Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons . No stimulation and low stimulation frequencies did not increase levels of DPR . The C9 DPR reporter , monitoring the GP DPR ( ORF2 ) , was cotransfected with ChR2 . Cortical neuron : n = 4 with m > 10 cells per n . ( * * * * P < 0 . 0001 ) .

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Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons. No stimulation and low stimulation frequencies did not increase levels of DPR. The C9 DPR reporter, monitoring the GP DPR (ORF2), was cotransfected with ChR2. Cortical neuron: n = 4 with m > 10 cells per n. (****P < 0.0001).


Filter trap binding assays show that ISR proteins are enhanced by all stressors . Significant increases following cellular stress treatments are seen in the ratio of phospho - eif2α to total eif2α as well as phospho - PERK and ATF4 levels compared to untreated controls . Changes in ISR proteins are normalized to GAPDH and shown relative to non - stressed or DMSO - only treated cells ( CTRL ) depending on the vehicle compounds were dissolved in . n = 4 .

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Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.


Fluorescent intensity measurements show the levels of two ISR - related proteins , PERK and relative phospho - eif2α ( phospho - eif2α / total eif2α ) , are increased following different excitotoxic stress treatments in cortical neurons . Representative images indicate fluorescent intensity differences for phospho - eif2α and phospho - PERK levels 24 h post treatment with different excitotoxic stressor paradigms . Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR - related protein levels , which parallels reduced DPR levels ( Figure 3B ) . Changes in ISR proteins are relative to non - stressed cells ( CTRL ) .

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Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).


Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR - related or translation - targeting pathways . Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) , which inhibits eif2α - P activity , had the most significant decreases in DPR production . Trazodone ( Traz ; 20 µM ) showed similar significant reduction in DPR levels as the PERK inhibitor , GSK2606414 ( 500 nM ) .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).


Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone ( Traz ; 20 µM ) or Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) . Similar to the treatments in NSC34 , 1 , 3DBM shows the most significant decrease in DPR production .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.


Analysis of ER architecture and dynamics as a function of RNF26 in U2OS cells ectopically expressing mCherry - KDEL and transfected with either control siRNA ( siC ) or siRNAs targeting RNF26 ( siRNF26 - 1 or siRNF26 - 2 ) . B . Time - lapse imaging . Top panels : representative confocal fluorescence images of Cherry - KDEL ( white ) at the start of time - lapse t = 0 ( top ) . Middle panels : time color coded overlays of 60x1s frames , each assigned a different color LUT ( white - same ER location across all frames ; color - ER movement between frames ) . Bottom panels : overlays of twice eroded ER sheet masks ( magenta ) with mCherry - KDEL signal ( green ) . Masks were generated using ImageJ software . Zoom - ins show select PN and PP regions . Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively .

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Analysis of ER architecture and dynamics as a function of RNF26 in U2OS cells ectopically expressing mCherry-KDEL and transfected with either control siRNA (siC) or siRNAs targeting RNF26 (siRNF26-1 or siRNF26-2). B. Time-lapse imaging. Top panels: representative confocal fluorescence images of Cherry-KDEL (white) at the start of time-lapse t=0 (top). Middle panels: time color coded overlays of 60x1s frames, each assigned a different color LUT (white - same ER location across all frames; color - ER movement between frames). Bottom panels: overlays of twice eroded ER sheet masks (magenta) with mCherry-KDEL signal (green). Masks were generated using ImageJ software. Zoom-ins show select PN and PP regions. Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively.


I . CLIMP63 abundance as a function of RNF26 . Top : representative immunoblot of U2OS cells transfected with the indicated siRNAs stained against endogenous CLIMP63 and Vinculin ( loading control ) . Bottom : quantification of CLIMP63 abundance , normalized to Vinculin and expressed relative to control ( siC = 1 . 0 ) , n = 4 independent experiments . Data information : Graphs report mean or median ( red line ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test with error bars reflecting + / - SD )

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I. CLIMP63 abundance as a function of RNF26. Top: representative immunoblot of U2OS cells transfected with the indicated siRNAs stained against endogenous CLIMP63 and Vinculin (loading control). Bottom: quantification of CLIMP63 abundance, normalized to Vinculin and expressed relative to control (siC=1.0), n=4 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Significance was assessed using Student's t-test with error bars reflecting +/- SD)


J , K . Effect of RNF26 depletion on basal ER stress using IRE1 - mediated XBP1 splicing as a readout . J . Relative abundance of RNF26 transcript normalized to GAPDH ; n = 3 independent experiments . All transcripts were detected by qPCR . Plots report values relative to control ( siC = 1 . 0 ) . K . Quantification of IRE1 - dependent XBP1 splicing . XBP - s and XBP1 transcripts isolated from U2OS cells transfected with the indicated siRNAs were detected by qPCR , normalized to GAPDH , and expressed as ratio , n = 3 independent experiments . Data information : Graphs report mean or median ( red line ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test J and K with error bars reflecting + / - SD ) p < 0 . 05 ; * * * p < 0 . 001 , ns : not significant .

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J, K. Effect of RNF26 depletion on basal ER stress using IRE1-mediated XBP1 splicing as a readout. J. Relative abundance of RNF26 transcript normalized to GAPDH; n=3 independent experiments. All transcripts were detected by qPCR. Plots report values relative to control (siC=1.0). K. Quantification of IRE1-dependent XBP1 splicing. XBP-s and XBP1 transcripts isolated from U2OS cells transfected with the indicated siRNAs were detected by qPCR, normalized to GAPDH, and expressed as ratio, n=3 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Significance was assessed using Student's t-test J and K with error bars reflecting +/- SD) p<0.05; *** p<0.001, ns: not significant.


C , D . Proximity biotinylation of endogenous vimentin by 2HA - TurboID - RNF26 , catalytically inactive point mutant ( I382R ) , or RING domain truncation mutant ( ∆ RING ) in HeLa cells following 30 min incubation with biotin prior to lysis . C . Representative immunoblots of neutravidin precipitates and lysate inputs stained against HA and vimentin . D . Quantifications of vimentin biotinylation by 2HA - TurboID - RNF26 and mutants normalized to auto biotinylated HA - tagged RNF26 species and expressed relative to RNF26 WT , n = 6 independent experiments performed in U2OS and HeLa cells . Data information : WCL : whole cell lysate . Graphs in D report mean ( red line ) and SEM ( error bars ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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C, D. Proximity biotinylation of endogenous vimentin by 2HA-TurboID-RNF26, catalytically inactive point mutant (I382R), or RING domain truncation mutant (∆RING) in HeLa cells following 30 min incubation with biotin prior to lysis. C. Representative immunoblots of neutravidin precipitates and lysate inputs stained against HA and vimentin. D. Quantifications of vimentin biotinylation by 2HA-TurboID-RNF26 and mutants normalized to auto biotinylated HA-tagged RNF26 species and expressed relative to RNF26 WT, n=6 independent experiments performed in U2OS and HeLa cells. Data information: WCL: whole cell lysate. Graphs in D report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.


E . In vitro co - precipitation of purified recombinant Rhodamine - labeled RING - I382R ( 304 - 433 ) with purified recombinant His - tagged vimentin on Ni - NTA beads . Rhodamine in - gel fluorescent scan and immunoblot against vimentin are shown , representative of three independent experiments .

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E. In vitro co-precipitation of purified recombinant Rhodamine-labeled RING-I382R (304-433) with purified recombinant His-tagged vimentin on Ni-NTA beads. Rhodamine in-gel fluorescent scan and immunoblot against vimentin are shown, representative of three independent experiments.


G . Proximity - based biotinylation of endogenous vimentin by 2HA - TurboID - I382R , - ∆ RING , and - I382R - ∆ C lacking amino acids 423 - 433 . Immunoblots of neutravidin precipitates and lysate inputs against HA and vimentin are shown and are representative of three independent experiments . Data information : WCL : whole cell lysate .

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G. Proximity-based biotinylation of endogenous vimentin by 2HA-TurboID-I382R, -∆RING, and -I382R-∆C lacking amino acids 423-433. Immunoblots of neutravidin precipitates and lysate inputs against HA and vimentin are shown and are representative of three independent experiments. Data information: WCL: whole cell lysate.


H . In vitro peptide binding assay . Rhodamine - linked C - terminal peptide of RNF26 ( Rho - RGILQTLNVYL ) or free Rhodamine were incubated at 10nM with increasing concentrations of His - vimentin and binding was measured using fluorescence polarization ; n = 3 independent experiments . Data information H report mean ( red line ) and SEM ( error bars ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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H. In vitro peptide binding assay. Rhodamine-linked C-terminal peptide of RNF26 (Rho-RGILQTLNVYL) or free Rhodamine were incubated at 10nM with increasing concentrations of His-vimentin and binding was measured using fluorescence polarization; n=3 independent experiments. Data information H report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.


A - E . Analysis of RNF26 sequence determinants with respect to the intracellular distribution of RNF26 and its colocalization with vimentin filaments . A . Representative confocal fluorescence images of U2OS cells ectopically expressing RFP - RNF26 ( green ) wild - type ( WT ) or mutants ( I382R , ∆ RING , ∆ C ) , fixed and immunostained against endogenous vimentin ( magenta ) and VAP - A ( magenta ) . Indicated single channels and their corresponding color overlays are shown . Zoom - ins highlight PN regions . B . Colocalization ( Manders ' ) analysis of RFP - RNF26 and mutants overlap with vimentin . Plot reports n WT = 38 , n I382R = 31 , n ΔRING = 34 , n ΔC = 22 cells analysed from three independent experiments . C . Colocalization ( Manders ' ) analysis of RFP - RNF26 and mutants with VAP - A . Plot reports n WT = 29 , n I382R = 21 , n ΔRING = 17 , n ΔC = 16 cells analysed from three independent experiments . D . Perinuclearity of RFP - RNF26 as a function of its RING domain determinants . Plot reports n WT = 72 , n I382R = 48 , n ΔRING = 37 , n ΔC = 28 cells analysed from three independent experiments . E . Intracellular distribution of vimentin as a function of RNF26 ( mutant ) overexpression . Plot reports n WT59 , n I382R = 58 , n ΔRING = 36 , n ΔC = 32 technical replicates from three independent experiments . Values capped at 10 . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm . Statistical analyses were performed using Students ' T - test ( B , D error bars indicate mean - / + SD ) or Mann - Whitney U test ( C , E error bars indicate median and 95 % confidence interval ) ; * * * p < 0 . 001 , ns : not significant .

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A-E. Analysis of RNF26 sequence determinants with respect to the intracellular distribution of RNF26 and its colocalization with vimentin filaments. A. Representative confocal fluorescence images of U2OS cells ectopically expressing RFP-RNF26 (green) wild-type (WT) or mutants (I382R, ∆RING, ∆C), fixed and immunostained against endogenous vimentin (magenta) and VAP-A (magenta). Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions. B. Colocalization (Manders') analysis of RFP-RNF26 and mutants overlap with vimentin. Plot reports n WT=38, n I382R=31, n ΔRING=34, n ΔC=22 cells analysed from three independent experiments. C. Colocalization (Manders') analysis of RFP-RNF26 and mutants with VAP-A. Plot reports n WT=29, n I382R=21, n ΔRING=17, n ΔC=16 cells analysed from three independent experiments. D. Perinuclearity of RFP-RNF26 as a function of its RING domain determinants. Plot reports n WT=72, n I382R=48, n ΔRING=37, n ΔC=28 cells analysed from three independent experiments. E. Intracellular distribution of vimentin as a function of RNF26 (mutant) overexpression. Plot reports n WT59, n I382R=58, n ΔRING=36, n ΔC=32 technical replicates from three independent experiments. Values capped at 10. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Statistical analyses were performed using Students' T-test (B, D error bars indicate mean -/+ SD) or Mann-Whitney U test (C, E error bars indicate median and 95% confidence interval); *** p<0.001, ns: not significant.


F , G . Analysis of vimentin cytoskeleton distribution as a function of RNF26 . F . Representative confocal fluorescence images of control cells , cells silenced for RNF26 ( si # 1 ) , or RNF26 KO cells . Zoom - ins highlight PN regions . G . Perinuclearity of vimentin in the presence or absence of RNF26 . Plot reports n control = 98 , n siRNF26 = 132 , n RNF26 KO = 41 technical replicates from three independent experiments . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm . Statistical analyses were performed using Students ' T - test Mann - Whitney U test error bars indicate median and 95 % confidence interval ) ; * * * p < 0 . 001 , ns : not significant .

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F, G. Analysis of vimentin cytoskeleton distribution as a function of RNF26. F. Representative confocal fluorescence images of control cells, cells silenced for RNF26 (si#1), or RNF26 KO cells. Zoom-ins highlight PN regions. G. Perinuclearity of vimentin in the presence or absence of RNF26. Plot reports n control=98, n siRNF26=132, n RNF26 KO=41 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Statistical analyses were performed using Students' T-test Mann-Whitney U test error bars indicate median and 95% confidence interval); *** p<0.001, ns: not significant.


H . Vimentin abundance as a function of RNF26 . Top : representative immunoblot of U2OS cells transfected with siRNF26 # 1 stained against endogenous vimentin and Vinculin ( loading control ) . Bottom : quantification of vimentin abundance , normalized to Vinculin and expressed relative to control ( siC = 1 . 0 ) , n = 4 independent experiments . Statistical analyses were performed using Students ' T - test error bars indicate mean - / + SD ) ; * * * p < 0 . 001 , ns : not significant .

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H. Vimentin abundance as a function of RNF26. Top: representative immunoblot of U2OS cells transfected with siRNF26#1 stained against endogenous vimentin and Vinculin (loading control). Bottom: quantification of vimentin abundance, normalized to Vinculin and expressed relative to control (siC=1.0), n=4 independent experiments. Statistical analyses were performed using Students' T-test error bars indicate mean -/+ SD) ; *** p<0.001, ns: not significant.


A - D . Effect of vimentin ablation on intracellular distribution of RNF26 . A . Representative confocal images of parental and vimentin knockout ( Vim KO ) U2OS cells ectopically expressing GFP - RNF26 ( green ) , fixed and immunostained against endogenous VAP - A ( magenta ) . B . Validation of vimentin knockout . Immunoblots against endogenous vimentin and Vinculin ( loading control ) of a Vim KO clonal cell line and their parental U2OS cells are shown , representative of three independent experiments . Molecular weight markers are as indicated . C . Quantification of GFP - RNF26 signal distribution expressed as perinuclearity ratio . Graph reports n Parental = 73 , n Vim KO = 43 technical replicates from three independent experiments . D . Colocalization ( Manders ' ) analysis of VAP - A overlapping with GFP - RNF26 ( left plot ) and vice versa ( right plot ) . Plots report on subset of cells from data set in ( B ) , same as in ( C ) . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Zoom - ins designate regions encompassing perinuclear and peripheral areas . All scale bars = 10µm . Graphs report mean ( red line ) of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test ( D Mann - Whitney U test ( C error bars indicate median and 95 % confidence interval ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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A-D. Effect of vimentin ablation on intracellular distribution of RNF26. A. Representative confocal images of parental and vimentin knockout (Vim KO) U2OS cells ectopically expressing GFP-RNF26 (green), fixed and immunostained against endogenous VAP-A (magenta). B. Validation of vimentin knockout. Immunoblots against endogenous vimentin and Vinculin (loading control) of a Vim KO clonal cell line and their parental U2OS cells are shown, representative of three independent experiments. Molecular weight markers are as indicated. C. Quantification of GFP-RNF26 signal distribution expressed as perinuclearity ratio. Graph reports n Parental = 73, n Vim KO = 43 technical replicates from three independent experiments. D. Colocalization (Manders') analysis of VAP-A overlapping with GFP-RNF26 (left plot) and vice versa (right plot). Plots report on subset of cells from data set in (B), same as in (C). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test (D Mann-Whitney U test (C error bars indicate median and 95% confidence interval); * p<0.05, ** p<0.01, *** p<0.001.


E , F . Effect of vimentin or RNF26 ablation on the intracellular distribution of endolysosomes . E . Representative confocal images of parental , RNF26 KO , vimentin KO # 1 U2OS cells , vimentin KO # 1 cells ectopically complemented with untagged wild type vimentin ( Vim KO + Rescue ) , fixed and immunostained against endogenous LAMP1 . F . Quantification of LAMP1 signal distribution expressed as perinuclearity ratio . Graph reports on n Parental = 145 , n R26 KO = 64 , n Vim KO # 1 = 119 , n Vim KO # 1 / Rescue = 32 technical replicates from three independent experiments . Values capped at 6 . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Zoom - ins designate regions encompassing perinuclear and peripheral areas . All scale bars = 10µm . Graphs report mean ( red line ) of sample values ( open circles ) . Mann - Whitney U test F error bars indicate median and 95 % confidence interval ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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E, F. Effect of vimentin or RNF26 ablation on the intracellular distribution of endolysosomes. E. Representative confocal images of parental, RNF26 KO, vimentin KO #1 U2OS cells, vimentin KO#1 cells ectopically complemented with untagged wild type vimentin (Vim KO + Rescue), fixed and immunostained against endogenous LAMP1. F. Quantification of LAMP1 signal distribution expressed as perinuclearity ratio. Graph reports on n Parental = 145, n R26 KO = 64, n Vim KO#1 = 119, n Vim KO#1/Rescue= 32 technical replicates from three independent experiments.Values capped at 6. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Mann-Whitney U test F error bars indicate median and 95% confidence interval); * p<0.05, ** p<0.01, *** p<0.001.


G , H . Effects of vimentin ablation on trafficking of extracellular materials to lysosomes . G . Time - lapse imaging . Representative confocal overlays of parental and vimentin KO # 2 U2OS cells , stained with SiR - Lysosome ( magenta ) to mark late endocytic compartments and incubated with the cell impermeable dye SR101 ( green ) , taken soon after the start ( t = 5 min ) and at the end ( t = 120 min ) of time - lapse are shown . Zoom - ins highlight select PN regions in overlay and single channels . H . Colocalization ( Manders ' ) of SiR - Lysosome overlapping SR101 at t = 120 minutes following SR101 addition . Graph reports on n Parental = 71 , n Vim KO = 69 technical replicates from 3 independent experiments . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Zoom - ins designate regions encompassing perinuclear and peripheral areas . All scale bars = 10µm . Graphs report mean ( red line ) of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test H error bars indicate mean - / + SD ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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G, H. Effects of vimentin ablation on trafficking of extracellular materials to lysosomes. G. Time-lapse imaging. Representative confocal overlays of parental and vimentin KO#2 U2OS cells, stained with SiR-Lysosome (magenta) to mark late endocytic compartments and incubated with the cell impermeable dye SR101 (green), taken soon after the start (t=5 min) and at the end (t=120 min) of time-lapse are shown. Zoom-ins highlight select PN regions in overlay and single channels. H. Colocalization (Manders') of SiR-Lysosome overlapping SR101 at t=120 minutes following SR101 addition. Graph reports on n Parental = 71, n Vim KO = 69 technical replicates from 3 independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test H error bars indicate mean -/+ SD) ; * p<0.05, ** p<0.01, *** p<0.001.


Effect of vimentin ablation on ER organization and dynamics . Analysis of parental versus vimentin KO # 1 U2OS cells ectopically expressing mCherry - KDEL . D . Time - lapse imaging . Left panels : representative confocal fluorescence images of Cherry - KDEL ( white ) at the start of time - lapse t = 0 ( top ) . Middle panels : time color coded ( TCC ) overlays of 60x1s frames . Right panels : overlays of twice eroded ER sheet masks ( magenta ) with mCherry - KDEL signal ( green ) . Masks were generated using ImageJ software . Zoom - ins show select PN and PP regions . Scale bar = 10µm .

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Effect of vimentin ablation on ER organization and dynamics. Analysis of parental versus vimentin KO#1 U2OS cells ectopically expressing mCherry-KDEL. D. Time-lapse imaging. Left panels: representative confocal fluorescence images of Cherry-KDEL (white) at the start of time-lapse t=0 (top). Middle panels: time color coded (TCC) overlays of 60x1s frames. Right panels: overlays of twice eroded ER sheet masks (magenta) with mCherry-KDEL signal (green). Masks were generated using ImageJ software. Zoom-ins show select PN and PP regions. Scale bar = 10µm.


D , Consequences of RNF26 , UBE2J1 , and vimentin loss on ERQC formation . D . Representative confocal images of fixed U2OS cells transfected or genetically modified as indicated , treated in the absence ( - ) or presence ( + ) of tunicamycin ( 5ug / mL , O / N ) , fixed and immunostained against endogenous calnexin ( white ) .

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D, Consequences of RNF26, UBE2J1, and vimentin loss on ERQC formation. D. Representative confocal images of fixed U2OS cells transfected or genetically modified as indicated, treated in the absence (-) or presence (+) of tunicamycin (5ug/mL, O/N), fixed and immunostained against endogenous calnexin (white).


F , G . Consequences of RNF26 , UBE2J1 , and vimentin loss on induction of PERK - mediated UPR signaling by tunicamycin . F . U2OS cells , transfected or genetically modified as indicated , were treated in the absence ( - ) or presence ( + ) of tunicamycin ( 2 . 5µg / mL , 5hrs ) . Immunoblots of whole cell lysates against endogenous PERK , phospho - eIF2a , ATF4 , HERP1 , and Vinculin ( loading control ) are shown , representative of three independent experiments . G . CHOP transcripts isolated from U2OS cells transfected or genetically modified as indicated and treated in the absence ( - ) or presence ( + ) of tunicamycin ( 2 . 5ug / mL , O / N ) were detected by qPCR and normalized to GAPDH ; n siC ( - ) = 4 , n siC ( + ) = 4 , n siRNF26 # 1 ( + ) = 3 , n siRNF26 # 2 ( + ) = 4 , n PARENTAL ( + ) = 4 , n Vim KO ( + ) = 4 independent experiments . Data information : Graphs report mean or median ( red line ) of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test G error bars indicate mean - / + SEM ) p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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F, G. Consequences of RNF26, UBE2J1, and vimentin loss on induction of PERK-mediated UPR signaling by tunicamycin. F. U2OS cells, transfected or genetically modified as indicated, were treated in the absence (-) or presence (+) of tunicamycin (2.5µg/mL, 5hrs). Immunoblots of whole cell lysates against endogenous PERK, phospho-eIF2a, ATF4, HERP1, and Vinculin (loading control) are shown, representative of three independent experiments. G. CHOP transcripts isolated from U2OS cells transfected or genetically modified as indicated and treated in the absence (-) or presence (+) of tunicamycin (2.5ug/mL, O/N) were detected by qPCR and normalized to GAPDH; n siC (-)=4, n siC (+)=4, n siRNF26#1 (+)=3, n siRNF26#2 (+)=4, n PARENTAL (+)=4, n Vim KO (+)=4 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test G error bars indicate mean -/+ SEM) p<0.05, ** p<0.01, *** p<0.001.


A , Analysis of localization of RNF26 sequence variants and HERP1 or HERP2 in the ER . A . Representative confocal images of U2OS cells transfected with GFP - RNF26 WT , RFP - RNF26 I382R , GFP - RNF26 ΔRING and HERP1 or GFP - RNF26 WT with HA - HERP2 that were stained for HA and endogenous VAP - A . Indicated single channels and their corresponding color overlays are shown . Zoom - ins highlight PN regions . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm .

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A, Analysis of localization of RNF26 sequence variants and HERP1 or HERP2 in the ER. A. Representative confocal images of U2OS cells transfected with GFP-RNF26 WT, RFP-RNF26 I382R, GFP-RNF26 ΔRING and HERP1 or GFP-RNF26 WT with HA-HERP2 that were stained for HA and endogenous VAP-A. Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.


C . Analysis of determinants for RNF26 complex formation with ERQC proteins HERP1 and HRD1 . U2OS cells were transfected with RFP - RNF26 variants ( WT , I382R , ΔRING ) or EV with either HA - HERP1 or HA - HRD1 before lysis in 1 % DMNG detergent , pulldown with RFP - TRAP beads and analysis by SDS - PAGE and WB . Representative images of three independent replicates .

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C. Analysis of determinants for RNF26 complex formation with ERQC proteins HERP1 and HRD1. U2OS cells were transfected with RFP-RNF26 variants (WT, I382R, ΔRING) or EV with either HA-HERP1 or HA-HRD1 before lysis in 1% DMNG detergent, pulldown with RFP-TRAP beads and analysis by SDS-PAGE and WB. Representative images of three independent replicates.


D , E . Analysis of localization of ERQC domains with vimentin . D . Representative confocal images of U2OS cells transfected with HA - HERP1 that were stained for endogenous vimentin . Indicated single channels and their corresponding color overlays are shown . Zoom - ins highlight PN regions where vimentin surrounds HERP1 - labeled ERQC domains . E . Line graph analysis of HA - HERP1 ( magenta ) and vimentin ( green ) signals from E ( bottom zoom image ) . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm .

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D, E. Analysis of localization of ERQC domains with vimentin. D. Representative confocal images of U2OS cells transfected with HA-HERP1 that were stained for endogenous vimentin. Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions where vimentin surrounds HERP1-labeled ERQC domains. E. Line graph analysis of HA-HERP1 (magenta) and vimentin (green) signals from E (bottom zoom image). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.


F , G . Analysis of HERP1 - induced ERQC domain localization as a function of vimentin and RNF26 . F . Representative confocal fluorescence images of control cells , RNF26 KO cells , or vimentin KO # 2 cells , transfected with HA - HERP1 and immunostained for HA . Zoom - ins highlight PN regions . G . Perinuclearity analysis of HA - HERP1 in the presence or absence of RNF26 or vimentin . Plot reports median ( red line ) and 95 % confidence interval ( error bar ) of sample values ( open circle ) , n Parental = 79 , n R26 KO = 51 , n Vim KO = 45 technical replicates from three independent experiments ( Mann - Whitney U test ; * * p < 0 . 01 , * * * p < 0 . 001 ) . Graphs report median ( red line ) and 95 % confidence interval ( error bars ) of sample values ( open circles ) . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm .

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F, G. Analysis of HERP1-induced ERQC domain localization as a function of vimentin and RNF26. F. Representative confocal fluorescence images of control cells, RNF26 KO cells, or vimentin KO#2 cells, transfected with HA-HERP1 and immunostained for HA. Zoom-ins highlight PN regions. G. Perinuclearity analysis of HA-HERP1 in the presence or absence of RNF26 or vimentin. Plot reports median (red line) and 95% confidence interval (error bar) of sample values (open circle), n Parental=79, n R26 KO=51, n Vim KO=45 technical replicates from three independent experiments (Mann-Whitney U test; ** p<0.01, *** p<0.001). Graphs report median (red line) and 95% confidence interval (error bars) of sample values (open circles). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.


C , D . Lysosomal processing of Sec62 - HALO - GFP over time in the presence or absence of RNF26 or vimentin . C . Parental , Vim KO # 2 or RNF26 KO U2OS cells were transfected with Sec62 - HALO - GFP overnight , incubated with 6 - chlorohexanol for 15 minutes , washed 3 times with PBS , pulsed with TAMRA HALOtag ligand ( 200nM ) for 1hr , and incubated with 6 - chlorohexanol with or without Bafilomycin A1 ( 100nM ) for the indicated times before cell lysis and analysis by SDS - PAGE . Shown is a representative in - gel fluorescence scan with indication of full - length Sec62 , protease - resistant HALO fragment and molecular weight markers ( MW ) , as well as a Vinculin blot ( loading control ) D . Quantification of Sec62 accumulation in LEs from ( B ) . Unprocessed and protease - resistant ( lysosomal ) band intensities in ( B ) were calculated with ImageJ and ratios of lysosomal Sec62 versus unprocessed Sec62 were normalized to transfer in parental cells after 10 hours . Error bars show mean + / - SD of n Parental = 6 , n R26 KO = 4 , n Vim KO = 6 independent experiments . Scale bar = 10µm . Graphs report mean ( in D of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test ( D ( paired ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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C, D. Lysosomal processing of Sec62-HALO-GFP over time in the presence or absence of RNF26 or vimentin. C. Parental, Vim KO#2 or RNF26 KO U2OS cells were transfected with Sec62-HALO-GFP overnight, incubated with 6-chlorohexanol for 15 minutes, washed 3 times with PBS, pulsed with TAMRA HALOtag ligand (200nM) for 1hr, and incubated with 6-chlorohexanol with or without Bafilomycin A1 (100nM) for the indicated times before cell lysis and analysis by SDS-PAGE. Shown is a representative in-gel fluorescence scan with indication of full-length Sec62, protease-resistant HALO fragment and molecular weight markers (MW), as well as a Vinculin blot (loading control) D. Quantification of Sec62 accumulation in LEs from (B). Unprocessed and protease-resistant (lysosomal) band intensities in (B) were calculated with ImageJ and ratios of lysosomal Sec62 versus unprocessed Sec62 were normalized to transfer in parental cells after 10 hours. Error bars show mean +/- SD of n Parental=6, n R26 KO=4, n Vim KO =6 independent experiments. Scale bar = 10µm. Graphs report mean (in D of sample values (open circles). Statistical analyses were performed using Students' T-test (D (paired) ; * p<0.05, ** p<0.01, *** p<0.001).


E , F . Sec62 - mediated transfer of ER to lysosomes as a function of RNF26 or vimentin . E . Representative Airyscan images of Parental , RNF26 KO or Vim KO # 2 U2OS cells that were transfected with Sec62 - HALO - GFP , incubated with 6 - chlorohexanol for 15 minutes , washed 3 times with PBS , pulsed with JF646 HALOtag ligand for 1hr , and incubated with 6 - chlorohexanol and Bafilomycin A1 for 8hrs before fixation and immunostaining for CD63 to visualize LEs . F . Colocalization ( Manders ' ) analysis of CD63 over HALO signals from ( D ) . Plot reports n Parental = 106 , n R26 KO = 69 , n Vim KO = 68 technical replicates from three independent experiments . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm . Graphs report mean ( in F ) of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test F ( unpaired ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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E, F. Sec62-mediated transfer of ER to lysosomes as a function of RNF26 or vimentin. E. Representative Airyscan images of Parental, RNF26 KO or Vim KO#2 U2OS cells that were transfected with Sec62-HALO-GFP, incubated with 6-chlorohexanol for 15 minutes, washed 3 times with PBS, pulsed with JF646 HALOtag ligand for 1hr, and incubated with 6-chlorohexanol and Bafilomycin A1 for 8hrs before fixation and immunostaining for CD63 to visualize LEs. F. Colocalization (Manders') analysis of CD63 over HALO signals from (D). Plot reports n Parental=106, n R26 KO=69, n Vim KO =68 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Graphs report mean (in F) of sample values (open circles). Statistical analyses were performed using Students' T-test F (unpaired) ; * p<0.05, ** p<0.01, *** p<0.001).


G , H . Sec62 - mediated transfer of ER to lysosomes as a function of RNF26 overexpression and activity . G . Representative spinning disc live cell images of RNF26 WT and I382R inactive mutant with Sec62 . U2OS cells were transfected overnight with Sec62 - HALO - GFP and RFP - RNF26 WT or I382R , incubated for 5hrs with JF646 and imaged by spinning disc microscopy . Images show overlay of GFP ( green ) , JF646 ( magenta ) and RFP signals , zoom - ins of overlay and single channels that show transfer of RNF26 into LEs ( WT RNF26 ) . H . Analysis of Sec62 transfer to proteolytic compartments as a function of RNF26 WT or I382R overexpression . Number of GFP - negative / HALO - positive signals was manually counted from cells in ( B ) and ( G ) . Plot reports n EV = 86 , n R26 WT = 35 , n R26 IR = 40 technical replicates from three independent experiments . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , median ( in H ) of sample values ( open circles ) . Mann - Whitney U test ( H ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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G, H. Sec62-mediated transfer of ER to lysosomes as a function of RNF26 overexpression and activity. G. Representative spinning disc live cell images of RNF26 WT and I382R inactive mutant with Sec62. U2OS cells were transfected overnight with Sec62-HALO-GFP and RFP-RNF26 WT or I382R, incubated for 5hrs with JF646 and imaged by spinning disc microscopy. Images show overlay of GFP (green), JF646 (magenta) and RFP signals, zoom-ins of overlay and single channels that show transfer of RNF26 into LEs (WT RNF26). H. Analysis of Sec62 transfer to proteolytic compartments as a function of RNF26 WT or I382R overexpression. Number of GFP-negative/HALO-positive signals was manually counted from cells in (B) and (G). Plot reports n EV=86, n R26 WT=35, n R26 IR =40 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, median (in H) of sample values (open circles). Mann-Whitney U test (H); * p<0.05, ** p<0.01, *** p<0.001).


( A ) Coomassie stained SDS - PAGE showing purified T . thermophila tubulin .

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(A) Coomassie stained SDS-PAGE showing purified T. thermophila tubulin.


( B ) A representative cryo - electron micrograph of paclitaxel - stabilized T . thermophila MTs .

(B) A representative cryo-electron micrograph of paclitaxel-stabilized T. thermophila MTs.


( C ) Kymographs obtained by TIRF microscopy showing the dynamics of T . thermophila MTs at the indicated concentrations in µM .

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(C) Kymographs obtained by TIRF microscopy showing the dynamics of T. thermophila MTs at the indicated concentrations in µM.


( D ) Plot showing the time course of T . thermophila cell growth in the presence of CA4 ( 20µM ) or parabulin ( 50µM ; error bars corresponding to the standard deviation of triplicate biological measurements ) . Control experiments are done with DMSO alone .

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(D) Plot showing the time course of T. thermophila cell growth in the presence of CA4 (20µM) or parabulin (50µM; error bars corresponding to the standard deviation of triplicate biological measurements). Control experiments are done with DMSO alone.


( A ) Plot showing quantification of the T . thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs . Error bars represent the mean + / - 95 % CI . For each concentration , data is obtained from at least 2 independent experiments . For growth rates and growth lengths , n = 41 ( DMSO ) , n = 4 ( 0 . 04uM parabulin ) , n = 34 ( 0 . 04 uM colchicine ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison test ; DMSO vs parabulin P < 0001 ( * * * * ) ; DMSO vs colchicine p = 0 . 40 ( n . s . , not significant ) . ( B ) T . thermophila MT growth rates in the presence of the indicated drugs ( 40 μM ) . Error bars represent the mean + / - 95 % CI ( n = 4 ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison test ; DMSO vs parabulin P = 0 . 25 ; DMSO vs colchicine P = 0 . 20 ( n . s . , not significant ) .

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(A) Plot showing quantification of the T. thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs. Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments. For growth rates and growth lengths, n=41(DMSO), n= 4 (0.04uM parabulin), n= 34 (0.04 uM colchicine). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P<0001 (****); DMSO vs colchicine p = 0.40 (n.s., not significant). (B) T. thermophila MT growth rates in the presence of the indicated drugs (40 μM). Error bars represent the mean +/- 95% CI (n=4). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P = 0.25; DMSO vs colchicine P = 0.20 (n.s., not significant).


( D ) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt - MTs .

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(D) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt-MTs.


( A ) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample . The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100 % growth . Sub - confluent human fibroblasts were used to visualize potential compound effects during proliferation . The results represent the average of 9 readings ( 3 biological replicates with 3 technical replicates of each treatment ) ± standard error of the mean .

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(A) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample. The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100% growth. Sub-confluent human fibroblasts were used to visualize potential compound effects during proliferation. The results represent the average of 9 readings (3 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.


( B ) Replication of GFP - expressing T . gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging . Since vacuoles containing more than eight tachyzoites are not always easy to score , we tallied vacuoles above eight as being 8 + in size . At 36 hours , both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites ( three or more doublings ) . Vacuoles in parabulin - treated cultures contain significantly fewer parasites , indicating that replication is slowed . The graph shows three biological replicates with values normalized to 100 % for each condition and each replicate , error bars represent standard error of the mean between experiments .

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(B) Replication of GFP-expressing T. gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging. Since vacuoles containing more than eight tachyzoites are not always easy to score, we tallied vacuoles above eight as being 8+ in size. At 36 hours, both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites (three or more doublings). Vacuoles in parabulin-treated cultures contain significantly fewer parasites, indicating that replication is slowed. The graph shows three biological replicates with values normalized to 100% for each condition and each replicate, error bars represent standard error of the mean between experiments.


( C ) The parabulin EC50 value for T . gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions . Error bars represent standard error of the mean . The results represent the average of 18 readings ( 6 biological replicates with 3 technical replicates of each treatment ) ± standard error of the mean .

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(C) The parabulin EC50 value for T. gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions. Error bars represent standard error of the mean. The results represent the average of 18 readings (6 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.


( D ) Phase contrast of extracellular parasite cultures . Serial passage of T . gondii in 40 - 80 μM parabulin induces accumulation of aberrant tachyzoite forms . These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding .

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(D) Phase contrast of extracellular parasite cultures. Serial passage of T. gondii in 40-80 μM parabulin induces accumulation of aberrant tachyzoite forms. These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding.


A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP - positive cells was determined 16 h later by flow cytometry .

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A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP-positive cells was determined 16 h later by flow cytometry.


B LDH release from HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . At 24 h post transfection , cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined . Graphs show mean and s . d . of quadruplicate wells .

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B LDH release from HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. At 24 h post transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells.


C - D . PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD in the presence of osmoprotectants . At 24 h post transfection , PEGs of the indicated molecular weights were added to a final concentration of 30 mM , cells were treated with 250 ng / ml doxycycline for 8 h and the level of PI staining ( C ) or LDH release ( D ) was determined .

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C-D. PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng/ml doxycycline for 8 h and the level of PI staining (C) or LDH release (D) was determined.


E - F . PI staining of and LDH release from LPS - primed primary BMDMs infected with log - phase S . typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight ( numbers on the X - axis , 30 mM final concentration ) . * p < 0 . 05 as determined by student ' s t test . Graphs show mean and s . d . of quadruplicate wells . Data are representative of at least three independent expeirments .

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E-F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight (numbers on the X-axis, 30 mM final concentration).* p<0.05 as determined by student's t test. Graphs show mean and s.d. of quadruplicate wells. Data are representative of at least three independent expeirments.


A . Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD , cleaved GSDMD and α - tubulin in the cell lysates of immortalized LPS - primed WT macrophages left uninfected ( NS ) or infected for 10 - 40 minutes with log phase S . typhimurium ( MOI = 50 ) .

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A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10-40 minutes with log phase S. typhimurium (MOI=50).


C . Fractionation and immunoblot analysis for GSDMD , Na + K + ATPase , VDAC ( Voltage - dependent anion channel ) , HDAC1 ( Histone deacetylase 1 ) and GAPDH ( Glycerinaldehyd - 3 - phosphat - Dehydrogenase ) of WT macrophages infected for 10 and 20 minutes with log phase S . typhimurium ( MOI = 50 ) . Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane .

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C. Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (Voltage-dependent anion channel), HDAC1 (Histone deacetylase 1) and GAPDH (Glycerinaldehyd-3-phosphat-Dehydrogenase) of WT macrophages infected for 10 and 20 minutes with log phase S. typhimurium (MOI=50). Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane.


D . Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S . typhimurium ( MOI = 50 ) . Extraction was carried in variable conditions as described in the Material and Methods section .

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D. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S. typhimurium (MOI=50). Extraction was carried in variable conditions as described in the Material and Methods section.


A . Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . The protein was cleaved in a time - dependent manner into two bands of 31 kDa ( GSDMDNterm ) and 22 kDa ( GSDMDCterm ) .

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A. Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. The protein was cleaved in a time-dependent manner into two bands of 31 kDa (GSDMDNterm) and 22 kDa (GSDMDCterm).


B . Cross - linking experiment of full - length and cleaved GSDMD . GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . After enzymatic cleavage , GSDMDNterm is highly cross - linked by DSS , resulting in the gel - impenetrating species highlighted by the arrow . GSDMDCterm is not cross - linked .

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B. Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked.


C . GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase - 1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E . coli . After 2 h , the lipid fraction ( L ) was separated from the supernatant ( S ) by ultracentrifugation at 4C for 1 h at 120 , 000g .

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C. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4C for 1 h at 120,000g.


A - I . Dye release time courses from liposomes as a percentage of maximal release . A . Five different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli polar lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 130 , 100 , 65 ( colored dark to light orange ) . The time point of 20 min is highlighted by a vertical dashed line .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. A. Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli polar lipid extract were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . B . Five different reactions , where 130 nM of GSDMD and 400μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli extract polar lipid were incubated with caspase - 1 concentration of ( nM ) : 15 , 8 , 5 , 2 . 5 , 1 . 2 ( colored dark to light blue ) . The time point of 20 min is highlighted by a vertical dashed line .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. B. Five different reactions, where 130 nM of GSDMD and 400μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli extract polar lipid were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . C . Three different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 100 ( colored dark to light green ) .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. C. Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of (nM): 520, 260,100 (colored dark to light green).


A - I . Dye release time courses from liposomes as a percentage of maximal release . D . Dye release at 20 minutes reaction as a function of GSDMD ( dark orange ) and caspase - 1 ( dark blue ) concentrations . Errors bars for three independent experiments are shown .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. D. Dye release at 20 minutes reaction as a function of GSDMD (dark orange) and caspase-1 (dark blue) concentrations. Errors bars for three independent experiments are shown.


A - I . Dye release time courses from liposomes as a percentage of maximal release . E . Two different sets of reactions , where wild type GSDMD ( dark to light orange ) and the mutant GSDMDI104N ( dark to light blue ) were independently incubated at the concentrations of 260 , 130 and 65 nM with 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes . The time point of 60 minutes is highlighted by a vertical dashed line .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. E. Two different sets of reactions, where wild type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130 and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes. The time point of 60 minutes is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . F . Dye release at 60 minutes of reaction as a function of GSDMD wild type ( dark orange ) and GSDMDI104N ( dark blue ) concentration . Error bars for three independent experiments are shown .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. F. Dye release at 60 minutes of reaction as a function of GSDMD wild type (dark orange) and GSDMDI104N (dark blue) concentration. Error bars for three independent experiments are shown.


A - I . Dye release time courses from liposomes as a percentage of maximal release . G - I . Dye release from 400 μM liposomes loaded with the 6 - carboxifluorescein - derivates FD - 20 , FD - 40 and FD - 150 , with variable Stokes diameters , as indicated . 130 nM of GSDMD and 5 nM caspase - 1 were incubated with the liposomes .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. G-I. Dye release from 400 μM liposomes loaded with the 6-carboxifluorescein-derivates FD-20, FD-40 and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes.


A - C . Cryo - EM micrographs of GSDMDNterm pores in E . coli extract polar lipids liposomes . The micrographs were acquired at protein / lipid molar ratios of 1 / 1000 , 1 / 500 and 1 / 100 , respectively . Black arrows indicate ring - shaped structures corresponding to oligomeric GSDMDNterm pore forms . Scale bars = 80 nm .

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A-C. Cryo-EM micrographs of GSDMDNterm pores in E. coli extract polar lipids liposomes. The micrographs were acquired at protein/lipid molar ratios of 1/1000, 1/500 and 1/100, respectively. Black arrows indicate ring-shaped structures corresponding to oligomeric GSDMDNterm pore forms. Scale bars = 80 nm.


D . Proteoliposome with protein / lipid molar ratio of 1 / 100 at higher magnification . Scale bar = 80 nm .

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D. Proteoliposome with protein/lipid molar ratio of 1/100 at higher magnification. Scale bar = 80 nm.


A . AFM topograph of GSDMDNterm bound to lipid membranes of E . coli extract polar lipids . Overview topograph showing arc - , slit - and ring - like GSDMD oligomers .

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A. AFM topograph of GSDMDNterm bound to lipid membranes of E. coli extract polar lipids. Overview topograph showing arc-, slit- and ring-like GSDMD oligomers.


B - E . High - resolution AFM topographs and height profiles of GSDMDNterm oligomers ( red curves ) . Height profiles were measured along the red lines in the topographs . Dashed zero lines ( 0 nm ) indicate the surface of the lipid membrane ; heights of < − 4 nm indicate that the oligomers formed transmembrane pores . Topographs were taken in buffer solution ( 150 NaCl , 20 mM Hepes , pH 7 . 8 ) at room temperature . The experiment was reproduced at least three times using independent liposome and GSDMD preparations .

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B-E. High-resolution AFM topographs and height profiles of GSDMDNterm oligomers (red curves). Height profiles were measured along the red lines in the topographs. Dashed zero lines (0 nm) indicate the surface of the lipid membrane; heights of <−4 nm indicate that the oligomers formed transmembrane pores. Topographs were taken in buffer solution (150 NaCl, 20 mM Hepes, pH 7.8) at room temperature. The experiment was reproduced at least three times using independent liposome and GSDMD preparations.


F - H . Analysis of GSDMDNterm oligomers bound to lipid membranes of E . coli extract polar lipids . F . GSDMDNterm oligomers assembled into arcs , slits and rings leading to transmembrane ( TM ) pores or not transmembrane aggregate ( n = 218 ) . G . Height of GSDMDNterm oligomers protruding from the lipid membrane . Bars represent mean and error bars represent s . e . m . ( n = 218 ) . H . Distribution of the diameters of rings formed by GSDMDNterm oligomers . The average distribution was 21 . 2 ± 5 . 6 nm ( n = 164 ; average ± s . d . ) and the bin size was 3 nm .

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F-H. Analysis of GSDMDNterm oligomers bound to lipid membranes of E. coli extract polar lipids. F. GSDMDNterm oligomers assembled into arcs, slits and rings leading to transmembrane (TM) pores or not transmembrane aggregate (n = 218). G. Height of GSDMDNterm oligomers protruding from the lipid membrane. Bars represent mean and error bars represent s.e.m. (n = 218). H. Distribution of the diameters of rings formed by GSDMDNterm oligomers. The average distribution was 21.2 ± 5.6 nm (n = 164; average ± s.d.) and the bin size was 3 nm.


Co - immunoprecipitation assays in HEK293T cells after co - transfection with Flag - TRIM27 and the indicated combinations of empty HA - vector , HA - tagged FIP200 , ULK1 , or ATG13 before immunoprecipitation with anti - HA antibodies . Immunoprecipitates ( top panel ) and input samples ( bottom panel ) were then subjected to Western blotting against anti - HA or Flag . Data information : the experiments were performed at least three times independently with similar results .

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Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and the indicated combinations of empty HA-vector, HA-tagged FIP200, ULK1, or ATG13 before immunoprecipitation with anti-HA antibodies. Immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to Western blotting against anti-HA or Flag. Data information: the experiments were performed at least three times independently with similar results.


C - F . WT ( C ) , FIP200 knockout ( KO ) ( D ) , ULK1 / 2 knockout ( E ) MEF cells or ATG13 knockout 4T1 cells ( F ) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G ( IgG ) or anti - TRIM27 before blotting against the indicated endogenous forms of TRIM27 , FIP200 , ULK1 , ATG13 , and USP7 . Data information : the experiments were performed at least three times independently with similar results .

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C-F. WT (C), FIP200 knockout (KO) (D), ULK1/2 knockout (E) MEF cells or ATG13 knockout 4T1 cells (F) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G (IgG) or anti-TRIM27 before blotting against the indicated endogenous forms of TRIM27, FIP200, ULK1, ATG13, and USP7. Data information: the experiments were performed at least three times independently with similar results.


G . Co - immunoprecipitation assays in HEK293T cells after co - transfection with Flag - TRIM27 and either full length ULK ( HA - ULK1 1 - 1051 ) or the indicated various truncated forms of ULK1 designated HA - ULK1 1 - 828 , HA - ULK1 1 - 600 , and HA - ULK1 1 - 500 ; AA , amino acids . Data information : , the experiments were performed at least three times independently with similar results .

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G. Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and either full length ULK (HA-ULK1 1-1051) or the indicated various truncated forms of ULK1 designated HA-ULK1 1-828, HA-ULK1 1-600, and HA-ULK1 1-500; AA, amino acids. Data information: , the experiments were performed at least three times independently with similar results.


H . Co - immunoprecipitation assays in HeLa cells stably expressing HA - ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated . Data information : the experiments were performed at least three times independently with similar results .

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H. Co-immunoprecipitation assays in HeLa cells stably expressing HA-ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated. Data information: the experiments were performed at least three times independently with similar results.


HeLa cells stably expressing Flag - TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium . Anti - Flag immunoprecipitates ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against Flag and ULK1 . Data information : the experiments were performed at least three times independently with similar results .

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HeLa cells stably expressing Flag-TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium. Anti-Flag immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to blotting against Flag and ULK1. Data information: the experiments were performed at least three times independently with similar results.


Purified recombinant GST or GST - TRIM27 was incubated with recombinant HA - ULK1 and bound ULK1 analyzed by Western blotting . Data information : the experiments were performed at least three times independently with similar results .

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Purified recombinant GST or GST-TRIM27 was incubated with recombinant HA-ULK1 and bound ULK1 analyzed by Western blotting. Data information: the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T cells after co - transfection with HA - ULK1 and His - Ub along with Flag - TRIM27 ( A ) Samples recovered with Ni - NTA ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against anti - HA , Flag or His as indicated . Data information : , the experiments were performed at least three times independently with similar results .

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Ubiquitination assays in HEK293T cells after co-transfection with HA-ULK1 and His-Ub along with Flag-TRIM27 (A) Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA, Flag or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs ( shTRIM27 - 1 and - 2 ) ( B ) . Samples recovered with Ni - NTA ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against anti - HA or His as indicated . Data information : , the experiments were performed at least three times independently with similar results .

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Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs (shTRIM27-1 and -2) (B). Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


C . In vitro ubiquitination assays performed with the indicated combinations of recombinant GST , GST - TRIM27 , affinity - purified HA - ULK1 and His - Ub in the presence or absence of E1 , E2 , and ATP . The reactions were analyzed with anti - Ub , anti - HA , or anti - GST . Data information : the experiments were performed at least three times independently with similar results .

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C. In vitro ubiquitination assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1 and His-Ub in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST. Data information: the experiments were performed at least three times independently with similar results.


Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs ( D ) Data information : , the experiments were performed at least three times independently with similar results .

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Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs (D) Data information: , the experiments were performed at least three times independently with similar results.


Western blot analysis of HEK293T cells transfected with empty Flag vector ( - ) or Flag - TRIM27 after treatment with or without 10 μM MG132 for 4 hours ( E ) . Data information : the experiments were performed at least three times independently with similar results .

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Western blot analysis of HEK293T cells transfected with empty Flag vector (-) or Flag-TRIM27 after treatment with or without 10 μM MG132 for 4 hours (E). Data information: the experiments were performed at least three times independently with similar results.


F . Fluorescence microscopy image of MEFs with stable expression of mCherry - ULK1 and GFP - TRIM27 after treating cells with DMSO ( MOCK ) or 10 μM MG132 for 4 hours . Scale bar , 10 μm . Data information : the experiments were performed at least three times independently with similar results .

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F. Fluorescence microscopy image of MEFs with stable expression of mCherry-ULK1 and GFP-TRIM27 after treating cells with DMSO (MOCK) or 10 μM MG132 for 4 hours. Scale bar, 10 μm. Data information: the experiments were performed at least three times independently with similar results.


HA - ULK1 levels in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T cells with or without co - expression of Flag - TRIM27 ( G , H ) Western blots against HA - ULK1 and actin control ( G , were subjected to densitometric quantitation ( H , Data information : the experiments were performed at least three times independently with similar results . All the statistical data are presented as mean ± s . d . of three independent experiments . Statistical analysis was performed using two - tailed unpaired Student ' s t - tests ( H , * * P < 0 . 01 .

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HA-ULK1 levels in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells with or without co-expression of Flag-TRIM27 (G, H) Western blots against HA-ULK1 and actin control (G, were subjected to densitometric quantitation (H, Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests (H , **P < 0.01.


endogenous ULK1 levels ( I , J ) in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T cells or without TRIM27 RNAi ( I , J ) . and actin control I ) were subjected to densitometric quantitation J ) . Data information : the experiments were performed at least three times independently with similar results . All the statistical data are presented as mean ± s . d . of three independent experiments . Statistical analysis was performed using two - tailed unpaired Student ' s t - tests J ) , * * P < 0 . 01 .

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endogenous ULK1 levels (I, J) in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells or without TRIM27 RNAi (I, J). and actin control I) were subjected to densitometric quantitation J). Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests J), **P < 0.01.


Ubiquitination assays were performed in HEK293T cells after co - transfection with full length HA - ULK1 or the indicated truncated constructs ( K ) in the presence or absence of Flag - TRIM27 . Data information : , the experiments were performed at least three times independently with similar results .

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Ubiquitination assays were performed in HEK293T cells after co-transfection with full length HA-ULK1 or the indicated truncated constructs (K) in the presence or absence of Flag-TRIM27. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays were performed in HEK293T cells after co - transfection with double ( K568 / K571R ) or single ( K568R , K571R ) lysine substitution mutants ( L ) in the presence or absence of Flag - TRIM27 . Data information : the experiments were performed at least three times independently with similar results .

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Ubiquitination assays were performed in HEK293T cells after co-transfection with double (K568/K571R) or single (K568R, K571R) lysine substitution mutants (L) in the presence or absence of Flag-TRIM27. Data information: the experiments were performed at least three times independently with similar results.


M . Ubiquitination of HA - ULK1 in cells co - expressing Flag - TRIM27 with either His - Ub WT , His - Ub K48 , or His - Ub K63 , respectively . Data information : the experiments were performed at least three times independently with similar results .

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M. Ubiquitination of HA-ULK1 in cells co-expressing Flag-TRIM27 with either His-Ub WT, His-Ub K48, or His-Ub K63, respectively. Data information: the experiments were performed at least three times independently with similar results.


N . In vitro ubiquitination assays against affinity - purified HA - ULK1 WT or the HA - ULK1 K568 / K571R mutant in the presence or absence of GST - TRIM27 . Data information : the experiments were performed at least three times independently with similar results .

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N. In vitro ubiquitination assays against affinity-purified HA-ULK1 WT or the HA-ULK1 K568/K571R mutant in the presence or absence of GST-TRIM27. Data information: the experiments were performed at least three times independently with similar results.


O . ULK1 / 2 knockout MEFs stably expressing the indicated HA - ULK1 constructs were subjected to Western blot analysis against HA or actin before ( 0h ) and after treatment with CHX for 12 hours . Data information : the experiments were performed at least three times independently with similar results .

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O. ULK1/2 knockout MEFs stably expressing the indicated HA-ULK1 constructs were subjected to Western blot analysis against HA or actin before (0h) and after treatment with CHX for 12 hours. Data information: the experiments were performed at least three times independently with similar results.


A . Western blot analysis of autophagy marker proteins ( p62 and Ser318 phosphorylated ATG13 ) in WT HeLa cells and two independent TRIM27 KO clones . ULK1 and TRIM27 levels were measured , along with actin as a loading control .

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A. Western blot analysis of autophagy marker proteins (p62 and Ser318 phosphorylated ATG13) in WT HeLa cells and two independent TRIM27 KO clones. ULK1 and TRIM27 levels were measured, along with actin as a loading control.


B . TRIM27 KO HeLa cells were reconstituted with Flag - TRIM27 and Western blotting was performed as per ( A ) .

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B. TRIM27 KO HeLa cells were reconstituted with Flag-TRIM27 and Western blotting was performed as per (A).


C . LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 ( BAF A1 ) .

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C. LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 (BAF A1).


D . Western blot analysis of indicated proteins in MEFs generated from Trim27 + / + or Trim27 - / - mice .

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D. Western blot analysis of indicated proteins in MEFs generated from Trim27 +/+ or Trim27 -/- mice.


E - H . Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27 + / + or Trim27 - / - mice cultured in fed ( E ) or starved conditions ( G ) . Quantification of LC3B puncta Fed conditions ( F ) , starved conditions ( H ) ( n = 50 cells per condition ) . Data are mean ± s . d . in F and H . Scale bar , 10 μm . Data information : ( F , H , Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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E-H. Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27+/+ or Trim27-/- mice cultured in fed (E) or starved conditions (G). Quantification of LC3B puncta Fed conditions (F), starved conditions (H) (n = 50 cells per condition). Data are mean ± s.d. in F and H. Scale bar, 10 μm. Data information: (F, H, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


I - J . Western blot analysis of autophagy markers in the kidneys of three - month - old Trim27 + / + and Trim27 - / - mice . Each lane represents a different mouse ( I ) . Quantification of p62 , LC3 - II / LC3 - I ratio , and LC3 - II / actin ratio in I ( J ) . Data are mean ± s . e . m . for three mice per genotype . Data information : J , Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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I-J. Western blot analysis of autophagy markers in the kidneys of three-month-old Trim27 +/+ and Trim27-/- mice. Each lane represents a different mouse (I). Quantification of p62, LC3-II/LC3-I ratio, and LC3-II/actin ratio in I (J). Data are mean ± s.e.m. for three mice per genotype. Data information: J, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.