Figure 5B . Protein lysates expressing NRC4AAA - Myc with Rpi - amr3 - HF or Rpi - amr1 - Flag in the presence and absence of cognate effector were loaded on blue native - PAGE . Proteins were transiently expressed in N . benthamiana nrc2 / 3 / 4 knockout plants . Non - specific bands are indicated with * .

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Figure 5B. Protein lysates expressing NRC4AAA-Myc with Rpi-amr3-HF or Rpi-amr1-Flag in the presence and absence of cognate effector were loaded on blue native-PAGE. Proteins were transiently expressed in N. benthamiana nrc2/3/4 knockout plants. Non-specific bands are indicated with *.


Figure 6B . NRC2EEE - Myc oligomerizes in response - dependent manner . Protein lysates from N . benthamiana nrc2 / 3 / 4 knockout transiently expressing NRC2EEE - Myc , Rpi - amr3 - HF and AVRamr3 alleles from P . infestans , P . capsici , and P . parasitica were loaded on blue native - PAGE and blotted for NRC2EEE ( anti - Myc ) . C . Recognition is correlated with interaction and protein complex formation of AVRamr3 with Rpi - amr3 . Protein extracts from Fig 5B were immunoprecipitated with anti - Flag antibody , separated on blue native - PAGE , and AVRamr3 - GFP proteins of P . infestans , P . capsici , and P . parasitica were visualized .

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Figure 6B. NRC2EEE-Myc oligomerizes in response-dependent manner. Protein lysates from N. benthamiana nrc2/3/4 knockout transiently expressing NRC2EEE-Myc, Rpi-amr3-HF and AVRamr3 alleles from P. infestans, P. capsici, and P. parasitica were loaded on blue native-PAGE and blotted for NRC2EEE (anti-Myc). C. Recognition is correlated with interaction and protein complex formation of AVRamr3 with Rpi-amr3. Protein extracts from Fig 5B were immunoprecipitated with anti-Flag antibody, separated on blue native-PAGE, and AVRamr3-GFP proteins of P. infestans, P. capsici, and P. parasitica were visualized.


Figure 1 ( A ) Nissl - stained sagittal brain sections reveal no change in hippocampal morphology between Clcn3unc / unc and Clcn3 + / + mice at P45 or after 20 months . In contrast , the hippocampus was absent ( indicated by asterisk ) in 3 months - old Clcn3 - / - mice ( scale bar : 200 µm ) . ( B ) Nissl - stained paraffin sections show intact retinal layers in 20 months old Clcn3unc / un mice . Neurodegeneration in Clcn3 - / - mice however , results in a loss of retinal structure already at 11 weeks of age ( scale bar : 100 µm ) . RPE , retinal pigment epithelium ; OS , photoreceptor outer segments ; IS , photoreceptor inner segments ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , ganglion cell layer . ( C ) Immunoblots for ClC - 3 , - 4 and - 5 of membrane fractions of WT ( + / + ) , Clcn3unc / unc ( unc / unc ) , Clcn3unc / + ( unc / + ) and Clcn3 - / - ( - / - ) mice . β - actin , loading control . ( D ) Representative Western blot for ClC - 4 of membrane fractions from brain and kidney of WT ( + / + ) , Clcn3 + / - ( + / - ) and Clcn3 - / - ( - / - ) mice . β - actin , loading control . ( E ) Quantification of ClC - 4 immunoblots including those shown in ( C and D ) ( normalized to actin ) . Mean values ± s . e . m . Average from ≥ 5 animals per genotype and at ≥ 2 immunoblots per animal . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 ( two - tailed unpaired t test ) .

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Figure 1(A) Nissl-stained sagittal brain sections reveal no change in hippocampal morphology between Clcn3unc/unc and Clcn3+/+ mice at P45 or after 20 months. In contrast, the hippocampus was absent (indicated by asterisk) in 3 months-old Clcn3-/- mice (scale bar: 200 µm).(B) Nissl-stained paraffin sections show intact retinal layers in 20 months old Clcn3unc/un mice. Neurodegeneration in Clcn3-/- mice however, results in a loss of retinal structure already at 11 weeks of age (scale bar: 100 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.(C) Immunoblots for ClC-3, -4 and -5 of membrane fractions of WT (+/+), Clcn3unc/unc (unc/unc), Clcn3unc/+ (unc/+) and Clcn3-/- (-/-) mice. β-actin, loading control.(D) Representative Western blot for ClC-4 of membrane fractions from brain and kidney of WT (+/+), Clcn3+/- (+/-) and Clcn3-/- (-/-) mice. β-actin, loading control. (E) Quantification of ClC-4 immunoblots including those shown in (C and D) (normalized to actin). Mean values ± s.e.m. Average from ≥5 animals per genotype and at ≥2 immunoblots per animal. *** p < 0.0005 , ** p < 0.005, * p < 0.05 (two-tailed unpaired t test).


Figure 2 ( A ) Co - immunoprecipitation reveals a ClC - 3 - ClC - 4 complex . 10 % of solubilized brain membranes of WT and Clcn3unc / unc mice were directly loaded on the gel ( input , or first immunoprecipitated ( IP ) with antibodies against ClC - 3 or ClC - 4 . Western blots were probed for ClC - 3 and ClC - 4 . Equivalent amounts of lysates and precipitates were loaded . * unspecific band / contamination . ( B ) FRET experiments show homo - and heteromerization of ClC - 3 and ClC - 4 constructs ( fused to yellow fluorescent protein ( YFP ) and cyan fluorescent protein ( CFP ) ) overexpressed in COS - 7 cells . Co - expressed constructs are indicated . Graph represents energy transfer efficiencies from acceptor photobleaching , depicted as bleach corrected values ( subtraction of ClC - 4CFP alone ) . Mean values ± s . e . m . N = 20 ( ClC - 3YFP / ClC - 4CFP ) ; 33 ( ClC - 3YFP / ClC - 3CFP ) ; 26 ( ClC - 4YFP / ClC - 4CFP ) ; 42 ( ClC - 3YFP / Lamp - 1CFP ) cells . * * * p < 0 . 0005 ( two - tailed unpaired t test ) ( C ) Immunolabelling shows subcellular localization of hClC - 4 ( top , green in merge ) , hClC - 3 ( middle , green in merge ) and hClC - 3unc ( bottom , green in merge ) of transiently transfected COS - 7 cells , in comparison to either PDI or Lamp - 1 ( both red ) as marker for the ER and late endosomes / lysosomes , respectively . ( D ) Co - localization of hClC - 3 and hClC - 4 in cytoplasmic vesicles of COS - 7 cells transfected with hClC - 4 cDNA ( hemagglutinin ( HA ) tagged ) together with either hClC - 3 or hClC - 3unc . Immunostaining used antibodies against ClC - 3 and HA tag ( green and red in merge , respectively ) .

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Figure 2(A) Co-immunoprecipitation reveals a ClC-3-ClC-4 complex. 10% of solubilized brain membranes of WT and Clcn3unc/unc mice were directly loaded on the gel (input, or first immunoprecipitated (IP) with antibodies against ClC-3 or ClC-4. Western blots were probed for ClC-3 and ClC-4. Equivalent amounts of lysates and precipitates were loaded. * unspecific band / contamination.(B) FRET experiments show homo- and heteromerization of ClC-3 and ClC-4 constructs (fused to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)) overexpressed in COS-7 cells. Co-expressed constructs are indicated. Graph represents energy transfer efficiencies from acceptor photobleaching, depicted as bleach corrected values (subtraction of ClC-4CFP alone). Mean values ± s.e.m. N = 20 (ClC-3YFP / ClC-4CFP); 33 (ClC-3YFP / ClC-3CFP); 26 (ClC-4YFP / ClC-4CFP); 42 (ClC-3YFP / Lamp-1CFP) cells. *** p < 0.0005 (two-tailed unpaired t test)(C) Immunolabelling shows subcellular localization of hClC-4 (top, green in merge), hClC-3 (middle, green in merge) and hClC-3unc (bottom, green in merge) of transiently transfected COS-7 cells, in comparison to either PDI or Lamp-1 (both red) as marker for the ER and late endosomes/lysosomes, respectively.(D) Co-localization of hClC-3 and hClC-4 in cytoplasmic vesicles of COS-7 cells transfected with hClC-4 cDNA (hemagglutinin (HA) tagged) together with either hClC-3 or hClC-3unc. Immunostaining used antibodies against ClC-3 and HA tag (green and red in merge, respectively).


Figure 3 ( A ) Clcn3unc / unc / Clcn4 - / - and Clcn3 - / - mice died within 3 - 4 weeks after birth . Approximately 20 % of the animals survived in either line ( Clcn3unc / unc / Clcn4 - / - , n = 136 and Clcn3 - / - , n = 187 ) . All Clcn3 - / - / Clcn4 - / - mice ( n = 4 ) died within 1 - 2 weeks after birth . ( B ) Nissl - stained paraffin sections show progressive neuronal cell loss ( arrows ) that begins in hippocampal CA1 region of P14 Clcn3unc / unc / Clcn4 - / - mice and results in a complete loss of the hippocampus at P28 . Neurodegeneration progresses slower in Clcn3 - / - mice Stobrawa et al , 2001 ( ) ( scale bar : 200 µm ) . ( C ) Semi - thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc / unc / Clcn4 - / - , but not of Clcn3unc / unc or Clcn4 - / - mice ( scale bar : 50 µm ) . RPE , retinal pigment epithelium ; OS , photoreceptor outer segments ; IS , photoreceptor inner segments ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , ganglion cell layer .

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Figure 3(A) Clcn3unc/unc/Clcn4-/- and Clcn3-/- mice died within 3-4 weeks after birth. Approximately 20% of the animals survived in either line (Clcn3unc/unc/Clcn4-/-, n = 136 and Clcn3-/-, n = 187). All Clcn3-/-/Clcn4-/- mice (n= 4) died within 1-2 weeks after birth.(B) Nissl-stained paraffin sections show progressive neuronal cell loss (arrows) that begins in hippocampal CA1 region of P14 Clcn3unc/unc/Clcn4-/- mice and results in a complete loss of the hippocampus at P28. Neurodegeneration progresses slower in Clcn3-/- mice Stobrawa et al, 2001() (scale bar: 200 µm).(C) Semi-thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc/unc/Clcn4-/-, but not of Clcn3unc/unc or Clcn4-/- mice (scale bar: 50 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


Figure 4 ( A ) GFP antibodies immunolabel VenusClC - 3 ( green ) in the somata of CA1 pyramidal neurons of Clcn3ven / ven , but not Clcn3 + / + brain sections . sp , stratum pyramidale ( scale bar : 20 µm ) . ( B ) Co - immunolabelling of VenusClC - 3 ( green ) with ClC - 4 ( red ) in the CA3 region of Clcn3ven / ven mice ( scale bar : 20 µm ) . sl , stratum lucidum ; sp , stratum pyramidale . ( C ) Co - immunostaining of VenusClC - 3 ( green ) with EEA1 ( red , left panel ) , Lamp - 1 ( red , middle panel ) or synaptophysin ( red , right panel ) in either CA1 or CA3 region of Clcn3ven / ven mice . Arrows indicate structures of overlap ( scale bar : 5 µm ) . ( D ) GFP antibody immunolabels VenusClC - 3 ( green ) in neurites of cultured neurons . EEA1 and VGLUT1 are co - stained ( both in red ; left and right panel , respectively ) . The dendritic marker MAP2 is stained in blue ( right panel ) ( scale bars : 10 µm ) . DNA stained with DAPI . ( E ) Immuno - blot analysis of fractions collected during preparation of SVs from brain of Clcn3ven / ven mice . VenusClC - 3 ( detected with GFP antibody ) co - purified with SV marker protein synaptophysin . ( F ) Fluorescence of immobilized Venus - tagged SVs imaged with TIRF microscopy . Scale bar , 2 µm . ( G , H ) Co - localization between the Venus - tagged vesicles with antibodies against VGLUT1 and synaptophysin was analyzed . ( G ) 11 . 4 ± 0 . 6 ( SD ) of VGLUT1 - labeled SVs and 8 . 2 ± 2 ( SD ) of synaptophysin - labeled SVs showed Venus fluorescence ( n = 6 ) . ( H ) 65 ± 5 . 4 ( SD ) and 79 . 4 ± 3 . 8 ( SD ) of Venus - tagged SVs showed co - localization with VGLUT1 and synaptophysin , respectively ( n = 6 ) . ( I ) Co - immunolabelling of VenusClC - 3 ( green ) with synaptophysin ( red ) of immobilized SVs imaged with TIRF microscopy . Vesicle in quadrant delimited by dashed line is shown in higher magnification in merge . Scale bar , 2 µm .

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Figure 4(A) GFP antibodies immunolabel VenusClC-3 (green) in the somata of CA1 pyramidal neurons of Clcn3ven/ven, but not Clcn3+/+ brain sections. sp, stratum pyramidale (scale bar: 20 µm).(B) Co-immunolabelling of VenusClC-3 (green) with ClC-4 (red) in the CA3 region of Clcn3ven/ven mice (scale bar: 20 µm). sl, stratum lucidum; sp, stratum pyramidale.(C) Co-immunostaining of VenusClC-3 (green) with EEA1 (red, left panel), Lamp-1 (red, middle panel) or synaptophysin (red, right panel) in either CA1 or CA3 region of Clcn3ven/ven mice. Arrows indicate structures of overlap (scale bar: 5 µm).(D) GFP antibody immunolabels VenusClC-3 (green) in neurites of cultured neurons. EEA1 and VGLUT1 are co-stained (both in red; left and right panel, respectively). The dendritic marker MAP2 is stained in blue (right panel) (scale bars: 10 µm). DNA stained with DAPI.(E) Immuno-blot analysis of fractions collected during preparation of SVs from brain of Clcn3ven/ven mice. VenusClC-3 (detected with GFP antibody) co-purified with SV marker protein synaptophysin.(F) Fluorescence of immobilized Venus-tagged SVs imaged with TIRF microscopy. Scale bar, 2 µm.(G, H) Co-localization between the Venus-tagged vesicles with antibodies against VGLUT1 and synaptophysin was analyzed. (G) 11.4 ± 0.6 (SD) of VGLUT1-labeled SVs and 8.2 ± 2 (SD) of synaptophysin-labeled SVs showed Venus fluorescence (n=6). (H) 65 ± 5.4 (SD) and 79.4 ± 3.8 (SD) of Venus-tagged SVs showed co-localization with VGLUT1 and synaptophysin, respectively (n=6).(I) Co-immunolabelling of VenusClC-3 (green) with synaptophysin (red) of immobilized SVs imaged with TIRF microscopy. Vesicle in quadrant delimited by dashed line is shown in higher magnification in merge. Scale bar, 2 µm.


Figure 5 ( A ) Immunoblots for ClC - 3 , ClC - 4 , VGLUT1 , VGLUT2 , VGAT , GluR4 and GABAA - R - α1 of whole lysates from brain of Clcn3 + / + and Clcn3 - / - mice at 2 , 6 and 12 weeks of age . ( B ) Acidification of synaptic vesicle LP2 fractions from Clcn3unc / unc and Clcn3 + / + mice at one year of age ( upper panel , n = 2 animals each and ≥ 3 measurements per animal ) , 10 - weeks - old Clcn4 - / - and WT mice ( middle panel , n = 3 animals each and ≥ 3 measurements per animal ) , and 4 - 6 - weeks - old Clcn3unc / unc / Clcn4 - / - and control mice ( lower panel , 6 animals each with ≥ 2 measurements per animal ) . A decrease in fluorescence reflects acidification . The protonophore FCCP dissipated the pH gradient . Mean values ± s . e . m . ( C ) Quantification of ATP - induced acidification of LP2 fractions derived from 2 , 6 , and 10 - 12 weeks - old Clcn3 - / - compared to wild - type mice . Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl . At least 2 animals in at least 2 independent experiments were pooled . Each measurement was performed at least 3 times . Mean ± s . e . m . is shown . * p < 0 . 05 , * * * p < 0 . 0005 ( two - tailed unpaired t test ) . ( D ) Immunoblot revealed strong reduction of VGLUT1 levels in the homogenate ( H ) and LP2 fraction of Clcn3unc / unc / Clcn4 - / - mice . ( E ) Endosomal pH of Clcn3 + / + and Clcn3 - / - primary hippocampal neurons determined with a pH - sensitive transferrin conjugate . N = 5 independent cell lines with at least 2 live cell dishes per cell line with about 10 images each and ≥ 10 Tfn - positive compartments per image and genotype were analysed . Mean ± s . e . m . is shown .

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Figure 5(A) Immunoblots for ClC-3, ClC-4, VGLUT1, VGLUT2, VGAT, GluR4 and GABAA-R-α1 of whole lysates from brain of Clcn3+/+ and Clcn3-/- mice at 2, 6 and 12 weeks of age.(B) Acidification of synaptic vesicle LP2 fractions from Clcn3unc/unc and Clcn3+/+ mice at one year of age (upper panel, n= 2 animals each and ≥3 measurements per animal), 10-weeks-old Clcn4-/- and WT mice (middle panel, n= 3 animals each and ≥3 measurements per animal), and 4-6-weeks-old Clcn3unc/unc/Clcn4-/- and control mice (lower panel, 6 animals each with ≥2 measurements per animal). A decrease in fluorescence reflects acidification. The protonophore FCCP dissipated the pH gradient. Mean values ± s.e.m.(C) Quantification of ATP-induced acidification of LP2 fractions derived from 2, 6, and 10-12 weeks-old Clcn3-/- compared to wild-type mice. Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl. At least 2 animals in at least 2 independent experiments were pooled. Each measurement was performed at least 3 times. Mean ± s.e.m. is shown.* p < 0.05, *** p < 0.0005 (two-tailed unpaired t test).(D) Immunoblot revealed strong reduction of VGLUT1 levels in the homogenate (H) and LP2 fraction of Clcn3unc/unc/Clcn4-/- mice.(E) Endosomal pH of Clcn3+/+ and Clcn3-/- primary hippocampal neurons determined with a pH-sensitive transferrin conjugate. N= 5 independent cell lines with at least 2 live cell dishes per cell line with about 10 images each and ≥10 Tfn-positive compartments per image and genotype were analysed. Mean ± s.e.m. is shown.


Figure 6Miniature inhibitory postsynaptic currents ( mIPSCs ) measured in CA1 pyramidal neurons in different mouse models at P14 - P16 . ( A ) example mIPSCs of WT ( left ) and Clcn3 - / - neurons . Mean values ± s . e . m . and cumulative histogram of amplitude ( top ) and frequency / interevent - interval ( bottom ) of mIPSCs in different mouse - models as indicated . Several cells of 3 mice of each genotype were analyzed . Compared genotypes were siblings . No difference in amplitude or frequency / interevent - interval distribution of mIPSCs was found in ( B ) Clcn3 - / - and Clcn3 + / + ( Clcn3 + / + : 16 cells , Clcn3 - / - : 15 cells ; mean amplitude : p > 0 . 8 , t test ; mean frequency : p > 0 . 4 , MW - test ) , in ( C ) Clcn3unc / unc and Clcn3 + / + ( Clcn3 + / + : 21 cells , Clcn3unc / unc : 13 cells ; mean amplitude : p > 0 . 7 ; mean frequency : p = 0 . 3 , t test ) , and in ( D ) Clcn4 - / - and Clcn3 + / + ( Clcn3 + / + : 18 cells , Clcn4 - / - : 18 cells ; mean amplitude : p > 0 . 3 , t - test ; mean frequency : p = 0 . 6 , MW - test ) . ( E ) No difference in mean amplitude ( p > 0 . 5 , t test ) , in cumulative amplitude and mean frequency of mIPSCs ( p > 0 . 1 , t test ) was found in Clcn3unc / unc / Clcn4 - / - and Clcn3 + / + / Clcn4 - / - . The interevent - interval distribution was shifted to the right , indicating a reduced frequency of mIPSCs in Clcn3unc / unc / Clcn4 - / - ( KS - test , p < 0 , 05 ) ( Clcn3 + / + / Clcn4 - / - : 16 cells , Clcn3unc / unc / Clcn4 - / - : 12 cells ) . ( F , G ) miniature excitatory postsynaptic currents ( mEPSCs ) . ( F ) example traces of mEPSCs in WT and Clcn3 - / - neurons ( G ) No significant difference between Clcn3unc / unc / Clcn4 - / - and Clcn3 + / + / Clcn4 - / - mEPSCs in mean amplitudes ( p > 0 . 9 , t test ) or in the distribution of amplitudes ( Clcn4 - / - : 12 cells , 2 mice ; Clcn3unc / unc / Clcn4 - / - : 8 cells , 2 mice ) . ( H ) Primary hippocampal neurons co - transfected with ClC - 3 and synaptobrevin ( syb2 ) , both luminally tagged with pHluorin or the pH - sensitive mOrange Ramirez et al , 2012 ( ) , respectively , were stimulated by 200 action potentials ( APs ) at 20 Hz ( indicated by bar ) . Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH . Note that only Syb2 and not ClC - 3 shows signs of exocytosis . Traces were corrected for photobleaching . The initial decay of the pHluorin signal is probably due to the high light exposure , which forces the fluorophores into a transient dark - state Dean et al , 2011 ( ) . Averaged fluorescence traces ( n = 10 ) , error bars , s . e . m .

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

Figure 6Miniature inhibitory postsynaptic currents (mIPSCs) measured in CA1 pyramidal neurons in different mouse models at P14-P16. (A) example mIPSCs of WT (left) and Clcn3-/- neurons.Mean values ± s.e.m. and cumulative histogram of amplitude (top) and frequency/interevent-interval (bottom) of mIPSCs in different mouse-models as indicated . Several cells of 3 mice of each genotype were analyzed. Compared genotypes were siblings. No difference in amplitude or frequency/ interevent-interval distribution of mIPSCs was found in (B) Clcn3-/- and Clcn3+/+ (Clcn3+/+: 16 cells, Clcn3-/-: 15 cells; mean amplitude: p > 0.8, t test; mean frequency: p > 0.4, MW-test), in (C) Clcn3unc/unc and Clcn3+/+ (Clcn3+/+: 21 cells, Clcn3unc/unc: 13 cells; mean amplitude: p > 0.7; mean frequency: p = 0.3, t test), and in (D) Clcn4-/- and Clcn3+/+ (Clcn3+/+: 18 cells, Clcn4-/-: 18 cells; mean amplitude: p > 0.3, t-test; mean frequency: p = 0.6, MW-test).(E) No difference in mean amplitude (p > 0.5, t test), in cumulative amplitude and mean frequency of mIPSCs (p > 0.1, t test) was found in Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/-. The interevent-interval distribution was shifted to the right, indicating a reduced frequency of mIPSCs in Clcn3unc/unc/Clcn4-/- (KS-test, p < 0,05) (Clcn3+/+/Clcn4-/-: 16 cells, Clcn3unc/unc/Clcn4-/-: 12 cells).(F,G) miniature excitatory postsynaptic currents (mEPSCs). (F) example traces of mEPSCs in WT and Clcn3-/- neurons (G) No significant difference between Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/- mEPSCs in mean amplitudes (p > 0.9, t test) or in the distribution of amplitudes (Clcn4-/-: 12 cells, 2 mice; Clcn3unc/unc/Clcn4-/-: 8 cells, 2 mice).(H) Primary hippocampal neurons co-transfected with ClC-3 and synaptobrevin (syb2), both luminally tagged with pHluorin or the pH-sensitive mOrange Ramirez et al, 2012(), respectively, were stimulated by 200 action potentials (APs) at 20 Hz (indicated by bar). Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH. Note that only Syb2 and not ClC-3 shows signs of exocytosis. Traces were corrected for photobleaching. The initial decay of the pHluorin signal is probably due to the high light exposure, which forces the fluorophores into a transient dark-state Dean et al, 2011(). Averaged fluorescence traces (n=10), error bars, s.e.m.


Figure 1 ( C ) day 20 ± 2 cerebral organoids derived from controls , patients and EML1 - heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD , counterstained with DAPI . Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines . ( D ) Quantification of VZ areas with ectopic neural rosettes ( 3 batches , 3 organoids analyzed per batch , significance based on Kruskal - Wallis - test , P = 0 . 0001 , Dunn ' s post hoc test for multiple comparisons performed to define statistical differences between genotypes , single data points are presented colored by batch ) . ( E ) Quantification of heterotopic , disorganized or organized cortical areas ( 3 batches , 3 organoids per batch , significance based on Kruskal - Wallis - test , P = 0 . 0001 for " organized " and " heterotopic " ; no significant difference for " disorganized " , Dunn ' s post hoc test performed for multiple comparisons to define statistical differences between genotypes ) . Data information : * marks statistical significance in relation to Control 1 , # in relation to Control 2 . P - values : * * * / # # # < 0 . 001 . VZ : ventricular zone ; Data in graphs are represented as means ± SD . Scale bars : 50µm .

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

Figure 1(C) day 20±2 cerebral organoids derived from controls, patients and EML1-heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD, counterstained with DAPI. Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines. (D) Quantification of VZ areas with ectopic neural rosettes (3 batches, 3 organoids analyzed per batch, significance based on Kruskal-Wallis-test, P=0.0001, Dunn's post hoc test for multiple comparisons performed to define statistical differences between genotypes, single data points are presented colored by batch). (E) Quantification of heterotopic, disorganized or organized cortical areas (3 batches, 3 organoids per batch, significance based on Kruskal-Wallis-test, P=0.0001 for "organized" and "heterotopic"; no significant difference for "disorganized", Dunn's post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P-values: ***/###<0.001. VZ: ventricular zone; Data in graphs are represented as means ± SD. Scale bars: 50µm.


Figure 2 ( A ) Hybrid organoids generated from controls mixed with control - EGFP + or EML1 - heKO - EGFP + cells ( day 20 ± 2 ) . Yellow dotted lines indicate VZ areas . ( B ) Quantification of control - EGFP + or EML1 - heKO - EGFP + cells within or at the basal side of VZ areas ( 3 batches , 3 organoids each , significance based on Mann Whitney test , * * * P = 0 . 0001 for " EGFP + cells at the basal side of VZ areas " ) . Data information : Data in graphs are represented as means ± SD . Scale bars : ( A ) 50µm ( C ) Representative images and scheme of cleavage plan orientation . Upper panel : cells immunostained for pVim ( red ) and TPX2 ( green ) , counterstained with DAPI , white dotted line indicates cleavage plane orientation , yellow dotted line VZ - surface . ( D ) Quantification of horizontal , vertical and oblique plane of cell divisions in control and EML1 - deficient organoids ( day 20 ± 2 ; 3 batches , 3 organoids each ) . Data information : Scale bars : 5µm ( F ) Electron microscopy of primary cilia derived from control or EML1 - deficient cortical progenitors in DMSO - control or following EpothiloneD ( EpoD ) exposure ( blue arrow : basal body , yellow arrow : appendages , red arrow : ciliary pockets ) . Data information : Scale bars : 0 , 5µm .

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

Figure 2(A) Hybrid organoids generated from controls mixed with control-EGFP+ or EML1-heKO-EGFP+ cells (day 20±2). Yellow dotted lines indicate VZ areas. (B) Quantification of control-EGFP+ or EML1-heKO-EGFP+ cells within or at the basal side of VZ areas (3 batches, 3 organoids each, significance based on Mann Whitney test, ***P= 0.0001 for "EGFP+ cells at the basal side of VZ areas"). Data information: Data in graphs are represented as means ± SD. Scale bars: (A) 50µm(C) Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for pVim (red) and TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, yellow dotted line VZ-surface. (D) Quantification of horizontal, vertical and oblique plane of cell divisions in control and EML1-deficient organoids (day 20±2; 3 batches, 3 organoids each). Data information: Scale bars: 5µm(F) Electron microscopy of primary cilia derived from control or EML1-deficient cortical progenitors in DMSO-control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, red arrow: ciliary pockets). Data information: Scale bars: 0,5µm.


Figure 3 ( E ) Control and EML1 - heKO - derived cerebral organoid stained for COL1A2 and MEIS2 . VZ areas are encircled , squares indicate areas enlarged in F . ( F ) Control and EML1 - heKO - derived cerebral organoid stained for COL1A2 , MEIS2 and MAPT . ( G ) Representative images showing EGFP - control and EGFP - EML1 - heKO hybrid cerebral organoid stained for EGFP , FAM107A and NCAD . Yellow dotted lines indicate morphology of EGFP + cells ( 4 independent batches each , 3 organoids per batch , at least 30 ectopic rosettes in total ) . Data information : Scale bars : ( E ) 50µm ; ( F ) 10µm ; ( G ) 50µm , enlarged 10µm .

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Figure 3(E) Control and EML1-heKO-derived cerebral organoid stained for COL1A2 and MEIS2. VZ areas are encircled, squares indicate areas enlarged in F. (F) Control and EML1-heKO-derived cerebral organoid stained for COL1A2, MEIS2 and MAPT. (G) Representative images showing EGFP-control and EGFP-EML1-heKO hybrid cerebral organoid stained for EGFP, FAM107A and NCAD. Yellow dotted lines indicate morphology of EGFP+ cells (4 independent batches each, 3 organoids per batch, at least 30 ectopic rosettes in total). Data information: Scale bars: (E) 50µm; (F) 10µm; (G) 50µm, enlarged 10µm.


Figure 4 ( D , E ) Immunofluorescence staining for ( D ) p - VIM and YAP1 or ( E ) Pax6 and YAP1 in control and EML1 - heKO - derived organoids ( day 20 ± 2 , squares indicate areas enlarged in adjacent part of the panel respectively ) . ( F ) Quantification of basally located pVim + cells with nuclear YAP1 signal per VZ area in control and EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . ( G ) Quantification of PAX6 + cells with nuclear YAP1 signal per VZ area in control and EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance based on Kruskal - Wallis - test Dunn ' s post hoc test for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( D and E ) 50µm , enlarged 10µm ( H ) Immunofluorescence staining for p - Vim in EML1 - heKO and control derived organoids in DMSO , Verteporfin or Fluvastatin treated conditions , counterstained with DAPI . Dotted line indicates VZ areas . ( I ) Quantification of p - VIM + cells located at the basal side of VZ areas in control or EML1 - heKO - derived organoids under DMSO , Verteporfin or Fluvastatin treated condition ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance based on two - way ANOVA ( on log10 normalized data ) Tukey post hoc test for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( H )

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

Figure 4(D, E) Immunofluorescence staining for (D) p-VIM and YAP1 or (E) Pax6 and YAP1 in control and EML1-heKO-derived organoids (day 20±2, squares indicate areas enlarged in adjacent part of the panel respectively). (F) Quantification of basally located pVim+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). (G) Quantification of PAX6+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on Kruskal-Wallis-test Dunn's post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (D and E) 50µm, enlarged 10µm(H) Immunofluorescence staining for p-Vim in EML1-heKO and control derived organoids in DMSO, Verteporfin or Fluvastatin treated conditions, counterstained with DAPI. Dotted line indicates VZ areas. (I) Quantification of p-VIM+ cells located at the basal side of VZ areas in control or EML1-heKO-derived organoids under DMSO, Verteporfin or Fluvastatin treated condition (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on two-way ANOVA (on log10 normalized data) Tukey post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (H) 50µm


Figure 1A . Binding of ubiquitin chains to immobilized GST - fusion proteins . Indicated proteins were purified from E . coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains , which were either K48 - or K63 - linked . The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS ‑ PAGE and fluorescence scan . Total fluorescence was quantified per lane and normalized to the first lane ( rel . fluo . ) . Asterisk ( * ) indicates lane from the same gel that was cropped and moved .

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Figure 1A. Binding of ubiquitin chains to immobilized GST-fusion proteins. Indicated proteins were purified from E. coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains, which were either K48- or K63-linked. The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS‑PAGE and fluorescence scan. Total fluorescence was quantified per lane and normalized to the first lane (rel. fluo.). Asterisk (*) indicates lane from the same gel that was cropped and moved.


Figure 2B . At indicated time points , samples were taken from the reaction mix and analyzed by SDS ‑ PAGE and fluorescence scan . Product fluorescence over total fluorescence per lane was quantified ( rel . fluo . ) . D . Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full ‑ length Ubc1 or Ubc1 - ΔUBA ( aa1 ‑ 150 ) . The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin , while acceptor sites were blocked by introducing the K48R substitution . Reactions were performed in triplicate .

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Figure 2B. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.). D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate.


Figure 3b . L143E and / or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC / UBA domain interface . Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63 ‑ Ub2 with Ub ( K48R ) at the distal position or monoubiquitin as acceptor . Experiment and initial rate determinationc . Ubc1 variants were generated with deletions in the linker region ( aa152 ‑ 167 ) , which connects the UBA domain and the UBC domain . Single turnover ubiquitination reaction mixes were incubated for 5 min with K63 - Ub2 or for 20 min with monoubiquitin as acceptor . Final product formation was analyzed by SDS - PAGE and fluorescence scan . Product fluorescence over total fluorescence ( rel . fluo . ) was quantified . d . C - terminally blocked M1 - linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full - length Ubc1 or Ubc1 - ΔUBA . Reactions with acceptor K63 - Ub2 were carried out controls . Initial rate

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

Figure 3b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determinationc. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified.d. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination


Figure 4e . CSPs of I44 were plotted over UBA domain concentration . f . KD values for binding of the UBA domain were determined from results in ( E ) assuming two independent but different binding sites .

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Figure 4e. CSPs of I44 were plotted over UBA domain concentration. f. KD values for binding of the UBA domain were determined from results in (E) assuming two independent but different binding sites.


Figure 5a . The activity of Ubc1 was compared with the K48 - linked chain building enzyme Ubc7 / Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin , K48 ‑ Ub2 or K63 - Ub2 . Initial rates were determinedb . The K63 - linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K ( h . sapiens ) were employed in single turnover ubiquitination experiments in presence of K63 - Ub2 with Ub ( K48R ) at the proximal or distal position or in presence of K48 ‑ Ub2 . Initial rate determination c . Experiment as in ( B ) but with the Ubc1 homologue ubc - 20 ( c . elegans ) .

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Figure 5a. The activity of Ubc1 was compared with the K48-linked chain building enzyme Ubc7/Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin, K48‑Ub2 or K63-Ub2. Initial rates were determinedb. The K63-linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K (h. sapiens) were employed in single turnover ubiquitination experiments in presence of K63-Ub2 with Ub(K48R) at the proximal or distal position or in presence of K48‑Ub2. Initial rate determination c. Experiment as in (B) but with the Ubc1 homologue ubc-20 (c. elegans).


Figure 6D . Alexa488 - labeled monoubiquitin and C - terminally capped K63 - Ub4 were used in a 4 : 1 molar ratio for in vitro ubiquitination reactions with full ‑ length Ubc1 or Ubc1 - ΔUBA . Where indicated , Alexa488 - labeled monoubiquitin was methylated prior to the reaction ( met - Ub ) to block de novo chain assembly . Reactions were incubated overnight , quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH ( lanes 4 - 6 ) . Samples were analyzed by SDS - PAGE and fluorescence scan .

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Figure 6D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.


Figure 7F . Ubc1 - EA was genomically integrated into various yeast strains . Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10 ‑ fold dilutions . Where noted cells were incubated at elevated temperature ( 37 ° C ) or in presence of hydroxyurea ( HU ) to induce DNA replication stress .

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Figure 7F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.


Figure 1A . 293T cells were co - transfected with CMV - renilla , 8X - TBS ( eight repeats of the TEAD - binding - sequence ) luciferase reporter , Flag - YAP , HA - TEAD2 and wild - type ( WT ) or catalytically inactive ( CS ) USP9X - V5 . One day after transfection , luciferase activity was measured and normalized with respect to renilla activity . The value for Flag - YAP / HA - TEAD - transfected cells ( 2nd column ) was adjusted to 1 . Shown below is a representative Western blot showing that the expression levels were comparable between samples ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired Student ' s t - test ) . B . 293T cells were transfected with control or USP9X - targeting siRNAs . One day after siRNA transfection , the cells were co - transfected with CMV - renilla , 8X - TBS luciferase , Flag - YAP and HA - TEAD2 . Reporter activity was quantified as described in ( A ) ( n = 4 ) . Error bars indicate the S . E . M . ( * * * p < 0 . 001 ; paired Student ' s t - test ) . C . RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re - seeded to either sparse or dense culture conditions . At 48 hours after siRNA transfection , the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins . L . E . , long exposure , S . E . , short exposure . D . Cells were treated as described in ( C ) , the indicated mRNAs were analyzed with RT - qPCR , and the results were normalized with respect to the β - actin mRNA ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired Student ' s t - test ) . E . RPE cells were transfected with the indicated siRNAs for 24 hours , and then co - transfected with CMV - renilla and 8X - TBS luciferase . One day after the latter transfection , the cells were re - seeded to either sparse or dense conditions , and reporter activity was measured at 24 hours after re - seeding ( n = 3 ) . Error bars indicate the S . E . M . ( * * p < 0 . 01 ; paired Student ' s t - test ) .

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Figure 1A. 293T cells were co-transfected with CMV-renilla, 8X-TBS (eight repeats of the TEAD-binding-sequence) luciferase reporter, Flag-YAP, HA-TEAD2 and wild-type (WT) or catalytically inactive (CS) USP9X-V5. One day after transfection, luciferase activity was measured and normalized with respect to renilla activity. The value for Flag-YAP/HA-TEAD-transfected cells (2nd column) was adjusted to 1. Shown below is a representative Western blot showing that the expression levels were comparable between samples (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student's t-test).C. RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re-seeded to either sparse or dense culture conditions. At 48 hours after siRNA transfection, the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins. L.E., long exposure, S.E., short exposure.D. Cells were treated as described in (C), the indicated mRNAs were analyzed with RT-qPCR, and the results were normalized with respect to the β-actin mRNA (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).E. RPE cells were transfected with the indicated siRNAs for 24 hours, and then co-transfected with CMV-renilla and 8X-TBS luciferase. One day after the latter transfection, the cells were re-seeded to either sparse or dense conditions, and reporter activity was measured at 24 hours after re-seeding (n=3). Error bars indicate the S.E.M. (**p < 0.01; paired Student's t-test).


Figure 2A . Representative anti - YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs . Green , YAP . Blue , DAPI . Scale bar , 10 μm . B . The nuclear YAP intensities of the cells shown in ( A ) were quantified using the Image J software . At least 70 cells from five or six random fields were analyzed . Quantified values were normalized by adjusting the average of " siControl Dense cells " to 1 . We performed two independent experiments and obtained similar results . Data from one representative experiment are presented . The red bar indicates the average value . S , Sparse . D , Dense ( * * * p < 0 . 001 ; paired Student ' s t - test ) . C . Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs , fractionated into cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins . D . Representative anti - YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids . Green , GFP or V5 . Red , YAP . Blue , DAPI . Scale bar , 10 μm . E . The nuclear YAP intensities of transfected cells in ( D ) were quantified using the Image J software . At least 70 cells from five to six random fields were analyzed . Quantified values were normalized by adjusting the average of " EGFP transfected cells " to 1 . Similar results were obtained from two independent experiments . Data from one representative experiment are presented . The red bar indicates the average value . ( * * * p < 0 . 001 ; paired Student ' s t - test ) . F . Densely cultured stable RPE clones integrated with empty vector ( Vec ) , USP9X - V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins . G . Densely cultured RPE cells transduced with CRISPR - SAM targeting USP9X were fractionated into cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins .

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Figure 2A. Representative anti-YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs. Green, YAP. Blue, DAPI. Scale bar, 10 μm.B. The nuclear YAP intensities of the cells shown in (A) were quantified using the Image J software. At least 70 cells from five or six random fields were analyzed. Quantified values were normalized by adjusting the average of "siControl Dense cells" to 1. We performed two independent experiments and obtained similar results. Data from one representative experiment are presented. The red bar indicates the average value. S, Sparse. D, Dense (***p < 0.001; paired Student's t-test).C. Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs, fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.D. Representative anti-YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids. Green, GFP or V5. Red, YAP. Blue, DAPI. Scale bar, 10 μm.E. The nuclear YAP intensities of transfected cells in (D) were quantified using the Image J software. At least 70 cells from five to six random fields were analyzed. Quantified values were normalized by adjusting the average of "EGFP transfected cells" to 1. Similar results were obtained from two independent experiments. Data from one representative experiment are presented. The red bar indicates the average value. (***p < 0.001; paired Student's t-test).F. Densely cultured stable RPE clones integrated with empty vector (Vec), USP9X-V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.G. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.


Figure 3A . RPE cells were analyzed by Western blotting for the indicated proteins . B . Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for the indicated proteins . C . Densely cultured RPE cells transduced with CRISPR - SAM targeting USP9X were analyzed by Western blotting for the indicated proteins . D . RPE or MCF10A cells expressing the control or USP9X shRNA were transfected with the LATS1 / 2 siRNA as indicated . Cells were re - seeded to low density and harvested after one day . E . RPE cells transduced with Flag - YAP WT or Flag - YAP 5SA were transfected with the indicated siRNAs . Cells were re - seeded to low density and harvested after one day . F . RT - qPCR of CTGF mRNAs in the cells described in ( E ) ( n = 3 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , paired Student ' s t - test ) .

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Figure 3A. RPE cells were analyzed by Western blotting for the indicated proteins.B. Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for the indicated proteins.C. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were analyzed by Western blotting for the indicated proteins.D. RPE or MCF10A cells expressing the control or USP9X shRNA were transfected with the LATS1/2 siRNA as indicated. Cells were re-seeded to low density and harvested after one day.E. RPE cells transduced with Flag-YAP WT or Flag-YAP 5SA were transfected with the indicated siRNAs. Cells were re-seeded to low density and harvested after one day.F. RT-qPCR of CTGF mRNAs in the cells described in (E) (n=3). Error bars indicate the S.E.M. (*p< 0.05, paired Student's t-test).


Figure 4A . RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re - seeded to low density , and cell extracts were analyzed by Western blotting for the indicated proteins . B . The cells described in ( A ) were analyzed by RT - qPCR for the indicated target genes ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired Student ' s t - test ) . C . Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti - AMOTL2 antibody and analyzed by Western blotting for the indicated proteins . WCL , whole cell lysates .

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Figure 4A. RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re-seeded to low density, and cell extracts were analyzed by Western blotting for the indicated proteins.B. The cells described in (A) were analyzed by RT-qPCR for the indicated target genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).C. Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti-AMOTL2 antibody and analyzed by Western blotting for the indicated proteins. WCL, whole cell lysates.


Figure 5A . 293T cells were transfected with the indicated siRNAs , cultured for 24 hours , and then transfected with the indicated DNAs . 48 hours after siRNA transfection , the cells were harvested and ubiquitination of Flag - AMOTL2 was examined . C , control siRNA . B . 293T cells were transfected with the indicated combinations of DNAs , cultured for 24 hours , and subjected to in vivo ubiquitination assays . C . RPE cells transduced with vector ( control ) or Flag - AMOTL2 were seeded under sparse ( S ) or dense ( D ) conditions , and an in vivo ubiquitination assay was performed . D . RPE cells transduced with Flag - AMOTL2 were transfected with a 1 : 1 mixture of the two USP9X siRNAs . One day after siRNA transfection , the cells were re - seeded to the sparse condition and an in vivo ubiquitination assay was performed after one day . E . 293T cells were transfected with the indicated combinations of DNAs , and a co - immunoprecipitation assay was performed . F . Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti - AMOTL2 antibody .

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Figure 5A. 293T cells were transfected with the indicated siRNAs, cultured for 24 hours, and then transfected with the indicated DNAs. 48 hours after siRNA transfection, the cells were harvested and ubiquitination of Flag-AMOTL2 was examined. C, control siRNA.B. 293T cells were transfected with the indicated combinations of DNAs, cultured for 24 hours, and subjected to in vivo ubiquitination assays.C. RPE cells transduced with vector (control) or Flag-AMOTL2 were seeded under sparse (S) or dense (D) conditions, and an in vivo ubiquitination assay was performed.D. RPE cells transduced with Flag-AMOTL2 were transfected with a 1:1 mixture of the two USP9X siRNAs. One day after siRNA transfection, the cells were re-seeded to the sparse condition and an in vivo ubiquitination assay was performed after one day.E. 293T cells were transfected with the indicated combinations of DNAs, and a co-immunoprecipitation assay was performed.F. Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti-AMOTL2 antibody.


Figure 6A . Flag - AMOTL2 constructs were designed to express regions spanning from the N - terminus to the coiled - coil domain ( AA 1 - 306 ) , the coiled - coil domain ( AA 307 - 581 ) , and the C - terminal portion ( AA 582 - 781 ) . These constructs were co - transfected into 293T cells with Myc - Ub , and an in vivo ubiquitination assay was conducted . * , non - specific band . B . The SBP - AMOTL2 coiled - coil domain was expressed in 293T cells , subjected to tandem affinity purification , fractionated by SDS - PAGE and stained with Coomassie blue . Bands corresponding to the AMOTL2 coiled - coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites ( K - G - G linkages ) . Shown are MS / MS spectra recoded using an LTQ - Orbitrap mass spectrometer for the doubly charged peptide , IEK * LESEIQR ( MH + = 1358 . 72 , z = 2 + ; upper ) , and the triply charged peptide , LASK * TQEAQAGSQDMVAK ( MH + = 1992 . 96 , z = 3 + ; lower ) . These peptides contain the K347 and K408 ubiquitination sites , respectively ( marked by asterisks ) . Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry . C . The indicated Flag - AMOTL2 WT or mutant proteins were subjected to an in vivo ubiquitination assay in 293T cells . D . RPE or MCF10A cells were transduced with Flag - AMOTL2 WT or Flag - AMOTL2 K347 / 408R , and immunoprecipitated with an anti - Flag antibody . Immunoprecipitated and WCL samples were analyzed by Western blotting for the indicated proteins . E . RPE or MCF10A cells were transduced with Flag - AMOTL2 WT or Flag - AMOTL2 Y213A , and processed as ( D ) .

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Figure 6A. Flag-AMOTL2 constructs were designed to express regions spanning from the N-terminus to the coiled-coil domain (AA 1-306), the coiled-coil domain (AA 307-581), and the C-terminal portion (AA 582-781). These constructs were co-transfected into 293T cells with Myc-Ub, and an in vivo ubiquitination assay was conducted. *, non-specific band.B. The SBP-AMOTL2 coiled-coil domain was expressed in 293T cells, subjected to tandem affinity purification, fractionated by SDS-PAGE and stained with Coomassie blue. Bands corresponding to the AMOTL2 coiled-coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites (K-G-G linkages). Shown are MS/MS spectra recoded using an LTQ-Orbitrap mass spectrometer for the doubly charged peptide, IEK*LESEIQR (MH+ = 1358.72, z = 2+; upper), and the triply charged peptide, LASK*TQEAQAGSQDMVAK (MH+ = 1992.96, z = 3+; lower). These peptides contain the K347 and K408 ubiquitination sites, respectively (marked by asterisks). Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry.C. The indicated Flag-AMOTL2 WT or mutant proteins were subjected to an in vivo ubiquitination assay in 293T cells.D. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 K347/408R, and immunoprecipitated with an anti-Flag antibody. Immunoprecipitated and WCL samples were analyzed by Western blotting for the indicated proteins.E. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 Y213A, and processed as (D).


Figure 7A . MCF10A cells expressing shRNA against AMOTL2 were complemented with shRNA - resistant Flag - AMOTL2 WT or the K347 / 408R mutant . The indicated cells were harvested and analyzed by Western blotting for the indicated proteins . B . mRNAs isolated from cells in ( A ) were analyzed by RT - qPCR against indicated EMT marker genes ( n = 4 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; paired Student ' s t - test ) . C . Transwell migration assay was performed using cells in ( A ) ( n = 8 ) . We analyzed 5 random fields for each sample . Error bars indicate the S . E . M . ( * * p < 0 . 01 , paired Student ' s t - test ) . D . Representative images of soft agar assay results using cells in ( A ) . Scale bar , 1 mm . E . Soft agar assay was performed using cells in ( A ) ( n = 8 ) . Error bars indicate the S . E . M . ( * * p < 0 . 01 , * * * p < 0 . 001 , paired Student ' s t - test ) .

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Figure 7A. MCF10A cells expressing shRNA against AMOTL2 were complemented with shRNA-resistant Flag-AMOTL2 WT or the K347/408R mutant. The indicated cells were harvested and analyzed by Western blotting for the indicated proteins.B. mRNAs isolated from cells in (A) were analyzed by RT-qPCR against indicated EMT marker genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01, ***p < 0.001; paired Student's t-test).C. Transwell migration assay was performed using cells in (A) (n=8). We analyzed 5 random fields for each sample. Error bars indicate the S.E.M. (**p< 0.01, paired Student's t-test).D. Representative images of soft agar assay results using cells in (A). Scale bar, 1 mm.E. Soft agar assay was performed using cells in (A) (n=8). Error bars indicate the S.E.M. (**p < 0.01, ***p < 0.001, paired Student's t-test).


Figure 8B , C . 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti - Flag antibody or pulled down with streptavidin - binding beads . D , E . 293T cells were transfected as indicated and immunoprecipitated with an anti - HA antibody . LATS2 immune complexes were subjected to a kinase assay with His - YAP and cold ATP . F . Lats1 - / - ; Lats2 fl / fl ; SV40 mouse embryonic fibroblasts complemented with vector , LATS2 WT or ΔUBA mutant were infected with empty ( control ) or Zeocin - Cre retroviruses and selected with 400 μg / ml of Zeocin for 4 days . Harvested cells were analyzed by Western blotting for the indicated proteins .

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Figure 8B, C. 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti-Flag antibody or pulled down with streptavidin-binding beads.D, E. 293T cells were transfected as indicated and immunoprecipitated with an anti-HA antibody. LATS2 immune complexes were subjected to a kinase assay with His-YAP and cold ATP.F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.


Figure 1 ( B , C ) ( B ) Growth inhibition assay of E . coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG induction . ( C ) Growth inhibition assay of E . coli DH10B cells co - expressing the Tde1 variants expressed from pTrc200 plasmid and Tdi1 immunity gene expressed from pRL662 plasmid . Growth curve was determined at OD600 . Graphs of panels B and C show mean ± SD of three biological replicates ( n = 3 ) , each averaged with 3 technical repeats . One way ANOVA was used for the analysis of statistical significance followed by the Tukey ' s multiple comparison . Different letters indicate statistically different groups of strains ( p value , 4 . 6x10 - 5 and 5 . 19x10 - 8 for panels B and C respectively ) .

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Figure 1(B, C) (B) Growth inhibition assay of E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG induction. (C) Growth inhibition assay of E. coli DH10B cells co-expressing the Tde1 variants expressed from pTrc200 plasmid and Tdi1 immunity gene expressed from pRL662 plasmid. Growth curve was determined at OD600. Graphs of panels B and C show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 4.6x10-5 and 5.19x10-8 for panels B and C respectively).


Figure 2 ( A ) Growth inhibition assay of E . coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG inducible expression . The growth of E . coli was monitored by CFU counting every 1 hr . Data information : Graphs of panels A , show mean ± SD of three biological replicates ( n = 3 ) , each averaged with 3 technical repeats . One way ANOVA was used for the analysis of statistical significance followed by the Tukey ' s multiple comparison . For membrane permeabilization assays , BW25113 WT alone or ∆ lacY ( pYTA - lacZ ) cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were carried out for ( C ) propidium iodide permeability with cells treated with Propidium iodide and Hoechst for detection by fluorescence microscope ( Scale bar = 5 μm ) . For quantification of cells with PI signals , a total of 6 randomly selected images obtained from two biological repeats were used to quantify the number of PI - stained cells / number of Hoechst - stained cells as indicated . ( D ) Bacteriostatic activity assay . E . coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were cultured with or without IPTG induction for 1 hr . The IPTG - induced cells were further centrifuged and resuspended in the fresh medium with or without IPTG . Cell density was measured again before continuous growth for additional 1 hr . Data information : Graphs of panels show mean ± SD of three biological replicates ( n = 3 ) , each averaged with 3 technical repeats . One way ANOVA was used for the analysis of statistical significance followed by the Tukey ' s multiple comparison . Different letters indicate statistically different groups of strains ( p value , 1 × 10 - 16 and 2 × 10 - 16 for panels D respectively ) .

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

Figure 2(A) Growth inhibition assay of E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG inducible expression. The growth of E. coli was monitored by CFU counting every 1 hr. Data information: Graphs of panels A, show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison.For membrane permeabilization assays, BW25113 WT alone or ∆lacY(pYTA-lacZ) cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were carried out for (C) propidium iodide permeability with cells treated with Propidium iodide and Hoechst for detection by fluorescence microscope (Scale bar = 5 μm). For quantification of cells with PI signals, a total of 6 randomly selected images obtained from two biological repeats were used to quantify the number of PI-stained cells / number of Hoechst-stained cells as indicated.(D) Bacteriostatic activity assay. E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were cultured with or without IPTG induction for 1 hr. The IPTG-induced cells were further centrifuged and resuspended in the fresh medium with or without IPTG. Cell density was measured again before continuous growth for additional 1 hr. Data information: Graphs of panels show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 1×10-16 and 2×10-16 for panels D respectively).


Figure 3 ( A ) Co - immunoprecipitation ( Co - IP ) in A . tumefaciens . A . tumefaciens C58 ∆ tde1 harbouring pTrc200 vector or its derivatives expressing HA - tagged Tde1 variants . Anti - HA resin was used to coprecipitate the Tde1 variants and Tap1 . ( B ) Secretion assay for HA - tagged Tde1 variants . Western blot for the cellular and extracellular fractions of A . tumefaciens C58 ∆ tdei and ∆ tdei ∆ tssK expressing the HA - tagged Tde1 variants . Hcp secretion was detected as a positive control for active T6SS secretion . Representative western blot results of three biological repeats were shown with antibody against HA , Hcp , or EF - Tu where EF - Tu serve as a loading and non - secreted protein control . Protein markers are indicated in kDa .

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Figure 3(A) Co-immunoprecipitation (Co-IP) in A. tumefaciens. A. tumefaciens C58 ∆tde1 harbouring pTrc200 vector or its derivatives expressing HA-tagged Tde1 variants. Anti-HA resin was used to coprecipitate the Tde1 variants and Tap1.(B) Secretion assay for HA-tagged Tde1 variants. Western blot for the cellular and extracellular fractions of A. tumefaciens C58 ∆tdei and ∆tdei∆tssK expressing the HA-tagged Tde1 variants. Hcp secretion was detected as a positive control for active T6SS secretion. Representative western blot results of three biological repeats were shown with antibody against HA, Hcp, or EF-Tu where EF-Tu serve as a loading and non-secreted protein control. Protein markers are indicated in kDa.


Figure 4 ( A ) Fluorescence microscopy for Tde1 translocation . A . tumefaciens C58 ∆ tdei expressing Tde1 variants fused with sfGFP ( in green ) and E . coli DH10B carrying mCherry ( false coloured in blue ) were co - cultured for 20 hrs . A cyan fluorescence with merged blue and green signals represented the translocation of Tde1 variants from A . tumefaciens to E . coli ( Scale bar = 5 μm ) . ( B ) The number of cells with overlayed GFP and mCherry fluorescence was quantified from a total of 6 randomly selected images obtained from three biological repeats ( number of cells with cyan fluorescence / total E . coli cells counted ) .

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Figure 4(A) Fluorescence microscopy for Tde1 translocation. A. tumefaciens C58 ∆tdei expressing Tde1 variants fused with sfGFP (in green) and E. coli DH10B carrying mCherry (false coloured in blue) were co-cultured for 20 hrs. A cyan fluorescence with merged blue and green signals represented the translocation of Tde1 variants from A. tumefaciens to E. coli (Scale bar = 5 μm).(B) The number of cells with overlayed GFP and mCherry fluorescence was quantified from a total of 6 randomly selected images obtained from three biological repeats (number of cells with cyan fluorescence/ total E. coli cells counted).


Figure 5 ( A ) Interbacterial competition of A . tumefaciens C58 ∆ tdei and ∆ tdei ∆ tssK expressing the Tde1 variants against E . coli cells was carried out on LB medium and E . coli survival rate was quantified by CFU counting . ( B ) Interbacterial competition between various A . tumefaciens C58 strains and A . tumefaciens 1D1609 on AK medium and the competition outcome was shown by competitive index . Data information : Data in panel A are mean ± SD of four biological repeats of two independent experiments ( n = 4 ) . Panels B show mean ± SD of three biological repeats ( n = 3 ) . One way ANOVA was used for the analysis of statistical significance followed by the Fishers Least Significant Difference ( LSD ) test for panel A and B Different letters indicate statistically different groups of strains ( p value , 3 . 63 × 10 - 4 , 2 . 70 × 10 - 3 , 2 . 3 × 10 - 15 for panels A , B respectively ) . ( C ) Secretion assay for Tde1 and its variants co - expressed with its immunity protein Tdi1 in A . tumefaciens C58 ∆ tdei and ∆ tdei ∆ tssK . Data information : Western blots were detected with specific antibody against Tde1 , Hcp , or EF - Tu serving as a loading and non - secreted protein control . Protein markers are indicated in kDa . Results in panel C are representative of three biological repeats .

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

Figure 5(A) Interbacterial competition of A. tumefaciens C58 ∆tdei and ∆tdei∆tssK expressing the Tde1 variants against E. coli cells was carried out on LB medium and E. coli survival rate was quantified by CFU counting. (B) Interbacterial competition between various A. tumefaciens C58 strains and A. tumefaciens 1D1609 on AK medium and the competition outcome was shown by competitive index. Data information: Data in panel A are mean ± SD of four biological repeats of two independent experiments (n=4). Panels B show mean ± SD of three biological repeats (n=3). One way ANOVA was used for the analysis of statistical significance followed by the Fishers Least Significant Difference (LSD) test for panel A and B Different letters indicate statistically different groups of strains (p value, 3.63×10-4, 2.70 × 10-3, 2.3 × 10-15 for panels A, B respectively).(C) Secretion assay for Tde1 and its variants co-expressed with its immunity protein Tdi1 in A. tumefaciens C58 ∆tdei and ∆tdei∆tssK. Data information: Western blots were detected with specific antibody against Tde1, Hcp, or EF-Tu serving as a loading and non-secreted protein control. Protein markers are indicated in kDa. Results in panel C are representative of three biological repeats.


Figure 1A microfluidic device for quantitative recovery time measurements . Each PDMS - fabricated chip consists of six independent channels , one of which is shown , from top view ( top row ) . Green circles indicate media inflow ; black circle indicates outflow . Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers ( gray circles ) , the height of which ensures that bacteria are imaged in a monolayer ( bottom row ) . Growth conditions ( e . g . antibiotic dose profile ) are controlled via programmable syringe pumps . Fluorescent images ( bottom row ) show representative growth of a monolayer of E . coli BW25113 cells constitutively expressing GFP over four hours . Representative time series fluorescence data showing dose - dependent population recovery in response to transient streptomycin treatment . Here , time zero corresponds to the end of treatment ( 120 minutes ) . Trajectories show mean and standard deviation for five replicates ; colors indicate increasing antibiotic concentration ( 2 , 4 , 6 , 8 , 10 , 12 μg / mL ) . Fluorescence values are normalized to those at time zero . Dotted line indicates a 2 - fold increase , corresponding to the recovery time for each population . Recovery time increases with total antibiotic exposure . Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations ( as in panel C ) . When plotted against total antibiotic exposure ( calculated as ) , all recovery time values collapse onto a single exponential function ( black line , R2 = 0 . 83 ) . Recovery time was measured in response to streptomycin concencentrations of 0 , 2 , 4 , 6 , 8 , 10 , and 12 μg / mL , and treatment durations 30 , 60 , 90 , and 120 minutes .

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Figure 1A microfluidic device for quantitative recovery time measurements. Each PDMS-fabricated chip consists of six independent channels, one of which is shown, from top view (top row). Green circles indicate media inflow; black circle indicates outflow. Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers (gray circles), the height of which ensures that bacteria are imaged in a monolayer (bottom row). Growth conditions (e.g. antibiotic dose profile) are controlled via programmable syringe pumps. Fluorescent images (bottom row) show representative growth of a monolayer of E. coli BW25113 cells constitutively expressing GFP over four hours.Representative time series fluorescence data showing dose-dependent population recovery in response to transient streptomycin treatment. Here, time zero corresponds to the end of treatment (120 minutes). Trajectories show mean and standard deviation for five replicates; colors indicate increasing antibiotic concentration (2, 4, 6, 8, 10, 12 μg/mL). Fluorescence values are normalized to those at time zero. Dotted line indicates a 2-fold increase, corresponding to the recovery time for each population.Recovery time increases with total antibiotic exposure. Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations (as in panel C). When plotted against total antibiotic exposure (calculated as ), all recovery time values collapse onto a single exponential function (black line, R2 = 0.83). Recovery time was measured in response to streptomycin concencentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL, and treatment durations 30, 60, 90, and 120 minutes.


Figure 3Streptomycin treatment ( closed data points , R2 = 0 . 83 ) results in significantly longer recovery times than chloramphenicol treatment ( open data points ) . Here , the difference between two responses is statistically significant ( p < 0 . 05 by ANOVA ) . The addition of efflux pump inhibitor ( CCCP ) ( closed data points , R2 = 0 . 91 ) increases population recovery time in response to chloramphenicol treatment ( open data points , R2 = 0 . 80 ) . Here , CCCP was added at subinhibitory concentrations ( 3μg / mL ) ; in the absence of antibiotic treatment , CCCP alone did not inhibit population recovery . The CCCP - mediated increase in recovery time is statistically significant ( p < 0 . 01 ) . The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0 . 01 % w / v ( closed data points ) to 0 . 05 % w / v ( open data points ) . Faster synthesis resulted in lower recovery times ( p < 0 . 001 ) in response to streptomycin treatment . In each of these panels , the color scheme indicates increasing antibiotic concentration , as in Figure 1C .

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

Figure 3Streptomycin treatment (closed data points, R2 = 0.83) results in significantly longer recovery times than chloramphenicol treatment (open data points). Here, the difference between two responses is statistically significant (p<0.05 by ANOVA).The addition of efflux pump inhibitor (CCCP) (closed data points, R2 = 0.91) increases population recovery time in response to chloramphenicol treatment (open data points, R2 = 0.80). Here, CCCP was added at subinhibitory concentrations (3μg/mL); in the absence of antibiotic treatment, CCCP alone did not inhibit population recovery. The CCCP-mediated increase in recovery time is statistically significant (p < 0.01).The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0.01% w/v (closed data points) to 0.05% w/v (open data points). Faster synthesis resulted in lower recovery times (p<0.001) in response to streptomycin treatment. In each of these panels, the color scheme indicates increasing antibiotic concentration, as in Figure 1C.


Figure 4Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop . Antibiotics are as follows : penicillin G ( PenG ) , spectinomycin ( Spec ) , gentamicin ( Gent ) , streptomycin ( Str ) , chloramphenicol ( Cm ) , tetracyclin ( Tet ) , and carbenicillin ( Carb ) . Antibiotic concentrations used have been scaled by their respective IC50 values ( solid points and open circles show response with no efflux inhibition and 2μg / mL CCCP , respectively ) . Colors correspond to the motifs of action shown in Figure 4A .

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Figure 4Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop. Antibiotics are as follows: penicillin G (PenG), spectinomycin (Spec), gentamicin (Gent), streptomycin (Str), chloramphenicol (Cm), tetracyclin (Tet), and carbenicillin (Carb). Antibiotic concentrations used have been scaled by their respective IC50 values (solid points and open circles show response with no efflux inhibition and 2μg/mL CCCP, respectively). Colors correspond to the motifs of action shown in Figure 4A.


Figure 1A Baseline blood counts of granulocytes . One - way ANOVA ; n = 8 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 . B Flow cytometry quantification of neutrophils in digested hearts . The representative dot plots show the gating strategy for cardiacneutrophils ( CD45 + Ly6G + CD11b + ) at ZT5 . One - way ANOVA ; n = 5 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 . C Representative immunostainings for neutrophils in the myocardium ( left ventricle ) , identified as Ly6G - positive ( 20x magnification ) . D Cardiac mRNA expression levels normalized to HPRT . One - way ANOVA ; n = 5 mice per ZT ; ZT5 vs . ZT17 : * P = 0 . 0064 ( CXCL1 ) , * P = 0 . 0007 ( CXCL2 ) , * P = 0 . 0007 ( CXCL5 ) , * P = 0 . 0001 ( ICAM - 1 ) , * P = 0 . 0009 ( VCAM - 1 ) , * P = 0 . 0181 ( CCL3 ) , * P = 0 . 0360 ( CCL5 ) . E Mean fluorescence intensity ( MFI ) of CXCR2 expression by neutrophils in blood and heart . One - way ANOVA ; n = 3 mice for ZT1 , ZT17 , ZT21 and n = 5 for ZT5 , ZT9 , ZT13 ; ZT5 vs . ZT13 : * P = 0 . 0425 ( blood ) , * P = 0 . 0078 ( heart ) .

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Figure 1A Baseline blood counts of granulocytes. One-way ANOVA; n = 8 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.B Flow cytometry quantification of neutrophils in digested hearts. The representative dot plots show the gating strategy for cardiacneutrophils (CD45+Ly6G+CD11b+) at ZT5. One-way ANOVA; n = 5 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.C Representative immunostainings for neutrophils in the myocardium (left ventricle), identified as Ly6G-positive (20x magnification).D Cardiac mRNA expression levels normalized to HPRT. One-way ANOVA; n = 5 mice per ZT; ZT5 vs. ZT17: *P = 0.0064 (CXCL1), *P = 0.0007 (CXCL2), *P = 0.0007 (CXCL5), *P = 0.0001 (ICAM-1), *P = 0.0009 (VCAM-1), *P = 0.0181 (CCL3), *P = 0.0360 (CCL5).E Mean fluorescence intensity (MFI) of CXCR2 expression by neutrophils in blood and heart. One-way ANOVA; n = 3 mice for ZT1, ZT17, ZT21 and n = 5 for ZT5, ZT9, ZT13; ZT5 vs. ZT13: *P = 0.0425 (blood), *P = 0.0078 (heart).