from wild - type controls by Student ' s t - test ( P < 0 . 01 ) .

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C mapkkk5 mutations reduce resistance to Alternaria brassiciicola. Lesions sizes were measured 6 days after inoculation. Values are mean ±S.E., n ≥92. Asterisks indicate significant difference from wild-type controls by Student's t-test (P < 0.01).


D Analysis of chitin - induced transcriptional reprogramming in mapkkk5 - 1 . Total RNA was extracted from seedlings treated with mock or 40 μM chitin for 3 hours and analyzed by RNA - seq . Genes that are significantly induced or suppressed by chitin in the wild type were selected ( q - value < 0 . 05 ; 12 , 992 genes ) . The log2 fold changes of the selected genes compared to mock in the wild type and mapkkk5 - 1 were plotted . Yellow and blue dots indicate genes that show reduced induction or suppression ( 506 genes ) or enhanced induction or suppression ( 151 genes ) in mapkkk5 - 1 compared to the wildtype , respectively . The slope of the linear regression line ( red ) indicates that the overall transcriptional response is weakened in mapkkk5 - 1 compared to the wild type . As a comparison , the line y = x ( black ) is shown . For more details , see the Methods . E A heatmap showing MAPKKK5 - dependent genes . Genes showing reduced induction or suppression in mapkkk5 - 1 compared to the wild type were selected as described in the Methods ( 339 genes ) . The log2 fold changes of the selected genes compared to mock were subjected to hierarchical clustering analysis . Yellow indicates positive values , blue indicates negative values and black indicates zero : see the color scale .

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D Analysis of chitin-induced transcriptional reprogramming in mapkkk5-1. Total RNA was extracted from seedlings treated with mock or 40 μM chitin for 3 hours and analyzed by RNA-seq. Genes that are significantly induced or suppressed by chitin in the wild type were selected (q-value < 0.05; 12,992 genes). The log2 fold changes of the selected genes compared to mock in the wild type and mapkkk5-1 were plotted. Yellow and blue dots indicate genes that show reduced induction or suppression (506 genes) or enhanced induction or suppression (151 genes) in mapkkk5-1 compared to the wildtype, respectively. The slope of the linear regression line (red) indicates that the overall transcriptional response is weakened in mapkkk5-1 compared to the wild type. As a comparison, the line y=x (black) is shown. For more details, see the Methods.E A heatmap showing MAPKKK5-dependent genes. Genes showing reduced induction or suppression in mapkkk5-1 compared to the wild type were selected as described in the Methods (339 genes). The log2 fold changes of the selected genes compared to mock were subjected to hierarchical clustering analysis. Yellow indicates positive values, blue indicates negative values and black indicates zero: see the color scale.


F Expression patterns of representative MAPKKK5 - dependent genes . qRT - PCR analysis of defense - related genes in 8 - day - old seedlings exposed to 40μM ( GlcNAc ) 7 for 3h . Data are shown as the average of three independent biological replicates ± SD . The asterisks indicate statistically significant differences from the WT controls by Student ' s t - test ( P < 0 . 05 ) .

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F Expression patterns of representative MAPKKK5-dependent genes. qRT-PCR analysis of defense-related genes in 8-day-old seedlings exposed to 40μM (GlcNAc)7 for 3h. Data are shown as the average of three independent biological replicates ± SD. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


A The MAPKKK5 - GFP protein was detected in the membrane fraction . The total ( T ) , soluble ( S ) , and microsomal membrane ( M ) fractions purified from Nb leaves expressing MAPKKK5 - GFP were used for immunoblots with antibodies against GFP , PM - localized aquaporin PIP1 and cytosolic ascorbate peroxidase EAP1 .

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A The MAPKKK5-GFP protein was detected in the membrane fraction. The total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5-GFP were used for immunoblots with antibodies against GFP, PM-localized aquaporin PIP1 and cytosolic ascorbate peroxidase EAP1.


B Subcellular localization of MAPKKK5 - GFP and GFP in Nb leaves . The results were photographed at 38 hpi . Scale bar = 50µm .

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B Subcellular localization of MAPKKK5-GFP and GFP in Nb leaves. The results were photographed at 38 hpi. Scale bar=50µm.


C Detection of MAPKKK5 - GFP and PBL27 - HA transiently expressed in Nb leaves . Total proteins were purified from Nb leaves at 38 hpi .

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C Detection of MAPKKK5-GFP and PBL27-HA transiently expressed in Nb leaves. Total proteins were purified from Nb leaves at 38 hpi.


D Localization of MAPKKK5K375M - GFP was analyzed by using total ( T ) , soluble ( S ) , and microsomal membrane ( M ) fractions purified from Nb leaves expressing MAPKKK5 K375M -

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D Localization of MAPKKK5K375M-GFP was analyzed by using total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5 K375M -GFP


E Co - immunoprecipitation assays show that MAPKKK5K375M - GFP forms the complex with PBL27 - HA in Nb leaves . The microsomal fractions purified from Nb leaves co - expressing MAPKKK5K375M - GFP and PBL27 - HA at 48 hpi were used for co - immunoprecipitation with α - GFP .

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E Co-immunoprecipitation assays show that MAPKKK5K375M-GFP forms the complex with PBL27-HA in Nb leaves. The microsomal fractions purified from Nb leaves co-expressing MAPKKK5K375M-GFP and PBL27-HA at 48 hpi were used for co-immunoprecipitation with α-GFP.


F Visualization of the interaction between PBL27 and MAPKKK5K375M by bimolecular fluorescence complementation ( BiFC ) analysis in Nb leaves . The results were photographed at 38 hpi . Venus fluorescence indicates the interaction between PBL27 and MAPKKK5K375M . Scale bar = 50µm .

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F Visualization of the interaction between PBL27 and MAPKKK5K375M by bimolecular fluorescence complementation (BiFC) analysis in Nb leaves. The results were photographed at 38 hpi. Venus fluorescence indicates the interaction between PBL27 and MAPKKK5K375M. Scale bar=50µm.


A Detection of MAPKKK5 - GFP and MAPKKK5K375M - GFP transiently expressed in Arabidopsis protoplasts .

A Detection of MAPKKK5-GFP and MAPKKK5K375M-GFP transiently expressed in Arabidopsis protoplasts.


B Expression of MAPKKK5 - GFP reduces the level of PBL27 . CBB , Coomassie brilliant blue .

B Expression of MAPKKK5-GFP reduces the level of PBL27. CBB, Coomassie brilliant blue.


C Arabidopsisprotoplasts expressing PBL27 - HA and MAPKKK5 - GFP were treated with 30 μM MG132 for 3 h and subjected to immunoblots with α - HA and α - GFP .

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C Arabidopsisprotoplasts expressing PBL27-HA and MAPKKK5-GFP were treated with 30 μM MG132 for 3 h and subjected to immunoblots with α-HA and α-GFP.


D The co - immunoprecipitation assay shows that PBL27 forms the complex with MAPKKK5 in Arabidopsis protoplasts . Total proteins purified from protoplasts co - expressing PBL27 and MAPKKK5 were subjected to immunoprecipitation with α - HA .

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D The co-immunoprecipitation assay shows that PBL27 forms the complex with MAPKKK5 in Arabidopsis protoplasts. Total proteins purified from protoplasts co-expressing PBL27 and MAPKKK5 were subjected to immunoprecipitation with α-HA.