B , Cells stably expressing NPC1 - mCherry were treated with control or OCRL siRNAs for 2 days , or transfected with GFP - ORP2 or - ORP2 - mHHK ( PI ( 4 , 5 ) P2 binding - deficient mutant ) for 1 day , stained with PI ( 4 , 5 ) P2 antibody and imaged by confocal microscopy . For FAK inhibition , GFP - ORP2 transfected cells were treated with 10 μM PF228 for 4 h . Dashed lines indicate cell edges . Arrowheads indicate colocalization of PI ( 4 , 5 ) P2 and NPC1 . C , Quantification of B . Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA / Vector ctrl . Mean ± SD , n = 20 - 43 cells pooled from 2 independent experiments . Student ' s t - test .

O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

B, Cells stably expressing NPC1-mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP-ORP2 or -ORP2-mHHK (PI(4,5)P2 binding-deficient mutant) for 1 day, stained with PI(4,5)P2 antibody and imaged by confocal microscopy. For FAK inhibition, GFP-ORP2 transfected cells were treated with 10 μM PF228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5)P2 and NPC1. C, Quantification of B. Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA/Vector ctrl. Mean ± SD, n = 20-43 cells pooled from 2 independent experiments. Student's t-test.


D , E , Cells stably expressing NPC1 - mCherry were transfected with GFP - ORP2 or GFP - ORP2 - mHHK for 1 day and were indicated , treated with 10 μM PF228 for 4 h . For degron ORP2 cells , NPC1 - mCherry was transiently transfected for 2 days and cells incubated without ( - IAA ; ORP2 present ) or with IAA ( + IAA ; ORP2 depleted ) for 1 h . Arrowheads indicate tubular NPC1 organelles . Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured . Mean ± SD , n = 102 - 122 cells . Students ' s t - test . Please see also ctrl ( MovieEV4 ) and PF228 treated cell ( MovieEV5 ) , both videos 1 min recordings with 1 s frame rate .

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

D, E, Cells stably expressing NPC1-mCherry were transfected with GFP-ORP2 or GFP-ORP2-mHHK for 1 day and were indicated, treated with 10 μM PF228 for 4 h. For degron ORP2 cells, NPC1-mCherry was transiently transfected for 2 days and cells incubated without (-IAA; ORP2 present) or with IAA (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured. Mean ± SD, n = 102-122 cells. Students's t-test. Please see also ctrl (MovieEV4) and PF228 treated cell (MovieEV5), both videos 1 min recordings with 1 s frame rate.


A , B , Control and degron ORP2 cells were co - plated , and after 1 day 5 % LPDS , treated with 50 μg / ml LDL and IAA ( + IAA ; ORP2 depletion ) for the indicated times and immunostained for pFAK to quantify its intensity . For rescue experiments , degron ORP2 cells were plated and transfected with GFP - ORP2 or - ORP2 - mHHK or - ORP2 - ∆ ELSK for 6 h . Cells expressing GFP - ORP2 constructs at levels similar to endo - GFP - ORP2 were used for quantifying pFAK intensity . Dashed lines indicate cell outlines . See also Fig EV6C . Asterisk indicates cells depleted of endogenous ORP2 . Images were acquired by confocal microscopy . Mean ± SD , n = 20 - 23 cells pooled from 2 independent experiments . Student ' s t - test .

O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Control and degron ORP2 cells were co-plated, and after 1 day 5 % LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-∆ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20-23 cells pooled from 2 independent experiments. Student's t-test.


C , Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA . Cells were starved overnight in LPDS and loaded with 50 µg / mL LDL + / - IAA for 2 h . The proportion of FAK signal in the membrane fraction ( of total FAK in cytosol + membranes ) is presented ± SD . For no IAA n = 13 and for IAA n = 11 , 2 independent experiments . Student ' s t - test .

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 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

C, Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol+membranes) is presented ± SD. For no IAA n=13 and for IAA n= 11, 2 independent experiments. Student's t-test.


( A ) Immunohistochemistry of Bcl6 ( red ) , Cux1 ( green ) , and Satb2 ( blue ) in P2 Bcl11aF / F ; Emx1IRESCre and control neocortex . Nuclei are stained with Dapi ( white ) . ( B ) Relative quantification of Bcl6 + , Satb2 + , and Cux1 + cells in Bcl11aF / F ; Emx1IRESCre and control neocortex ( n = 4 ) . ( C ) Numbers of Cux1 + or Satb2 + cells that coexpress Bcl6 are reduced in Bcl11aF / F ; Emx1IRESCre compared to control neocortex ( n = 4 ) . Data information : All graphs represent the mean ± s . e . m . ; Student ' s t test ; * * * p < 0 . 001 . Scale bar , 50 µm .

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(A) Immunohistochemistry of Bcl6 (red), Cux1 (green), and Satb2 (blue) in P2 Bcl11aF/F;Emx1IRESCre and control neocortex. Nuclei are stained with Dapi (white). (B) Relative quantification of Bcl6+, Satb2+, and Cux1+ cells in Bcl11aF/F;Emx1IRESCre and control neocortex (n = 4). (C) Numbers of Cux1+ or Satb2+ cells that coexpress Bcl6 are reduced in Bcl11aF/F;Emx1IRESCre compared to control neocortex (n = 4). Data information: All graphs represent the mean ± s.e.m.; Student's t test; *** p < 0.001. Scale bar, 50 µm.


( C - E ) Immunohistochemistry of electroporated P2 Bcl11aF / F neurons in superficial cortical layers with GFP ( green ) and Bcl6 ( red , C ) , Cux1 ( red , D ) , or Satb2 ( red , E ) antibodies . Bcl6 expression is specifically downregulated Bcl11aF / F neocortex upon electroporation of CAG - CreGFP in comparison to CAG - CtlGFP control plasmid . Nuclei are stained with Dapi ( white ) . Data information : White arrowheads point at GFP + cells that also express Bcl6 , Cux1 , Satb2 indicate cells expressing only GFP + . Scale bars , 20 µm ( C - E ) ,

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(C-E) Immunohistochemistry of electroporated P2 Bcl11aF/F neurons in superficial cortical layers with GFP (green) and Bcl6 (red, C), Cux1 (red, D), or Satb2 (red, E) antibodies. Bcl6 expression is specifically downregulated Bcl11aF/F neocortex upon electroporation of CAG-CreGFP in comparison to CAG-CtlGFP control plasmid. Nuclei are stained with Dapi (white). Data information: White arrowheads point at GFP+ cells that also express Bcl6, Cux1, Satb2 indicate cells expressing only GFP+. Scale bars, 20 µm (C-E),


( F ) Quantification of the percentage of electroporated cells expressing Bcl6 ( n = 3 ) , Satb2 ( n = 3 ) , and Cux1 ( n = 5 ) . Results are expressed as mean ± s . e . m . ; Student ' s t test ; * * * p < 0 . 001 .

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(F) Quantification of the percentage of electroporated cells expressing Bcl6 (n = 3), Satb2 (n = 3), and Cux1 (n = 5). Results are expressed as mean ± s.e.m.; Student's t test; *** p < 0.001.


( H ) Immunohistochemistry of electroporated P5 Bcl11a ∆ / F and Bcl11a + / F neurons in superficial cortical layers with GFP ( green ) and cleaved caspase 3 ( CC3 , magenta ) antibodies . Electroporation of Neurod - CreGFP plasmid together with CAG - LSL - Bcl6GFP into Bcl11aF / F neocortex reduces the number of CC3 + cells to control levels . ( I ) Quantification of the experiment shown in ( H ) ( n = 4 ) . Results are expressed as mean ± s . e . m . ; one - way ANOVA followed by Tukey ' s post hoc test ; * * * p < 0 . 001 . Data information : White arrowheads point at GFP + cells that express CC3 . Black arrowheads indicate cells expressing only GFP + . Scale bars , , 50 µm ( H ) .

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(H) Immunohistochemistry of electroporated P5 Bcl11a∆/F and Bcl11a+/F neurons in superficial cortical layers with GFP (green) and cleaved caspase 3 (CC3, magenta) antibodies. Electroporation of Neurod-CreGFP plasmid together with CAG-LSL-Bcl6GFP into Bcl11aF/F neocortex reduces the number of CC3+ cells to control levels. (I) Quantification of the experiment shown in (H) (n = 4). Results are expressed as mean ± s.e.m.; one-way ANOVA followed by Tukey's post hoc test; *** p < 0.001. Data information: White arrowheads point at GFP+ cells that express CC3. Black arrowheads indicate cells expressing only GFP+. Scale bars, , 50 µm (H).


( A ) Immunohistochemistry of cleaved caspase 3 ( CC3 ) shows that the number of CC3 + cells ( marked by black arrowheads ) is increased in P5 Bcl6F / F ; NexCre compared to control neocortex . Insets are enlargements of the boxed areas in corresponding panels . Data information : Scale bars , 500 µm ( A )

(A) Immunohistochemistry of cleaved caspase 3 (CC3) shows that the number of CC3+ cells (marked by black arrowheads) is increased in P5 Bcl6F/F;NexCre compared to control neocortex. Insets are enlargements of the boxed areas in corresponding panels. Data information: Scale bars, 500 µm (A)


( C ) Quantification of the experiment shown in ( A ) ( n = 3 ) . Results are expressed as mean ± s . e . m . ; Student ' s t test ; * p < 0 . 05 .

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(C) Quantification of the experiment shown in (A) (n = 3). Results are expressed as mean ± s.e.m.; Student's t test; * p < 0.05.


( J ) Immunohistochemistry of electroporated P5 Bcl11aF / F neurons in superficial cortical layers with GFP ( green ) and cleaved caspase 3 ( CC3 , magenta ) antibodies . Electroporation of CAG - CreGFP plasmid together with Foxo1 - shRNAGFP # 4 into Bcl11aF / F neocortex reduces the number of CC3 + cells to control levels . White and black arrowheads indicate GFP + CC3 + and GFP + cells , respectively . ( K ) Quantification of the experiment shown in ( J ) ( n = 4 , CAG - CtlGFP / Ctl - shRNAGFP ; n = 3 , CAG - CreGFP / Ctl - shRNAGFP ; n = 5 , CAG - CreGFP / Foxo1 - shRNAGFP # 4 ) . Results are expressed as mean ± s . e . m . ; one - way ANOVA followed by Tukey ' s post hoc test ; * p < 0 . 05 . Data information : Scale bars , , 50 µm J ) .

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(J) Immunohistochemistry of electroporated P5 Bcl11aF/F neurons in superficial cortical layers with GFP (green) and cleaved caspase 3 (CC3, magenta) antibodies. Electroporation of CAG-CreGFP plasmid together with Foxo1-shRNAGFP#4 into Bcl11aF/F neocortex reduces the number of CC3+ cells to control levels. White and black arrowheads indicate GFP+ CC3+ and GFP+ cells, respectively. (K) Quantification of the experiment shown in (J) (n = 4, CAG-CtlGFP/Ctl-shRNAGFP; n = 3, CAG-CreGFP/Ctl-shRNAGFP; n = 5, CAG-CreGFP/Foxo1-shRNAGFP#4). Results are expressed as mean ± s.e.m.; one-way ANOVA followed by Tukey's post hoc test; * p < 0.05. Data information: Scale bars, , 50 µm J).


Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in ( B - F ) . B - F Immunolabeling results of Kcnq1 ( green ) in cochlear cryosections are shown for uninjected WT ( B ) , untreated Kcnq1 − / − ( C ) , injected WT ( D ) , and Kcnq1 − / − mice given injections into the ST ( E ) or SM ( F ) . Cell nuclei were outlined by counterstaining with either DAPI ( B - D , F ) or Qnuclear deep red ( E ) . Scale bars represent approximately 100 μm . Meaning of white arrows are given in the text .

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Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in (B-F).B-F Immunolabeling results of Kcnq1 (green) in cochlear cryosections are shown for uninjected WT (B), untreated Kcnq1−/− (C), injected WT (D), and Kcnq1−/− mice given injections into the ST (E) or SM (F). Cell nuclei were outlined by counterstaining with either DAPI (B-D, F) or Qnuclear deep red (E). Scale bars represent approximately 100 μm. Meaning of white arrows are given in the text.


Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were , respectively , labeled green and purple . The SV consists of three layers of cells ; marginal cells are the first layer of cells on the side of the endolymphatic space . Cryosection through the SV of WT mice . The large arrow points to the native Kcnq1 ( labeled green ) in WT mice . Smaller arrows show nuclei of the marginal cells . Cryosection through the SV of a Kcnq1 − / − mouse injected with AAV expressing Kcnq1 . Labeled in green ( bigger arrow ) is the AAV1 - expressed Kcnq1 , found only in the apical membrane of the marginal cells . Smaller arrows show nuclei of the marginal cells . Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV . Cryosection through the lateral wall of Kcnq1 − / − mice showing intracellular distribution of Kcnq1immunolabeling ( green ) in fibrocytes . Cryosection through the spiral ganglia of cochlea of treated Kcnq1 − / − mice showing immunolabeling ( green ) in the cells of spiral ganglia ( arrows ) . Data information : Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei . Scale bars represent appropriately 50 μm .

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Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were, respectively, labeled green and purple. The SV consists of three layers of cells; marginal cells are the first layer of cells on the side of the endolymphatic space.Cryosection through the SV of WT mice. The large arrow points to the native Kcnq1 (labeled green) in WT mice. Smaller arrows show nuclei of the marginal cells.Cryosection through the SV of a Kcnq1−/−mouse injected with AAV expressing Kcnq1. Labeled in green (bigger arrow) is the AAV1-expressed Kcnq1, found only in the apical membrane of the marginal cells. Smaller arrows show nuclei of the marginal cells. Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV.Cryosection through the lateral wall of Kcnq1−/−mice showing intracellular distribution of Kcnq1immunolabeling (green) in fibrocytes.Cryosection through the spiral ganglia of cochlea of treated Kcnq1−/−mice showing immunolabeling (green) in the cells of spiral ganglia (arrows).Data information: Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei. Scale bars represent appropriately 50 μm.


Quantification of Kcnq1 - positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled ( red ) by phalloidin conjugated with rhodamine . A Immunolabeling results ( Kcnq1 labeled in green ) in WT mice . B , C Immunolabeling results ( Kcnq1 labeled in green ) of treated Kcnq1 − / − mice , middle ( B ) and apical ( C ) turns , respectively . D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT ( gray bars , left ) , untreated Kcnq1 − / − ( middle ) , and treated Kcnq1 − / − mice ( black bars , right ) . Data are given as mean ± SD ( n = 6 ) . E , F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine . Results from WT ( E ) and untreated Kcnq1 − / − mice ( F ) are compared . Data information : Scale bars represent approximately 50 μm .

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Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.A Immunolabeling results (Kcnq1 labeled in green) in WT mice.B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/−mice, middle (B) and apical (C) turns, respectively.D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/−mice (black bars, right). Data are given as mean ± SD (n = 6).E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/−mice (F) are compared.Data information: Scale bars represent approximately 50 μm.


Comparison of cochlear morphologyA - C Cochlear sections obtained from WT ( A ) , nontreated Kcnq1 − / − ( B ) , and treated Kcnq1 − / − mice ( C ) were compared . Major landmarks of the cochlear sections are labeled and pointed by arrows . Scale bars represent approximately 50 μm .

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Comparison of cochlear morphologyA-C Cochlear sections obtained from WT (A), nontreated Kcnq1−/− (B), and treated Kcnq1−/− mice (C) were compared. Major landmarks of the cochlear sections are labeled and pointed by arrows. Scale bars represent approximately 50 μm.


Waveforms of ABRs are compared in WT , untreated Kcnq1 − / − , and treated Kcnq1 − / − mice , as labeled above data traces . Series of averaged ABR data traces were evoked from tone - burst sounds with intensities ranging from 20 to 90 dB SPL .

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Waveforms of ABRs are compared in WT, untreated Kcnq1−/−, and treated Kcnq1−/− mice, as labeled above data traces. Series of averaged ABR data traces were evoked from tone-burst sounds with intensities ranging from 20 to 90 dB SPL.


Summary of averaged ABR thresholds at various frequencies for different groups of mice ; untreated Kcnq1 − / − ( open triangles ) , treated Kcnq1 − / − ( filled triangles ) , injected WT ( filled circles ) , and uninjected WT ( filled squares ) . Plot legends are given in the figure . The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1 − / − mice . Error bars represent standard error of the mean .

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Summary of averaged ABR thresholds at various frequencies for different groups of mice; untreated Kcnq1−/− (open triangles), treated Kcnq1−/− (filled triangles), injected WT (filled circles), and uninjected WT (filled squares). Plot legends are given in the figure. The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1−/− mice. Error bars represent standard error of the mean.


Click - evoked ABR thresholds of WT ( filled squares ) , treated Kcnq1 − / − mice ( filled triangles ) , untreated Kcnq1 − / − mice ( open squares ) measured 4 - 30 weeks after mice were born . Error bars represent standard error of the mean . Upward arrows indicate that click - ABR thresholds were at the maximal sound level that could be reliably measured by the system .

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Click-evoked ABR thresholds of WT (filled squares), treated Kcnq1−/− mice (filled triangles), untreated Kcnq1−/− mice (open squares) measured 4-30 weeks after mice were born. Error bars represent standard error of the mean. Upward arrows indicate that click-ABR thresholds were at the maximal sound level that could be reliably measured by the system.


Western blot verification of knockout of FASN , ACC1 , and / or ACC2 in MDA - MB - 231 cells .

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Western blot verification of knockout of FASN, ACC1, and/or ACC2 in MDA-MB-231 cells.


Cell proliferation rate of MDA - MB - 231 and HeLa cells with knockout of FASN , ACC1 , and / or ACC2 .

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Cell proliferation rate of MDA-MB-231 and HeLa cells with knockout of FASN, ACC1, and/or ACC2.


Colony formation ability of MDA - MB - 231 cells with knockout of FASN , ACC1 , and / or ACC2 .

Colony formation ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2.


Invasion ability of MDA - MB - 231 cells with knockout of FASN , ACC1 , and / or ACC2 . The right panel shows the pictures of invasive cells , and the cell numbers are quantified in the left panel .

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Invasion ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2. The right panel shows the pictures of invasive cells, and the cell numbers are quantified in the left panel.


Tumor formation ability in nude mice of MDA - MB - 231 and HeLa cells with gene knockout as indicated .

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Tumor formation ability in nude mice of MDA-MB-231 and HeLa cells with gene knockout as indicated.


Western blot verification of knockout of KAR or TECR in MDA - MB - 231 cells .

Western blot verification of knockout of KAR or TECR in MDA-MB-231 cells.


B , C . Cell proliferation rate and colony formation ability of MDA - MB - 231 cells with knockout of KAR or TECR . Error bars represent mean ± SD . Data are from triplicate experiments , and all experimental data were verified in at least two independent experiments . * * P < 0 . 01 ( t - test ) ; asterisk shows comparison with wild type group .

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B,C. Cell proliferation rate and colony formation ability of MDA-MB-231 cells with knockout of KAR or TECR. Error bars represent mean ± SD. Data are from triplicate experiments, and all experimental data were verified in at least two independent experiments. **P < 0.01 (t-test); asterisk shows comparison with wild type group.


Tumor formation ability in nude mice of MDA - MB - 231 cells with gene knockout , as indicated .

Tumor formation ability in nude mice of MDA-MB-231 cells with gene knockout, as indicated.


Relative cellular levels of fatty acids in MDA - MB - 231 cells with knockout of KAR or TECR . Each colored datum shows the mean from three independent cultures .

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Relative cellular levels of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. Each colored datum shows the mean from three independent cultures.


G . Relative cellular levels of lipid - containing fatty acids in MDA - MB - 231 cells with knockout of KAR or TECR . The top curve plot indicates the fraction of the corresponding fatty acid . Each colored datum shows the mean from three independent cultures .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

G. Relative cellular levels of lipid-containing fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. The top curve plot indicates the fraction of the corresponding fatty acid. Each colored datum shows the mean from three independent cultures.


H . Mass isotopomer analysis of fatty acids in MDA - MB - 231 cells with knockout of KAR or TECR cultured with 10 mM of 13C6 - glucose for 48 h . Error bars represent mean ± SD . Data are from three independent cultures .

O O O O O O B-MEASURED_VAR I-MEASURED_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

H. Mass isotopomer analysis of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR cultured with 10 mM of 13C6-glucose for 48 h. Error bars represent mean ± SD. Data are from three independent cultures.


MDA - MB - 231 Wt cells or ACC - DKO cells were pretreated for 24 h and then cultured in FBS medium or DFBS medium with the indicated treatment for 5 days . Treatments were as follows : z - VAD - FMK ( z - VAD , 10 μM ) , necrostain - 1 ( Nec - 1 , 2 μM ) , Disulfiram ( 0 . 4 μM ) , ferrostain - 1 ( Ferr - 1 , 10 μM ) , Trolox ( 100 μM ) , N - Acetyl - L - cysteine ( NAC , 3 mM ) , and deferoxamine mesylate ( DFOM , 10 μM ) . Data are presented relative to the values obtained for the control cells cultured in FBS medium . Dotted line shows the survival level of cells in DFBS medium without treatment ( control ) .

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

MDA-MB-231 Wt cells or ACC-DKO cells were pretreated for 24 h and then cultured in FBS medium or DFBS medium with the indicated treatment for 5 days. Treatments were as follows: z-VAD-FMK (z-VAD, 10 μM), necrostain-1 (Nec-1, 2 μM), Disulfiram (0.4 μM), ferrostain-1 (Ferr-1, 10 μM), Trolox (100 μM), N-Acetyl-L-cysteine (NAC, 3 mM), and deferoxamine mesylate (DFOM, 10 μM). Data are presented relative to the values obtained for the control cells cultured in FBS medium. Dotted line shows the survival level of cells in DFBS medium without treatment (control).


Relative cellular levels of ROS in MDA - MB - 231 / ACC - DKO and Wt cells cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement .

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Relative cellular levels of ROS in MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement.


The inhibitory effects of H2O2 and PMS on MDA - MB - 231 ACC - DKO or Wt cells cultured in FBS medium for 24 h with indicated concentrations .

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The inhibitory effects of H2O2 and PMS on MDA-MB-231 ACC-DKO or Wt cells cultured in FBS medium for 24 h with indicated concentrations.


The protein levels of PARP1 and caspase - 3 in MDA - MB - 231 / ACC - DKO and Wt cultured in FBS or DFBS medium .

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The protein levels of PARP1 and caspase-3 in MDA-MB-231/ACC-DKO and Wt cultured in FBS or DFBS medium.


Annexin - V & Dead cell ( 7 - AAD ) flow cytometry analysis of MDA - MB - 231 / ACC - DKO and Wt cultured in DFBS medium . The right panel shows the quantification of annexin V - positive cells in the left panels .

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

Annexin-V & Dead cell (7-AAD) flow cytometry analysis of MDA-MB-231/ACC-DKO and Wt cultured in DFBS medium. The right panel shows the quantification of annexin V-positive cells in the left panels.


The relative cell survival of MDA - MB - 231 / ACC - DKO or Wt with control vector or Bcl - 2 or Bcl - xL overexpression cultured in FBS medium or DFBS medium for 5 days . The left panels show the cell survival , and the right panels verify the overexpression of Bcl - 2 and Bcl - xL .

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The relative cell survival of MDA-MB-231/ACC-DKO or Wt with control vector or Bcl-2 or Bcl-xL overexpression cultured in FBS medium or DFBS medium for 5 days. The left panels show the cell survival, and the right panels verify the overexpression of Bcl-2 and Bcl-xL.


The transmembrane potential in mitochondria of MDA - MB - 231 / ACC - DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis . The bar graphs show the percentage of JC - 1 monomer .

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

The transmembrane potential in mitochondria of MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis. The bar graphs show the percentage of JC-1 monomer.


MDA - MB - 231 / ACC - DKO and Wt cells were stained for Mito Tracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial morphology was scored in the cell types indicated . Data represent three independent experiments , and at least 100 cells were counted per cell type .

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

MDA-MB-231/ACC-DKO and Wt cells were stained for Mito Tracker to visualize mitochondria. Scale bar, 10 μm. Mitochondrial morphology was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type.


MDA - MB - 231 / ACC - DKO cells were in FBS medium with or without exogenous LCFA or VLCFA supplementation , and were stained for MitoTracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial morphology in ( D ) was scored in the cell types indicated . Data represent three independent experiments , and at least 100 cells were counted per cell type . Error bars represent mean ± SD , and data are from triplicate experiments .

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

MDA-MB-231/ACC-DKO cells were in FBS medium with or without exogenous LCFA or VLCFA supplementation, and were stained for MitoTracker to visualize mitochondria. Scale bar, 10 μm.   Mitochondrial morphology in (D) was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type. Error bars represent mean ± SD, and data are from triplicate experiments.


A . Equal aliquots ( 2 mg ) of a Triton X - 100 solubilized lysate from rat brain were incubated with GST or GST - ROC . Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins .

A. Equal aliquots (2 mg) of a Triton X-100 solubilized lysate from rat brain were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


B . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb . Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins .

O O O O O O O O O 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. Brain lysate as in (A) was incubated with GST or GST-nCLCb. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


C . Full - length GST - nCLCb ( 1 - 228 ) and various truncation mutants were purified from bacteria . Equal aliquots of purified protein were incubated with GST or GST - ROC . Specifically bound proteins were detected by Western blot with antibody against CLCs .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Full-length GST-nCLCb (1-228) and various truncation mutants were purified from bacteria. Equal aliquots of purified protein were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibody against CLCs.


D . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb proteins with boundaries as indicated . Specifically bound proteins were detected by Western blot with antibody against CLCs .

O O O O O O O O O O O O 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. Brain lysate as in (A) was incubated with GST or GST-nCLCb proteins with boundaries as indicated. Specifically bound proteins were detected by Western blot with antibody against CLCs.


F . Highly enriched triskelia were stripped from purified CCVs . The triskelia were incubated with GST or GST - ROC . Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins . For A - D / F , aliquots of the starting material ( SM ) equal to 10 % of that added to the beads , was analyzed in parallel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Highly enriched triskelia were stripped from purified CCVs. The triskelia were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. For A-D/F, aliquots of the starting material (SM) equal to 10% of that added to the beads, was analyzed in parallel.


PCR results of LRRK2 - WT from clone E1 ( 1 ) using primers that detect endogenous LRRK2 . Clone E1 is positive for 3xHA - LRRK2 ( 2 ) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2 . ( 3 ) Control unedited COS - 7 cells using the same primer combination as in ( 2 ) . ( 4 ) 1Kb marker .

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

PCR results of LRRK2-WT from clone E1 (1) using primers that detect endogenous LRRK2. Clone E1 is positive for 3xHA-LRRK2 (2) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2. (3) Control unedited COS-7 cells using the same primer combination as in (2). (4) 1Kb marker.


COS - 7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488 - EGF , after which the cells were fixed and processed for immunofluorescence using HA antibody . Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels .

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COS-7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488-EGF, after which the cells were fixed and processed for immunofluorescence using HA antibody. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


COS - 7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies . Scale bar = 10 m .

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

COS-7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies. Scale bar = 10 m.


COS - 7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies . Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels .

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

COS-7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


Lysates from COS - 7 cells transfected with siRNA as indicated were processed for Western blot with antibodies recognizing the indicated proteins . Band intensities of blots as in A are presented as % control siRNA . Two - tailed Student t - test , Mann - Whiteney post hoc test , * * * p < 0 . 001 , N = 3 .

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

Lysates from COS-7 cells transfected with siRNA as indicated were processed for Western blot with antibodies recognizing the indicated proteins.Band intensities of blots as in A are presented as % control siRNA. Two-tailed Student t-test, Mann-Whiteney post hoc test, ***p < 0.001, N = 3.


NP - 40 soluble lysates were prepared from COS - 7 cells transfected with siRNA as indicated and equal protein aliquots ( 1 . 5 mg ) were incubated with GST - PAK - CRIB domain . Specifically bound proteins were detected by Western blot with antibody recognizing Rac1 . An aliquot of the starting material ( SM ) equal to 5 % of that added to GST - PAK - CRIB was analyzed in parallel .

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

NP-40 soluble lysates were prepared from COS-7 cells transfected with siRNA as indicated and equal protein aliquots (1.5 mg) were incubated with GST-PAK-CRIB domain. Specifically bound proteins were detected by Western blot with antibody recognizing Rac1. An aliquot of the starting material (SM) equal to 5% of that added to GST-PAK-CRIB was analyzed in parallel.


Band intensities of blots as in A were presented as % control siRNA . Bars represent mean sem . One - way ANOVA , Bonferroni ' s post hoc test , * * * p < 0 . 001 , N = 3 .

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

Band intensities of blots as in A were presented as % control siRNA. Bars represent mean sem. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


COS - 7 cells transfected with siRNA as indicated were fixed and processed for immunofluorescence using Phalloidin - 647 and antibody recognizing CLCs . Scale bar = 10 m .

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

COS-7 cells transfected with siRNA as indicated were fixed and processed for immunofluorescence using Phalloidin-647 and antibody recognizing CLCs. Scale bar = 10 m.


Perimeter and area of cells as in A were measured and a perimeter : area ratio was plotted as a % of the control siRNA treated cells . One - way ANOVA , Bonferroni ' s post hoc test , * * * p < 0 . 001 , N = 3 .

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

Perimeter and area of cells as in A were measured and a perimeter:area ratio was plotted as a % of the control siRNA treated cells. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus ( at 7 days in vitro ) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa , CLCb , or both viruses in combination . The lysates were processed for Western blot with antibody that recognizes all forms of CLCs .

Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus (at 7 days in vitro) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa, CLCb, or both viruses in combination. The lysates were processed for Western blot with antibody that recognizes all forms of CLCs.


Hippocampal neurons transduced as in G were fixed at 21 days in vitro and processed for immunofluorescence with polyclonal antibody specific for the pre - synaptic protein synapsin and monoclonal antibody specific for the post - synaptic protein PSD95 . The lentivirus drives expression of GFP . The areas indicated by white boxes in the low power images ( squares ) are shown enlarged immediately below . The scale bars = 10 µm for the low mag and 2 . 5 µm for the high mag .

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

Hippocampal neurons transduced as in G were fixed at 21 days in vitro and processed for immunofluorescence with polyclonal antibody specific for the pre-synaptic protein synapsin and monoclonal antibody specific for the post-synaptic protein PSD95. The lentivirus drives expression of GFP. The areas indicated by white boxes in the low power images (squares) are shown enlarged immediately below. The scale bars = 10 µm for the low mag and 2.5 µm for the high mag.


Ai - x . Lateral view of the adult fly head . Scale bar = 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused ( arrows ) and disorganized but still separated ommatidia ( arrowheads ) . ii / iii . Rac1 overexpression with KD of dlrrk ( UAS - dlrrk - RNAi ( v22139 or v22140 ) / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused . iv / v . Rac1 overexpression with KD of Clc ( UAS - Clc - RNAi ( v22318 or v106632 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , results in smaller eyes with some ommatidia fused . vi . Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 ( irk3 ) , a non - relevant RNAi control ( UAS - irk3 - RNAi ( v101174 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , has no effect . vii / viii . KD of dlrrk with GMR - GAL4 / + ; UAS - dlrrk - RNAi ( v22139 or 22140 ) / + , respectively as control have no effect on eye morphology . ix / x . KD of Clc with GMR - GAL4 / UAS - Clc - RNAi ( v22318 or v106632 ) , respectively as control have no effect on eye morphology .

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

Ai-x. Lateral view of the adult fly head. Scale bar = 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused (arrows) and disorganized but still separated ommatidia (arrowheads).ii/iii. Rac1 overexpression with KD of dlrrk (UAS-dlrrk-RNAi (v22139 or v22140)/longGMR-GAL4, UAS-Rac1W), results in smaller eyes where most ommatidia are fused.iv/v. Rac1 overexpression with KD of Clc (UAS-Clc-RNAi (v22318 or v106632)/+;longGMR-GAL4,UAS-Rac1W/+), results in smaller eyes with some ommatidia fused.vi. Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 (irk3), a non-relevant RNAi control (UAS-irk3-RNAi(v101174)/+; longGMR-GAL4,UAS-Rac1W/+), has no effect.vii/viii. KD of dlrrk with GMR-GAL4/+; UAS-dlrrk-RNAi(v22139 or 22140)/+, respectively as control have no effect on eye morphology.ix/x. KD of Clc with GMR-GAL4/UAS-Clc-RNAi (v22318 or v106632), respectively as control have no effect on eye morphology.


Bi - ii . Lateral view of the adult fly head . Scale bar : 100 μm . i . Rac1 overexpression with removing one copy of dlrrk ( dlrrke03680 / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused together . ii . Removing one copy of dlrrke03680 in GMR - GAL4 background , as control has no effect on eye morphology ( GMR - GAL4 / + ; dlrrke03680 / + ) .

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

Bi-ii. Lateral view of the adult fly head. Scale bar: 100 μm.i. Rac1 overexpression with removing one copy of dlrrk (dlrrke03680/longGMR-GAL4,UAS-Rac1W), results in smaller eyes where most ommatidia are fused together.ii. Removing one copy of dlrrke03680 in GMR-GAL4 background, as control has no effect on eye morphology (GMR-GAL4/+;dlrrke03680/+).


Ci - viii . Lateral view of the adult fly head . Scale bar : 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia ( as explained in Ai ) . ii . Rac1 co - expression with dlrrk - WT using UAS - dlrrk - WT / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . iii . Rac1 co - expression with Clc using UAS - GFP - Clc / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . iv . Rac1 co - expression with dlrrk - WT - 3KD using UAS - dlrrk - 3KD / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . v . A wild - type eye of the longGMR - GAL4 promoter as a control . vi - viii . Overexpression of dlrrk - WT , dlrrk - 3KD , or Clc alone with GMR - GAL4 as control , have no effect on eye morphology .

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

Ci-viii. Lateral view of the adult fly head. Scale bar: 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia (as explained in Ai).ii. Rac1 co-expression with dlrrk-WT using UAS-dlrrk-WT/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iii. Rac1 co-expression with Clc using UAS-GFP-Clc/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iv. Rac1 co-expression with dlrrk-WT-3KD using UAS-dlrrk-3KD/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.v. A wild-type eye of the longGMR-GAL4 promoter as a control.vi-viii. Overexpression of dlrrk-WT, dlrrk-3KD, or Clc alone with GMR-GAL4 as control, have no effect on eye morphology.


( B ) flg22 - induced ROS production in Col - 0 and fer - 4 mutant roots under Pi - starvation conditions . Seedlings grown in either HP or LP media were treated with 1 μM flg22 , RALF23 or both flg22 and RALF23 for 15 mins . Roots were harvested and stained with H2DCFDA . One representative root was shown . ( C ) Average H2DCFDA signal intensity in roots shown in ( B ) . The fluorescence intensity was quantified with ImageJ . The data shown indicate the means ± SDs ( n = 30 , n refers to the number of roots per group ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

O O O O O O B-MEASURED_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

(B) flg22-induced ROS production in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22, RALF23 or both flg22 and RALF23 for 15 mins. Roots were harvested and stained with H2DCFDA. One representative root was shown. (C) Average H2DCFDA signal intensity in roots shown in (B). The fluorescence intensity was quantified with ImageJ. The data shown indicate the means ± SDs (n = 30, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( F ) RALF23 inhibits flg22 - induced MAMP - responsive FRK1 gene expression in a Pi content - dependent manner . Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested . The relative expression of FRK1 was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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(F) RALF23 inhibits flg22-induced MAMP-responsive FRK1 gene expression in a Pi content-dependent manner. Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested. The relative expression of FRK1 was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( A ) The PSR induces the cleavage of PRORALF23 - GFP into mature RALF23 - GFP . The 35S promoter - driven RALF23 - GFP line was grown in either HP or LP media for 5 days and roots were harvested . Premature and mature RALF23 levels were determined with anti - GFP . β - actin was used as a loading control . Data information : All experiments were repeated three times with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The PSR induces the cleavage of PRORALF23-GFP into mature RALF23-GFP. The 35S promoter-driven RALF23-GFP line was grown in either HP or LP media for 5 days and roots were harvested. Premature and mature RALF23 levels were determined with anti-GFP. β-actin was used as a loading control. Data information: All experiments were repeated three times with similar results.


( E ) The PSR activates RALF expression in a PHR1 - dependent manner . Col - 0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested . The relative expression of RALFs was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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(E) The PSR activates RALF expression in a PHR1-dependent manner. Col-0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( G ) PHR1 directly binds to RALF promoters , as shown via ChIP - PCR . The Myc - labelled PHR1 overexpression line was immunoprecipitated with anti - Myc . ChIP analysis was performed on Col - 0 , CRY1 - Myc and PHR1 - Myc transformed plants , with Col - 0 and CRY1 - Myc transformed plants as negative controls . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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(G) PHR1 directly binds to RALF promoters, as shown via ChIP-PCR. The Myc-labelled PHR1 overexpression line was immunoprecipitated with anti-Myc. ChIP analysis was performed on Col-0, CRY1-Myc and PHR1-Myc transformed plants, with Col-0 and CRY1-Myc transformed plants as negative controls. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( B ) PHR1 negatively regulates FLS2 - BAK1 complex formation under Pi - starvation conditions . Col - 0 , phr1 mutant seedlings were grown in either HP or LP media for 5 days , then lysing roots into protoplasts , transformed with water or PHR1 - Myc plasmid and treated with flg22 for 6 hours . The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti - FLS2 and anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as negative control . Data information : All experiments were repeated three times with similar results .

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(B) PHR1 negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, phr1 mutant seedlings were grown in either HP or LP media for 5 days, then lysing roots into protoplasts, transformed with water or PHR1-Myc plasmid and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as negative control. Data information: All experiments were repeated three times with similar results.


( D ) Application of RALF23 / 34 disrupted FLS2 - BAK1 complex formation in phr1 mutants . phr1 mutants grown in LP media were treated with water , 1 μM flg22 , 1 μM RALF23 , 1 μM RALF34 or both flg22 and RALF for 6 hours . The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti - FLS2 and anti - BAK1 antibodies , respectively . IgG was used as a negative control . Data information : All experiments were repeated three times with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Application of RALF23/34 disrupted FLS2-BAK1 complex formation in phr1 mutants. phr1 mutants grown in LP media were treated with water, 1 μM flg22, 1 μM RALF23, 1 μM RALF34 or both flg22 and RALF for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. IgG was used as a negative control. Data information: All experiments were repeated three times with similar results.


( E ) RALF23 / 34 promoted the growth of Pto DC3000 in phr1 mutants . Col - 0 and phr1 mutant seedlings grown in LP media were root inoculated ( by soaking ) with a bacterial suspension ( OD600 = 0 . 2 ) for 2 min , and the number of bacteria from individual root was quantified at 5 dpi . The data shown indicate the means ± SDs ( n = 18 , n refers to the number of roots per group ) . The different lowercase letters indicate statistical significance ( one - way ANOVA ) . Data information : All experiments were repeated three times with similar results .

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(E) RALF23/34 promoted the growth of Pto DC3000 in phr1 mutants. Col-0 and phr1 mutant seedlings grown in LP media were root inoculated (by soaking) with a bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual root was quantified at 5 dpi. The data shown indicate the means ± SDs (n = 18, n refers to the number of roots per group). The different lowercase letters indicate statistical significance (one-way ANOVA). Data information: All experiments were repeated three times with similar results.


( F ) FER negatively regulates FLS2 - BAK1 complex formation under Pi - starvation conditions . Col - 0 , fer - 4 mutant and FER / fer - 4 complementation lines seedlings were grown in either HP or LP media for 5 days and treated with flg22 for 6 hours . The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 was determined using anti - FLS2 and anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as a negative control . Data information : All experiments were repeated three times with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FER negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, fer-4 mutant and FER/fer-4 complementation lines seedlings were grown in either HP or LP media for 5 days and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 was determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as a negative control. Data information: All experiments were repeated three times with similar results.


( A - B ) Relative Pseudomonas abundance in rhizosphere samples after ( A ) , Pi - stress ( LP ) or ( B ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * * , p < 0 . 01 ( Student ' s t test ) . ( C - D ) Relative Bacillus abundance in rhizosphere samples after ( C ) , Pi - stress ( LP ) or ( D ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . ( E - F ) Relative Geodermatophilus abundance in rhizosphere samples after ( E ) , Pi - stress ( LP ) or ( F ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . ( G - H ) Relative Methylobacillus abundance in rhizosphere samples after ( G ) , Pi - stress ( LP ) or ( H ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * * , p < 0 . 01 ( Student ' s t test ) .

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(A-B) Relative Pseudomonas abundance in rhizosphere samples after (A), Pi-stress (LP) or (B) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test). (C-D) Relative Bacillus abundance in rhizosphere samples after (C), Pi-stress (LP) or (D) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (E-F) Relative Geodermatophilus abundance in rhizosphere samples after (E), Pi-stress (LP) or (F) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (G-H) Relative Methylobacillus abundance in rhizosphere samples after (G), Pi-stress (LP) or (H) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test).


Bacterial inoculation promotes plant growth and alleviates LP stress . Arabidopsis seeds were germinated on 1 / 2 - strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days . The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B . subtilis . ( A ) , Images were taken 4 weeks after inoculation . Data information : All experiments were repeated three times with similar results .

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

Bacterial inoculation promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B. subtilis. (A), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.


( C ) Pto DC3000 and B . subtilis upregulate phosphate absorption gene expression . Col - 0 , phr1 and fer - 4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B . subtilis in Pi - sufficient or Pi - deficient condition , and the relative expression of Pi - absorbing genes was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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(C) Pto DC3000 and B. subtilis upregulate phosphate absorption gene expression. Col-0, phr1 and fer-4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B. subtilis in Pi-sufficient or Pi-deficient condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


The fer - 4 rhizosphere microbiome promotes plant growth and alleviates LP stress . Arabidopsis seeds were germinated on 1 / 2 - strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days . The seedlings were then transferred to vermiculite and inoculated with Col - 0 or fer - 4 rhizosphere microbiomes . ( B ) , Images were taken 4 weeks after inoculation . Data information : All experiments were repeated three times with similar results .

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

The fer-4 rhizosphere microbiome promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Col-0 or fer-4 rhizosphere microbiomes. (B), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.


( D ) The fer - 4 rhizosphere microbiome upregulated phosphate absorption gene expression . The roots of Col - 0 were harvested after 4 weeks of inoculation with the Col - 0 or fer - 4 rhizosphere microbiome under LP condition , and the relative expression of Pi - absorbing genes was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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(D) The fer-4 rhizosphere microbiome upregulated phosphate absorption gene expression. The roots of Col-0 were harvested after 4 weeks of inoculation with the Col-0 or fer-4 rhizosphere microbiome under LP condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( A ) Left quadriceps muscle biopsy performed at age 10 months ( i - iii ) demonstrating a dystrophic process with variation in fiber size ( black arrow ) , increased connective tissue ( blue arrow ) and rare internalized nuclei ( white arrow ) on hematoxylin and eosin ( H & E ) staining ( i ) and Gömöritrichrome staining ( ii ) and type 1 fiber atrophy on nicotinamide adenine dinucleotide ( NADH ) staining ( iii ) . Muscle biopsy in an unaffected ( control ) individual ( iv - vi ) . Scale bar : 100μm .

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(A) Left quadriceps muscle biopsy performed at age 10 months (i-iii) demonstrating a dystrophic process with variation in fiber size (black arrow), increased connective tissue (blue arrow) and rare internalized nuclei (white arrow) on hematoxylin and eosin (H&E) staining (i) and Gömöritrichrome staining (ii) and type 1 fiber atrophy on nicotinamide adenine dinucleotide (NADH) staining (iii). Muscle biopsy in an unaffected (control) individual (iv-vi). Scale bar: 100μm.


( B ) Western blot data ( n = 1 ) with quantification demonstrated reduced α - Dystroglycan ( α - DG ) in P1 ' s muscle compared to control . ( C ) Western blot data with quantification demonstrated reduced α - Dystroglycan in fibroblasts from P1 and P2 compared to control ( c ) , although not to the extent of the reduction observed in fibroblasts from a patient with a confirmed α - dystroglycanopathy ( α - DG pt ) due to biallelic pathogenic variants in LARGE ( n = 1 ) .

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(B) Western blot data (n=1) with quantification demonstrated reduced α-Dystroglycan (α-DG) in P1's muscle compared to control. (C) Western blot data with quantification demonstrated reduced α-Dystroglycan in fibroblasts from P1 and P2 compared to control (c), although not to the extent of the reduction observed in fibroblasts from a patient with a confirmed α-dystroglycanopathy (α-DG pt) due to biallelic pathogenic variants in LARGE (n=1).


( D ) Brain MRI findings in P1 at 28 months of age ( top ) revealing normal appearing cortex , cerebellum and pons on T1 - weighted images and T2 - weighted image . There is evidence of mild thinning of the corpus callosum ( arrow ) . Brain MRI in an unaffected ( control ) individual at 30 months of age ( bottom ) demonstrating normal sized corpus callosum .

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(D) Brain MRI findings in P1 at 28 months of age (top) revealing normal appearing cortex, cerebellum and pons on T1-weighted images and T2-weighted image. There is evidence of mild thinning of the corpus callosum (arrow). Brain MRI in an unaffected (control) individual at 30 months of age (bottom) demonstrating normal sized corpus callosum.


( B ) Sashimi plots comparing muscle RNA sequencing reads in P1 ( red ) and two control muscle samples ( blue and green ) at the exons 3 - 5 of BET1 .

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(B) Sashimi plots comparing muscle RNA sequencing reads in P1 (red) and two control muscle samples (blue and green) at the exons 3-5 of BET1.


( A ) Immunoblot of patient fibroblast cell lines ( P1 and P2 ) and three independent controls ( C1 - 3 ) . Immunoblots were probed with the indicated primary antibodies . Quantification of BET1 protein levels normalized to GAPDH . Data represented are mean ± SD of three independent biological experiments . Significant differences of P1 BET1 - levels were checked compared to each control ( * * * = p < 0 . 001 , one - way - ANOVA followed by Tukey ` s post - hoc test ) .

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(A) Immunoblot of patient fibroblast cell lines (P1 and P2) and three independent controls (C1-3). Immunoblots were probed with the indicated primary antibodies. Quantification of BET1 protein levels normalized to GAPDH. Data represented are mean ± SD of three independent biological experiments. Significant differences of P1 BET1-levels were checked compared to each control (***=p<0.001, one-way-ANOVA followed by Tukey`s post-hoc test).


( B ) Subcellular localization of BET1 in control and patient fibroblasts by indirect immunofluorescent confocal microscopy . Representative immunofluorescence images of BET1 ( red ) and cis - Golgi marker GM130 ( green ) . Scale bar : 20µm . Colocalization of BET1 with GM130 was determined using Pearson ' s correlation coefficient ( PCC ) measured per cell ( right panel ; C1 : n = 63 , C2 : n = 50 , C3 : n = 49 and P2 : n = 33 ) ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Means are shown ± SEM . ( C ) Representative immunofluorescence images of BET1 ( red ) and ER marker PDI ( green ) . Scale bar : 20 µm . Right panel shows PCCs for BET1 versus PDI ( C1 : n = 61 , C2 : n = 42 , C3 : n = 59 and P2 : n = 32 of two biological replicates ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Means are shown ± SEM .

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(B) Subcellular localization of BET1 in control and patient fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of BET1 (red) and cis-Golgi marker GM130 (green). Scale bar: 20µm.Colocalization of BET1 with GM130 was determined using Pearson's correlation coefficient (PCC) measured per cell (right panel; C1: n=63, C2: n=50, C3: n=49 and P2: n=33) (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM. (C) Representative immunofluorescence images of BET1 (red) and ER marker PDI (green). Scale bar: 20 µm. Right panel shows PCCs for BET1 versus PDI (C1: n=61, C2: n=42, C3: n=59 and P2: n=32 of two biological replicates). (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM.


( D ) HeLa - cells stably expressing the pC4 - reporter were transfected with control siRNA ( ctrl ) or two independent siRNAs ( # 1 and # 2 ) directed against BET1 ( siRNA # 1 is shown in figure EV 2B ) . 2d post - transfection the pC4 - secretion assay was performed for 0 and 10 min post induction of secretion . Representative images are shown ( upper panel ctrl and lower panel # 2 siRNA , respectively . Scale bar : 20 µm . Right panels represent magnifications of dashed areas .

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(D) HeLa-cells stably expressing the pC4-reporter were transfected with control siRNA (ctrl) or two independent siRNAs (#1 and #2) directed against BET1 (siRNA #1 is shown in figure EV 2B). 2d post-transfection the pC4-secretion assay was performed for 0 and 10 min post induction of secretion. Representative images are shown (upper panel ctrl and lower panel #2 siRNA, respectively. Scale bar: 20 µm. Right panels represent magnifications of dashed areas.


( B ) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas . Parameter occupancy could be more than 100 % if several stable hydrogen bonds could be established by residue . Bars represent average fraction of the simulated time the residue is involved in a hydrogen bond ( s ) . Data represented are mean ± SD .

O O O O O B-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

(B) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas. Parameter occupancy could be more than 100% if several stable hydrogen bonds could be established by residue. Bars represent average fraction of the simulated time the residue is involved in a hydrogen bond(s). Data represented are mean ± SD.


( A ) Volcano plot of AP - MS data using HA - BET1 or Ile51Ser .

(A) Volcano plot of AP-MS data using HA-BET1 or Ile51Ser.


( B ) Co - IPs using HA - tagged constructs expressed in HEK293 cells . As baits HA - tagged proteins were immunoprecipitated with anti - HA beads . IP samples and cell lysates were subjected to immunoblotting with α - HA , α - ERGIC - 53 , α - BET1 , α - SEC22b , α - GOSR2 and α - GAPDH antibodies , respectively .

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(B) Co-IPs using HA-tagged constructs expressed in HEK293 cells. As baits HA-tagged proteins were immunoprecipitated with anti-HA beads. IP samples and cell lysates were subjected to immunoblotting with α-HA, α-ERGIC-53, α-BET1, α-SEC22b, α-GOSR2 and α-GAPDH antibodies, respectively.


( E ) Subcellular localization of ERGIC - 53 in fibroblasts by indirect immunofluorescent confocal microscopy . Representative immunofluorescence images of ERGIC - 53 ( green ) and GM130 ( red ) in fibroblasts from P1 , P2 and a control cell line . Scale bar : 20 µm . PCC ( right panel ) for GM130 versus ERGIC - 53 ( C1 : n = 74 , C2 : n = 95 , C3 : n = 59 , P1 : n = 72 and P2 : n = 76 ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Shown are means ± SEM . Immunofluorescence images shown are one example of a total of two biological replicates .

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(E) Subcellular localization of ERGIC-53 in fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of ERGIC-53 (green) and GM130 (red) in fibroblasts from P1, P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for GM130 versus ERGIC-53 (C1: n=74, C2: n=95, C3: n=59, P1: n=72 and P2: n=76). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Shown are means ± SEM. Immunofluorescence images shown are one example of a total of two biological replicates.


( F ) Colocalization of ERGIC - 53 and BET1 in control and P2 - derived fibroblasts . Representative immunofluorescence images of ERGIC - 53 ( green ) and BET1 ( red ) in fibroblasts from P2 and a control cell line . Scale bar : 20 µm . PCC ( right panel ) for BET1 versus ERGIC - 53 ( C1 : n = 72 , C2 : n = 46 , C3 : n = 56 and P2 : n = 37 ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Data represented are mean ± SEM of two independent experiments .

(F) Colocalization of ERGIC-53 and BET1 in control and P2-derived fibroblasts. Representative immunofluorescence images of ERGIC-53 (green) and BET1 (red) in fibroblasts from P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for BET1 versus ERGIC-53 (C1: n=72, C2: n=46, C3: n=56 and P2: n=37). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Data represented are mean ± SEM of two independent experiments.


( H ) The number of cells with intact Golgi was divided by the number of total cells per image and plotted against time ( three biological replicates ) . Dots are means ± SD . For quantification , a sigmoidal fit was performed with the software GraphPad Prism 7 . Dashed lines indicate extrapolated plateaus . LogIC50 ± 95 % confidence interval : C1 = 59 . 38 ± 2 . 31 min , P1 = 72 . 89 ± 1 . 65 min , P2 = 68 . 57 ± 1 . 929 min . Significant differences were assumed when 95 % confidence interval do not overlap .

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(H) The number of cells with intact Golgi was divided by the number of total cells per image and plotted against time (three biological replicates). Dots are means ±SD. For quantification, a sigmoidal fit was performed with the software GraphPad Prism 7. Dashed lines indicate extrapolated plateaus. LogIC50 ±95% confidence interval: C1 = 59.38±2.31 min, P1 = 72.89±1.65 min, P2 = 68.57±1.929 min. Significant differences were assumed when 95% confidence interval do not overlap.


( A ) Heat map representation of the TNF - α - up - regulated genes . Genes were classified based on the TNF - α - treated time points , using the algorithm " HOPACH " .

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(A) Heat map representation of the TNF-α-up-regulated genes. Genes were classified based on the TNF-α-treated time points, using the algorithm "HOPACH".


( B ) Heat map showing down - regulated miRNAs at 4 hrs , but not 24 hrs , after TNF - α treatment . The middle column indicates the fold - change values ( control / TNF ) at 4 hrs . The right column indicates the relative expression level of each miRNA from the FANTOM5 database ( Consortium et al , 2014 ) . Asterisks indicate the top three most down - regulated miRNAs which were reported to be expressed in human endothelial cells ( ECs ) according to the FANTOM5 database .

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(B) Heat map showing down-regulated miRNAs at 4 hrs, but not 24 hrs, after TNF-α treatment. The middle column indicates the fold-change values (control/TNF) at 4 hrs. The right column indicates the relative expression level of each miRNA from the FANTOM5 database (Consortium et al, 2014). Asterisks indicate the top three most down-regulated miRNAs which were reported to be expressed in human endothelial cells (ECs) according to the FANTOM5 database.


( C ) Bar plots showing mean mRNA levels of vascular cell adhesion molecule - 1 ( VCAM1 ) , intracellular adhesion molecule - 1 ( ICAM1 ) , and E - selectin ( SELE ) , measured 4 hrs after stimulation of human ECs with or without TNF - α ± miR - 3679 - 5p . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 and ‡ P = 0 . 203 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(C) Bar plots showing mean mRNA levels of vascular cell adhesion molecule-1 (VCAM1), intracellular adhesion molecule-1 (ICAM1), and E-selectin (SELE), measured 4 hrs after stimulation of human ECs with or without TNF-α ± miR-3679-5p. Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 and ‡ P = 0.203 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( D ) Western blots for VCAM1 , ICAM , SELE , and β - actin ( ACTB ) in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± miR - 3679 - 5p .

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(D) Western blots for VCAM1, ICAM, SELE, and β-actin (ACTB) in lysates from ECs treated with or without TNF-α (4 hrs) ± miR-3679-5p.


( E ) Representative images ( top ) and bar plot quantification ( bottom ) showing the adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± miR - 3679 - 5p . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(E) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± miR-3679-5p. Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) The scatter plot shows the correlation of RNA immunoprecipitation ( RIP ) followed by microarray data from two replicate screens for the identification of miR - 3679 - 5p target genes . Genes up - and down - regulated by miR - 3679 - 5p treatment ( | average Log2FC | > 1 ) are indicated in red and blue , respectively .

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(F) The scatter plot shows the correlation of RNA immunoprecipitation (RIP)　followed by microarray data from two replicate screens for the identification of miR-3679-5p target genes. Genes up- and down-regulated by miR-3679-5p treatment (|average Log2FC|>1) are indicated in red and blue, respectively.


( J and K ) Effect of miR - 3679 - 5p mimics ( J ) or miR - 3679 - 5p inhibitor ( K ) on luciferase activity in pmirGLO - transfected ECs expressing the 3 ′ UTR of KDM7A . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by the Student ' s t test .

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(J and K) Effect of miR-3679-5p mimics (J) or miR-3679-5p inhibitor (K) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of KDM7A. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( M and N ) The effect of miR - 3679 - 5p mimics ( M ) or miR - 3679 - 5p inhibitor ( N ) on luciferase activity in pmirGLO - transfected ECs expressing the 3 ′ UTR of UTX was measured . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by the Student ' s t test .

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(M and N) The effect of miR-3679-5p mimics (M) or miR-3679-5p inhibitor (N) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of UTX was measured. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( A ) Heat map representation of RNA - seq results . The p65 - dependent genes ( total 123 ; defined as genes up - regulated more than 2 - fold via TNF - α treatment and down - regulated more than 0 . 75 - fold via BAY 11 - 7082 treatment ) were classified based on the alteration patterns by the siRNA knockdown of KDM7A , UTX , and both , using the algorithm " HOPACH " .

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(A) Heat map representation of RNA-seq results. The p65-dependent genes (total 123; defined as genes up-regulated more than 2-fold via TNF-α treatment and down-regulated more than 0.75-fold via BAY 11-7082 treatment) were classified based on the alteration patterns by the siRNA knockdown of KDM7A, UTX, and both, using the algorithm "HOPACH".


( C - E ) Bar plot showing mean mRNA levels of VCAM1 , ICAM1 , and SELE measured 4 hrs after stimulation of ECs with or without TNF - α ± ( siKDM7A + siUTX ) . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(C-E) Bar plot showing mean mRNA levels of VCAM1, ICAM1, and SELE measured 4 hrs after stimulation of ECs with or without TNF-α ± (siKDM7A+siUTX). Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) Western blots for VCAM1 , ICAM , SELE , and ACTB in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± ( siKDM7A + siUTX ) .

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(F) Western blots for VCAM1, ICAM, SELE, and ACTB in lysates from ECs treated with or without TNF-α (4 hrs) ± (siKDM7A+siUTX).


( G ) Representative images ( top ) and bar plot quantification ( bottom ) showing the adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± ( siKDM7A + siUTX ) . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(G) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± (siKDM7A+siUTX). Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A and B ) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF - α - stimulated ECs . The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads ( FPKM ) values and the ChIP - seq signals ( TNF - α 60min / 0min ) for KDM7A ( A ) and UTX ( B ) , respectively . Genes with more than a 1 . 5 - fold increase in FPKM values ( light red dots ) satisfying a 0 . 5 - fold increase in ChIP - seq signals are indicated as dark red dots . Genes with a 1 . 5 - fold decrease in FPKM values ( light blue dots ) satisfying a 0 . 5 - fold decrease in ChIP - seq signals are indicated as dark blue dots . These genes are defined as the target genes for KDM7A ( A ) and UTX ( B ) , respectively . The names of representative genes are shown in the graphs .

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(A and B) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF-α-stimulated ECs. The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads (FPKM) values and the ChIP-seq signals (TNF-α 60min/0min) for KDM7A (A) and UTX (B), respectively. Genes with more than a 1.5-fold increase in FPKM values (light red dots) satisfying a 0.5-fold increase in ChIP-seq signals are indicated as dark red dots. Genes with a 1.5-fold decrease in FPKM values (light blue dots) satisfying a 0.5-fold decrease in ChIP-seq signals are indicated as dark blue dots. These genes are defined as the target genes for KDM7A (A) and UTX (B), respectively. The names of representative genes are shown in the graphs.


( C and D ) Metagene representations of KDM7A ( C ) and UTX ( D ) ChIP - seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF - α - up - regulated genes .

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(C and D) Metagene representations of KDM7A (C) and UTX (D) ChIP-seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF-α-up-regulated genes.


( F ) Heat map of H3K27ac ( green ) , p65 ( yellow ) , KDM7A ( red ) , and UTX ( purple ) levels in resting and TNF - α - stimulated ECs . Each row shows ± 10 kb centered on the KDM7A ( top ) or UTX ( bottom ) peak . Rows are ordered by max KDM7A or UTX signal in each region . The ChIP - seq signal is depicted by color scaled intensities .

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(F) Heat map of H3K27ac (green), p65 (yellow), KDM7A (red), and UTX (purple) levels in resting and TNF-α-stimulated ECs. Each row shows ± 10 kb centered on the KDM7A (top) or UTX (bottom) peak. Rows are ordered by max KDM7A or UTX signal in each region. The ChIP-seq signal is depicted by color scaled intensities.


( G and H ) Gene tracks of ChIP - seq signals for H3K27Ac , p65 , KDM7A , and UTX , and RNA - seq signals around the VCAM1 ( G ) and SELE ( H ) loci in TNF - α ( - ) and TNF - α ( + ) ECs . ChIP - Seq and RNA - seq signals are visualized by Integrated Genome Viewer ( http : / / software . broadinstitute . org / software / igv / ) .

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(G and H) Gene tracks of ChIP-seq signals for H3K27Ac, p65, KDM7A, and UTX, and RNA-seq signals around the VCAM1 (G) and SELE (H) loci in TNF-α (-) and TNF-α (+) ECs. ChIP-Seq and RNA-seq signals are visualized by Integrated Genome Viewer (http://software.broadinstitute.org/software/igv/).


( I and J ) ChIP - qPCR of p65 ( I ) and KDM7A ( J ) at the VCAM1 TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(I and J) ChIP-qPCR of p65 (I) and KDM7A (J) at the VCAM1 TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-). Statistical differences were analyzed by the Tukey-Kramer test.


( K and L ) ChIP - qPCR of p65 ( K ) and UTX ( L ) at the SELE TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(K and L) ChIP-qPCR of p65 (K) and UTX (L) at the SELE TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A and B ) Boxplots of the log2 fold change of ChIP - seq enrichment of H3K9me2 in KDM7A target genes ( A ) and that of H3K27me3 in UTX target genes ( B ) with TNF - α treatment ( TNF + ) and control ( TNF - ) . The horizontal line within the box represents the median sample value . The ends of the box represent the 3rd and 1st quantiles . The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed as follows : 3rd quartile + 1 . 5 * ( interquartile range ) and 1st quartile - 1 . 5 * ( interquartile range ) . Each dot in the box plot shows data for one single target gene . Genes colored red represent the top five downregulated target genes with TNF - α - treatment , as compared to control . Graphs are representative of two independent experiments .

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(A and B) Boxplots of the log2 fold change of ChIP-seq enrichment of H3K9me2 in KDM7A target genes (A) and that of H3K27me3 in UTX target genes (B) with TNF-α treatment (TNF+) and control (TNF-). The horizontal line within the box represents the median sample value. The ends of the box represent the 3rd and 1st quantiles. The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed as follows: 3rd quartile + 1.5*(interquartile range) and 1st quartile - 1.5*(interquartile range). Each dot in the box plot shows data for one single target gene. Genes colored red represent the top five downregulated target genes with TNF-α-treatment, as compared to control. Graphs are representative of two independent experiments.