Figure 5C ) Violin and boxplots of Pol II density at enhancer regions ( n = 8 , 144 ) as a means of quantifying eRNA production . p - values were calculated using a two - sided Wilcoxon rank - sum test . Box plots consist of the median ( central line ) , the 25th and 75th percentiles ( box ) and the highest / lowest value within 1 . 5 * interquartile range of the box ( whiskers ) . Data information : n = 3 - 4 biological replicates per age group for tNET - seqD ) Western Blots of cellular fractionations of young and aged liver samples probed with the indicated antibodies . E ) Heatmaps and average intensity profiles of RNA Pol II and its S2 and S5 phosphorylated forms in young and aged animals . Data information : n = 2 for ChIP - seqF ) Heatmaps and average intensity profiles of SPT4 ChIP - seq signal at TSSs of tNET genes in young and aged animals . Data information : n = 2 for ChIP -

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Figure 5C) Violin and boxplots of Pol II density at enhancer regions (n = 8,144) as a means of quantifying eRNA production. p-values were calculated using a two-sided Wilcoxon rank-sum test. Box plots consist of the median (central line), the 25th and 75th percentiles (box) and the highest/lowest value within 1.5 * interquartile range of the box (whiskers). Data information: n=3-4 biological replicates per age group for tNET-seqD) Western Blots of cellular fractionations of young and aged liver samples probed with the indicated antibodies.E) Heatmaps and average intensity profiles of RNA Pol II and its S2 and S5 phosphorylated forms in young and aged animals. Data information: n=2 for ChIP-seqF) Heatmaps and average intensity profiles of SPT4 ChIP-seq signal at TSSs of tNET genes in young and aged animals. Data information: n=2 for ChIP-seq


Figure 1A ) BW5147 reporter cells stably expressing human FcγR - ζ chain chimeras or BW5147 parental cells ( grey / dashed ) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry . Bar graphs show means of three independent experiments performed in technical duplicates . Error bars = SD . C ) Immobilized IC , immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs . Response curves of human FcγRs activated by opsonized cells ( 293T cells stably expressing CD20 + Rituximab [ Rtx ] ) , immobilized IC ( rec . soluble CD20 + Rtx ) and immobilized IgG ( Rtx ) . sICs formed using monovalent antigen ( rec . soluble CD20 + Rtx ) do not activate human FcγRs . X - Axis shows sample concentration determined by antibody molarity . Y - Axis shows FcγR activation determined by reporter cell mouse IL - 2 production ( OD 450nm ) . Two independent experiments performed in technical duplicates . Error bars = SD .

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Figure 1A) BW5147 reporter cells stably expressing human FcγR-ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD.C) Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X-Axis shows sample concentration determined by antibody molarity. Y-Axis shows FcγR activation determined by reporter cell mouse IL-2 production (OD 450nm). Two independent experiments performed in technical duplicates. Error bars = SD.


Figure 2Ultra - pure antigen ( Ag , TNF - α ) mixed with therapy - grade mAb ( infliximab , Ifx ) was used to generate sICs . X - Axis : concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC ( expressed as mAb molarity ) at a mAb : Ag ratio of 1 : 2 . Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs . Immobilized IgG ( Rtx ) or immobilized FcγR - specific mAbs served as positive controls . Two independent experiments performed in technical duplicates . Error bars = SD . Error bars smaller than symbols are not shown . Left panel : IL - 2 quantification 16 h after reporter cell activation . Background ( blank ) was subtracted ( dashed line ) . IL - 2 was measured via anti - IL - 2 ELISA ( A450nm ) and IL - 2 concentrations were calculated from an IL - 2 standard . Right panel : Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry . MFI were normalized to untreated cells ( ctrl . ) and are presented as fold - change increase .

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Figure 2Ultra-pure antigen (Ag, TNF-α) mixed with therapy-grade mAb (infliximab, Ifx) was used to generate sICs. X-Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR-specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL-2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL-2 was measured via anti-IL-2 ELISA (A450nm) and IL-2 concentrations were calculated from an IL-2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. MFI were normalized to untreated cells (ctrl.) and are presented as fold-change increase.


Figure 3A ) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry . NK cells were incubated with immobilized IgG ( mepolizumab , Mpz ) , soluble IC ( Mpz : IL - 5 = 1 : 1 ) , soluble Mpz or soluble IL - 5 ( all at 200 nM , 106 cells ) . Incubation with PMA and Ionomycin ( Iono ) served as a positive control . Incubation with medium alone served as a negative control . Each dot represents one donor measured in technical duplicates . Error bars = SD . One - way ANOVA ( Tukey ) ; * p < 0 . 05 , * * * * p < 0 . 0001 . B ) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry . Neutrophils were incubated with immobilized IgG ( Mpz ) , soluble IC ( Mpz : IL - 5 = 1 : 1 ) , soluble Mpz or soluble IL - 5 ( all at 200 nM , 2 * 105 cells ) . Incubation with PMA or immobilized rituximab served as positive controls . Incubation with medium served as a negative control . Immobilized FcγRII and FcγRIII specific mAbs served as functional controls . Mean florescence intensity ( MFI ) values at t = 30 minutes of incubation are presented as increase over t = 0 min . Each dot represents one donor measured in technical duplicates . Error bars = SD . Two - way ANOVA compared to medium ( Dunnett ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Figure 3A) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry. NK cells were incubated with immobilized IgG (mepolizumab, Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 106 cells). Incubation with PMA and Ionomycin (Iono) served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one donor measured in technical duplicates. Error bars = SD. One-way ANOVA (Tukey); *p<0.05, ****p<0.0001.B) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry. Neutrophils were incubated with immobilized IgG (Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 2*105 cells). Incubation with PMA or immobilized rituximab served as positive controls. Incubation with medium served as a negative control. Immobilized FcγRII and FcγRIII specific mAbs served as functional controls. Mean florescence intensity (MFI) values at t=30 minutes of incubation are presented as increase over t=0 min. Each dot represents one donor measured in technical duplicates. Error bars = SD. Two-way ANOVA compared to medium (Dunnett); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Figure 4B ) sICs of different size were generated by cross - titration according to the AF4 measurement . Reporter cells were incubated with fixed amounts of either mAb ( infliximab , left ) or Ag ( rhTNF - α , right ) and titrated amounts of antigen or antibody , respectively . X - Axis shows titration of either antigen or antibody , respectively ( TNF - α calculated as monomer ) . Two independent experiments performed in technical duplicates . Error bars = SD . C ) Purified primary NK cells from three different donors were incubated with cross - titrated sICs as in B . NK cells were measured for MIP - 1β expression ( % positivity ) . Incubation with PMA and Ionomycin served as a positive control . Incubation with medium alone served as a negative control . Each dot represents one experimental replicate , the dashed line represents the mean values .

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Figure 4B) sICs of different size were generated by cross-titration according to the AF4 measurement. Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNF-α, right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either antigen or antibody, respectively (TNF-α calculated as monomer). Two independent experiments performed in technical duplicates. Error bars = SD.C) Purified primary NK cells from three different donors were incubated with cross-titrated sICs as in B. NK cells were measured for MIP-1β expression (% positivity). Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one experimental replicate, the dashed line represents the mean values.


Figure 5A ) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals . Dose - dependent reactivity of FcγRs IIIA and IIB / C was observed only for SLE patient sera and not for sera from healthy individuals . Each titration represents one donor measured in technical duplicates . B ) Activation of FcγRs IIB / C and IIIA by patient serum is mediated by serum derived sICs . Patient serum samples were depleted of sICs by PEG precipitation and the supernatant ( SN ) was compared to untreated serum regarding FcγR activation ( left ) . Shown is one exemplary experiment performed in technical duplicates . Error bars indicate reproducibility as range . IgG concentration in the precipitate ( PC ) , supernatant ( SN ) or unfractionated serum respectively is shown in the bar graph ( right ) . C - D ) Correlation of FcγR activation data from A with ( C ) patient SLEDAI or ( D ) conventional SLE disease markers ( α - dsDNA levels indicated as IU / ml or C3d concentrations indicated as mg / L ) . Left panel : FcγRIIIA ; right panel : FcγRIIB / C . FcγR activation from a dose - response curve as in A was calculated as area under curve ( AUC ) for each SLE patient represented by individual dots . AUC from SLE patients are expressed as fold change compared to the healthy control mean ( n = 4 ) . SLE patients with α - dsDNA levels below 50 IU / ml and C3d values below 6 mg / L were excluded . One - tailed Spearman ' s . Dotted lines show 95 % confidence bands .

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Figure 5A) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals. Dose-dependent reactivity of FcγRs IIIA and IIB/C was observed only for SLE patient sera and not for sera from healthy individuals. Each titration represents one donor measured in technical duplicates.B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right).C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.


Figure 6A ) Reporter cells expressing mFcγRIII , mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF - α and mouse - anti - hTNF - α at a 1 : 1 ratio by mass . One experiment performed in technical duplicates . Error bars indicate reproducibility as range . B ) Titrations of three mouse sera per group ( C57BL / 6 , K / BxN or NZB / WF1 ) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above . Sera from BL / 6 mice served as negative control . Each symbol represents mean values of one mouse measured in technical duplicates . Error bars = Mean with SD .

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Figure 6A) Reporter cells expressing mFcγRIII, mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF-α and mouse-anti-hTNF-α at a 1:1 ratio by mass. One experiment performed in technical duplicates. Error bars indicate reproducibility as range.B) Titrations of three mouse sera per group (C57BL/6, K/BxN or NZB/WF1) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above. Sera from BL/6 mice served as negative control. Each symbol represents mean values of one mouse measured in technical duplicates. Error bars = Mean with SD.


Figure 1D . Northern blot analysis of V . cholerae wild - type strain examined for the expression of VcdRP monitored over bacterial growth in the indicated media . The solid triangle represents the band corresponding to the full length primary vcdRP transcript , whereas the open triangles correspond to the different processed isoforms . Probing with 5S rRNA confirmed equal loading .

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Figure 1D. Northern blot analysis of V. cholerae wild-type strain examined for the expression of VcdRP monitored over bacterial growth in the indicated media. The solid triangle represents the band corresponding to the full length primary vcdRP transcript, whereas the open triangles correspond to the different processed isoforms. Probing with 5S rRNA confirmed equal loading.


Figure 2A . Northern blot analysis of the expression of VcdRP and Spot42 sRNA monitored at low cell density ( OD600 of 0 . 1 ) and high cell density ( OD600 of 2 . 0 ) , in the absence ( - ) or presence ( + ) of cAMP ( 5mM f . c . ) supplemented externally in LB medium . The solid triangle indicates the band corresponding to the full - length primary transcript . Probing with 5S rRNA confirmed equal loading .

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Figure 2A. Northern blot analysis of the expression of VcdRP and Spot42 sRNA monitored at low cell density (OD600 of 0.1) and high cell density (OD600 of 2.0), in the absence (-) or presence (+) of cAMP (5mM f.c.) supplemented externally in LB medium. The solid triangle indicates the band corresponding to the full-length primary transcript. Probing with 5S rRNA confirmed equal loading.


Figure 3B . Western blot analysis ( top ) of CTX levels detected in secreted protein fractions of V . cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids . Coomassie - stained SDS gel ( bottom ) confirmed equal loading of the protein fractions . The normalized band intensities are indicated for each expression plasmid relative to pCtrl , which was set to 100 % .

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Figure 3B. Western blot analysis (top) of CTX levels detected in secreted protein fractions of V. cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids. Coomassie-stained SDS gel (bottom) confirmed equal loading of the protein fractions. The normalized band intensities are indicated for each expression plasmid relative to pCtrl, which was set to 100%.


Figure 4I - L . Relative fluorescence intensities ( y - axis ) of E . coli strains harboring the gene - specific translational reporters as in ( A - D ) or the corresponding M2 * variant for each gene , combined with an empty control plasmid or vcdRP expression plasmids ( pVcdRP and pVcdRP M2 ) measured using plate reader ( I - K ) or by Western blot analysis ( L ) . The fluorescence of pCtrl ( for each reporter fusion ) was set to 1 .

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Figure 4I-L. Relative fluorescence intensities (y-axis) of E. coli strains harboring the gene-specific translational reporters as in (A-D) or the corresponding M2* variant for each gene, combined with an empty control plasmid or vcdRP expression plasmids (pVcdRP and pVcdRP M2) measured using plate reader (I-K) or by Western blot analysis (L). The fluorescence of pCtrl (for each reporter fusion) was set to 1.


Figure 5A . Immunoprecipitation of chromosomally tagged GltA : : HA combined with either an empty vector control ( pCtrl ) or SPA - tagged vcdP over - expression plasmid , grown in LB medium to exponential phase ( OD600 of 0 . 5 ) . The SPA epitope contains the 3 × FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site ( Zeghouf et al , 2004 ) . Therefore , protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti - Flag antibody were loaded on a SDS - PAGE gel . Western blotting with anti - HA and anti - Flag antibodies confirmed the interaction of GltA with VcdP in vivo . RNAP served as loading control . The solid triangles indicate the corresponding protein sizes . B . Citrate synthase enzyme activity measurements performed on cellular lysates . An empty vector control ( pCtrl ) or vcdP expression plasmids ( pVcdP and pVcdP * ) were conjugated into V . cholerae wild - type , ΔvcdRP , ΔgltA or F383A GltA backgrounds . Cells were grown in LB medium to stationary phase and subsequently lysed . The protein concentrations of these cellular extracts were measured , and served as input for the assay . The resulting colorimetric product at 412nm was proportional to the enzymatic activity of citrate synthase present . The enzyme kinetics were assayed at 15 second intervals for at least 30 minutes and the activity per milligram of lysate was determined from the initial velocities .

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Figure 5A. Immunoprecipitation of chromosomally tagged GltA::HA combined with either an empty vector control (pCtrl) or SPA-tagged vcdP over-expression plasmid, grown in LB medium to exponential phase (OD600 of 0.5). The SPA epitope contains the 3×FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004). Therefore, protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti-Flag antibody were loaded on a SDS-PAGE gel. Western blotting with anti-HA and anti-Flag antibodies confirmed the interaction of GltA with VcdP in vivo. RNAP served as loading control. The solid triangles indicate the corresponding protein sizes.B. Citrate synthase enzyme activity measurements performed on cellular lysates. An empty vector control (pCtrl) or vcdP expression plasmids (pVcdP and pVcdP*) were conjugated into V. cholerae wild-type, ΔvcdRP, ΔgltA or F383A GltA backgrounds. Cells were grown in LB medium to stationary phase and subsequently lysed. The protein concentrations of these cellular extracts were measured, and served as input for the assay. The resulting colorimetric product at 412nm was proportional to the enzymatic activity of citrate synthase present. The enzyme kinetics were assayed at 15 second intervals for at least 30 minutes and the activity per milligram of lysate was determined from the initial velocities.


Figure 1A , B After grown in nutrient rich medium to log phase , wild - type ( WT ) cells were treated with rapamycin ( 10 μM ) or subject to nitrogen starvation ( SD - N ) for 3 h and 16 h and degradation of autophagic marker GFP - Atg8 was detected . The cycloheximide ( CHX , 100 μg / mL , ) or thiolutin ( 10 μg / mL ) was simultaneously used to block protein synthesis or gene transcription . Yeast cells were treated with thiolutin ( 10 μg / mL ) together with rapamycin or nitrogen starvation in different order for 3 h , 6h and 16 h and degradation of autophagic marker GFP - Atg8 was detected . Yeast cells were treated with thiolutin ( 10 μg / mL ) together with rapamycin or nitrogen starvation in different order for 3 h , 6h and 16 h and degradation of autophagic marker GFP - Atg8 was detected .

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Figure 1A, B After grown in nutrient rich medium to log phase, wild-type (WT) cells were treated with rapamycin (10 μM) or subject to nitrogen starvation (SD-N) for 3 h and 16 h and degradation of autophagic marker GFP-Atg8 was detected. The cycloheximide (CHX, 100 μg/mL,) or thiolutin (10 μg/mL) was simultaneously used to block protein synthesis or gene transcription.Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.


Figure 2B N - terminally GFP - tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1 , 4 and 16 h starvation . Yeast cells with ATG1 deletion were used as positive controls . H Vacuole localization and degradation of endocytosis substrate GFP - Sna3 in WT , atg1 ∆ and rpb9 ∆ cells . Scale bars : 5 μm . I Carboxypeptidases Y ( Cpy1 ) , a vacuolar - resident hydrolases , was analyzed for its transporting to vacuoles by the secretory pathway in WT , atg1 ∆ and rpb9 ∆ cells .

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Figure 2B N-terminally GFP-tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4 and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls.H Vacuole localization and degradation of endocytosis substrate GFP-Sna3 in WT, atg1∆ and rpb9∆ cells. Scale bars: 5 μm.I Carboxypeptidases Y (Cpy1), a vacuolar-resident hydrolases, was analyzed for its transporting to vacuoles by the secretory pathway in WT, atg1∆ and rpb9∆ cells.


Figure 3E - I Overexpression of ATG1 could partially restore autophagy in rpb9 ∆ cells . ATG1 , ATG9 , ATG13 , ATG17 and ATG5 were overexpressed in rpb9 ∆ yeast cells , GFP - 50Q was checked for its autophagic degradation by GFP processing assays after 1 , 4 and 16 h starvation .

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Figure 3E-I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1, ATG9, ATG13, ATG17 and ATG5 were overexpressed in rpb9∆ yeast cells, GFP-50Q was checked for its autophagic degradation by GFP processing assays after 1, 4 and 16 h starvation.


Figure 4B Indicated Rpb9 truncates and mutants were expressed in rpb9 ∆ cells and autophagic degradation of GFP - 50Q was checked by GFP - processing assays .

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Figure 4B Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP-50Q was checked by GFP-processing assays.


Figure 5E Purified GST - Gcn4 and His - Rpb9 proteins from E . coli cells were mixed and GST pulldown assays were performed . F - G Neither overexpression of Gcn4 in rpb9 ∆ yeast cells nor overexpression of Rpb9 in gcn4 ∆ yeast cells can restore autophagy . Gcn4 or Rpb9 were overexpressed in rpb9 ∆ or gcn4 ∆ yeast cells respectively and autophagic degradation of GFP - 50Q was detected .

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Figure 5E Purified GST-Gcn4 and His-Rpb9 proteins from E. coli cells were mixed and GST pulldown assays were performed.F-G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP-50Q was detected.


Figure 6C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9 ∆ yeast cells . Indicated Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected . D N - terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9 ∆ yeast cells . Indicated truncates of Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected .

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Figure 6C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.D N-terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


Figure 1Growth inhibitory concentration ( GI50 ) of CY and bendamustine in the NCI60 cell line panel . Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells . Images were taken at 48 h posttreatment . Cpd A augments DNA damage caused by bendamustine . H1650 cells were treated with 100 μM Bend , 50 μM Cpd A , 100 μM Bend plus 50 μM Cpd A , or 10 μM CY for 12 h , and cell lysates were blotted for γ - H2AX . Actin was used as a loading control .

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Figure 1Growth inhibitory concentration (GI50) of CY and bendamustine in the NCI60 cell line panel.Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment.Cpd A augments DNA damage caused by bendamustine. H1650 cells were treated with 100 μM Bend, 50 μM Cpd A, 100 μM Bend plus 50 μM Cpd A, or 10 μM CY for 12 h, and cell lysates were blotted for γ-H2AX. Actin was used as a loading control.


Figure 2Upper panel , in vitro HDAC1 inhibition assay using bendamustine and CY . Lower panel , summary of CY IC50 against other HDACs . Upper panel , in vitro HDAC1 inhibition assay using bendamustine and CY . Lower panel , summary of CY IC50 against other HDACs . CY and CpdA , but not bendamustine or CpdB , inhibited HDAC activities in cancer cells . H1650 cells were treated with 20 μM CY , 20 μM CpdA , 200 μM CpdB , 200 μM Bend , or 10 μM SAHA for 12 h , and cell lysates were blotted for histone acetylation markers . Actin was used as a loading control . Alteration of CY side chain ( Cpd B ) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage . Cells were treated for 12 h . Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ - H2AX . Actin was used as a loading control . Summary of lethal dose 50 ( LD50 ) of bendamustine , CY , Cpd A ( HDAC inhibition only ) , and Cpd B ( nitrogen mustard only ) against 11 human cancer cell lines . Cpd A enhances the anticancer activity of bendamustine . H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A ( nontoxic dose , see Fig1C and Supplementary Fig S2 ) for 48 h and subjected to MTT viability assays .

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Figure 2Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control.Alteration of CY side chain (Cpd B) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage. Cells were treated for 12 h. Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ-H2AX. Actin was used as a loading control.Summary of lethal dose 50 (LD50) of bendamustine, CY, Cpd A (HDAC inhibition only), and Cpd B (nitrogen mustard only) against 11 human cancer cell lines.Cpd A enhances the anticancer activity of bendamustine. H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A (nontoxic dose, see Fig1C and Supplementary Fig S2) for 48 h and subjected to MTT viability assays.


Figure 3CY , but not bendamustine treatment , led to suppression of CBP , TIP60 , MORF , and MSL1 in H1650 cells . Cells were treated with DMSO , bendamustine ( 350 μM ) , CY ( 15 μM ) , or SAHA ( 10 μM ) for 6 h , and mRNA was extracted for qPCR analysis . Data represent mean ± SEM from three independent experiments , and statistical significance was determined by unpaired two - tailed t - test . * * * P < 0 . 01 . Drug treatment up to 10 h at the concentrations indicated in ( A ) did not cause apoptosis as judged by PARP cleavage . Actin was used as a loading control . ShRNA - mediated suppression of CBP , TIP60 , MORF , and MSL1 at mRNA level . Data represent mean ± SEM from three independent experiments , and statistical significance was determined by unpaired two - tailed t - test . * P < 0 . 1 , * * P < 0 . 05 , * * * P < 0 . 01 . Suppression of CBP , TIP60 , MORF , and MSL1 sensitized cells to bendamustine . Y - axis represents relative resistance calculated from results of GFP competition assays , and statistical significance was determined by unpaired two - tailed t - test . Data represent mean ± SEM from two independent experiments . * * P < 0 . 05 , * * * P < 0 . 01 .

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Figure 3CY, but not bendamustine treatment, led to suppression of CBP, TIP60, MORF, and MSL1 in H1650 cells. Cells were treated with DMSO, bendamustine (350 μM), CY (15 μM), or SAHA (10 μM) for 6 h, and mRNA was extracted for qPCR analysis. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. ***P < 0.01.Drug treatment up to 10 h at the concentrations indicated in (A) did not cause apoptosis as judged by PARP cleavage. Actin was used as a loading control.ShRNA-mediated suppression of CBP, TIP60, MORF, and MSL1 at mRNA level. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. *P < 0.1, **P < 0.05, ***P < 0.01.Suppression of CBP, TIP60, MORF, and MSL1 sensitized cells to bendamustine. Y-axis represents relative resistance calculated from results of GFP competition assays, and statistical significance was determined by unpaired two-tailed t-test. Data represent mean ± SEM from two independent experiments. **P < 0.05, ***P < 0.01.


Figure 4A Determination of maximally tolerated dose ( MTD ) of CY at 60 mg / kg . Other drugs were used at MTD according to the literature . For each drug , n = 5 and all mice survived treatment . Data represent mean ± SEM . B CY exhibited enhanced activity against BCR - ABL Arf − / − murine ALL cells in vitro . C , D CY showed superior antitumor activity compared with bendamustine , SAHA , and other chemotherapeutic drugs in mice transplanted with BCR - ABL ALL cells . P - values of CY versus other drugs : NT ( P = 5 . 02E - 06 ) ( C ) , SAHA ( P = 4 . 45E - 06 ) , Bend ( P = 5 . 18E - 06 ) , NT ( P = 1 . 20E - 05 ) ( D ) , VCR ( P = 1 . 13E - 05 ) , Dox ( P = 8 . 08E - 06 ) , AraC ( P = 7 . 62E - 06 ) , and CTX ( P = 1 . 13E - 05 ) . Survival statistical analysis was done with the Mantel - Cox ( log - rank ) test of GraphPad Prism . E Therapeutic effects of CY ( weekly dose ) and targeted drugs PP242 ( daily dose ) and dasatinib ( daily dose ) in mice transplanted with BCR - ABL ALL cells . P - values of CY versus other drugs : NT ( P = 3 . 94E - 05 ) , PP242 ( P = 1 . 20E - 05 ) , and dasatinib ( P = 0 . 99 ) . Survival statistical analysis was done with the Mantel - Cox ( log - rank ) test of GraphPad Prism . F , G Antitumor activity of CY and bendamustine in nude mice models . Mice were inoculated with human NSCLC cell line H460 . When tumor volume reached 200 mm3 , mice were treated with single dose of bendamustine ( 40 mg / kg , MTD ) or CY ( 20 , 40 , 60 mg / kg ) . In ( G ) , tumors were dissected out after 15 days posttreatment .

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Figure 4A Determination of maximally tolerated dose (MTD) of CY at 60 mg/kg. Other drugs were used at MTD according to the literature. For each drug, n = 5 and all mice survived treatment. Data represent mean ± SEM.B CY exhibited enhanced activity against BCR-ABL Arf−/− murine ALL cells in vitro.C, D CY showed superior antitumor activity compared with bendamustine, SAHA, and other chemotherapeutic drugs in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 5.02E-06) (C), SAHA (P = 4.45E-06), Bend (P = 5.18E-06), NT (P = 1.20E-05) (D), VCR (P = 1.13E-05), Dox (P = 8.08E-06), AraC (P = 7.62E-06), and CTX (P = 1.13E-05). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.E Therapeutic effects of CY (weekly dose) and targeted drugs PP242 (daily dose) and dasatinib (daily dose) in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 3.94E-05), PP242 (P = 1.20E-05), and dasatinib (P = 0.99). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.F, G Antitumor activity of CY and bendamustine in nude mice models. Mice were inoculated with human NSCLC cell line H460. When tumor volume reached 200 mm3, mice were treated with single dose of bendamustine (40 mg/kg, MTD) or CY (20, 40, 60 mg/kg). In (G), tumors were dissected out after 15 days posttreatment.


Figure 5Dose curve of CY , fludarabine , and bendamustine using fresh CLL samples . Data represent mean ± SD . CY preferentially killed CLL cells over healthy B cells . Data represent mean ± SDToxicity of CY190602 in patients harboring deletion of chromosome 17p ( including p53 locus ) compared to patients without del17p . Viability of samples was normalized to background apoptosis for individual patient samples . For all experiments , cell viability was determined after 72 h of drug treatment . Data represent mean ± SD .

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Figure 5Dose curve of CY, fludarabine, and bendamustine using fresh CLL samples. Data represent mean ± SD.CY preferentially killed CLL cells over healthy B cells. Data represent mean ± SDToxicity of CY190602 in patients harboring deletion of chromosome 17p (including p53 locus) compared to patients without del17p. Viability of samples was normalized to background apoptosis for individual patient samples. For all experiments, cell viability was determined after 72 h of drug treatment. Data represent mean ± SD.


figf1 ( a - d ) Rotenone ( Rot ) increased the number of GFP - LC3 puncta and the level of co - localization with mitochondria ( arrows ) in SH - SY5Y cells ( a - c ; 1 μM ) and primary cortical neurons ( d ; 250 nM ) , quantified in Fig . 3f and Supplementary Fig . S1b , c . Veh , vehicle . ( e , f ) Rotenone increased delivery of MitoTracker Green - stained mitochondria ( mt ) to LysoTracker Red ( LTR ) - stained lysosomes , inhibited by siRNA knockdown of ATG7 or LC3 in SH - SY5Y cells . Ctrl , control . Right : RNAi knockdown . ( g ) Rotenone decreased IMM ( COXIV ) , OMM ( TOM40 ) and matrix ( MnSOD ) protein expression levels in primary neurons , reversed by bafilomycin ( Baf ) inhibition of autolysosomal degradation , quantified in Supplementary Fig . S1e . Mean ± s . d . of n = 7 independent experiments for b , c , and n = 3 independent experiments for f , g ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control ; † P0 . 05 versus Rot / control siRNA . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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figf1(a-d) Rotenone (Rot) increased the number of GFP-LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a-c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle.(e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown.(g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control; †P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.


figf2 ( a , b ) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC - MS analysis . ( a ) MS spectra demonstrated increased cardiolipin ( CL ) content of the OMM after rotenone ( Rot ) treatment , with diversification of the cluster distribution to the 7 clusters exhibited by the IMM . Veh , vehicle . ( b ) Pie charts showing the CL distribution between IMM and OMM fractions from toxin - treated neurons , normalized to mitochondrial lipid phosphorus . ( c , d ) Treatment with rotenone caused significant increases in PLA2 - hydrolysable ( surface accessible ) CL assessed by LC - MS in primary cortical neurons ( c ; 250 nM ) and SH - SY5Y cells ( d ; 1 μM ) . ( e , f ) Treatment with 6 - OHDA and CCCP increased surface exposure of CL probed with annexin V in SH - SY5Y ( e , 120 μM ) and parkin - expressing HeLa cells ( f , 20 μM ) , respectively . Mean ± s . d . of n = 3 independent experiments for c - f ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control .

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figf2(a,b) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC-MS analysis. (a) MS spectra demonstrated increased cardiolipin (CL) content of the OMM after rotenone (Rot) treatment, with diversification of the cluster distribution to the 7 clusters exhibited by the IMM. Veh, vehicle. (b) Pie charts showing the CL distribution between IMM and OMM fractions from toxin-treated neurons, normalized to mitochondrial lipid phosphorus.(c,d) Treatment with rotenone caused significant increases in PLA2-hydrolysable (surface accessible) CL assessed by LC-MS in primary cortical neurons (c; 250 nM) and SH-SY5Y cells (d; 1 μM).(e,f) Treatment with 6-OHDA and CCCP increased surface exposure of CL probed with annexin V in SH-SY5Y (e, 120 μM) and parkin-expressing HeLa cells (f, 20 μM), respectively. Mean ± s.d. of n = 3 independent experiments for c-f (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control.


figf3 ( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( a ) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647 - annexin V binding . ( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( b - d ) PLS3 knockdown had no effect on rotenone - induced autophagy ( b ) , but decreased mitophagy in SH - SY5Y cells treated with rotenone ( c ; 1 μM ) or 6 - OHDA ( d ; 120 μM ) . ( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( d , e ) The effects of PLS3 siRNA were recapitulated using a second siRNA # 433 ( d , Supplementary Fig . S3b , g ) , and reversed by transfection with an RNAi - resistant mouse PLS3 vector ( e ) . Inset : PLS3 overexpression . ( f - i ) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) and analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization analysis ( f ) ( f - i ) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) and analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization analysis ( f ) , or with mitochondrial protein immunoblotting ( g ) and densitometry ( h , i and Supplementary Fig . S4c ) . Knockdown efficiencies are shown in Supplementary Figs S3 and S4 . Mean ± s . d . of n = 4 independent experiments for b , c , and n = 3 independent experiments for a , d - f , h - i ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle ; † P0 . 05 versus toxin - treated control siRNA ; * * P0 . 05 versus Rot / PLS3 siRNA . See Supplementary Fig . S7 for uncropped blots .

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figf3(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647-annexin V binding.(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (b-d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM).(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression.(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f)(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of n = 4 independent experiments for b,c, and n = 3 independent experiments for a,d-f,h-i (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle; †P0.05 versus toxin-treated control siRNA; **P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.


figf4 ( a ) Western blot analysis showed beclin 1 cleavage products ( Mr49 K ) were not observed in primary neurons exposed to mitophagy - inducing sublethal concentrations of rotenone ( 250 nM × 2 h : Rot low ) or STS ( 100 nM × 2 h ) . However , beclin 1 was cleaved on exposure to a lethal dose of rotenone ( 1 mM × 24 h : Rot high ) . Veh , vehicle ; CTD , C - terminal domain . ( b , c ) Likewise , SH - SY5Y cells did not show beclin 1 cleavage on exposure to rotenone ( b ) or 6 - OHDA ( c ; duplicate lanes ) . Data are representative of 2 - 3 independent experiments per toxin . ( d ) Cell lysates from stable PINK1 - Flag - expressing SH - SY5Y cells were treated with 6 - OHDA ( 120 μM ) , rotenone ( 1 μM ) or FCCP ( 2 μM ) , and analysed by Flag immunoblot for PINK1 - Flag levels . ( e ) SH - SY5Y cells transfected with HA - parkin were treated with the indicated toxins , immunolabelled for HA ( green ) and mitochondrial p60 antigen ( red ) and analysed for HA - parkin redistribution to mitochondria . FCCP elicited translocation of parkin to mitochondria ( arrows ) , which was not seen with the other mitophagy stimuli . ( f ) Cytosolic and mitochondrial pellets were prepared from SH - SY5Y cells treated with vehicle ( V ) , 6 - OHDA ( O , 120 μM ) or rotenone ( R , 1 μM ) . Gels were loaded with cytosolic or mitochondrial proteins , and immunoblotted for p62 / SQSMT1 , and the indicated fractionation markers . ( g , h ) Confocal analysis of p62 / SQSMT1 distribution in SH - SY5Y cells ( g ) and in primary cortical neurons ( h ) that were co - transfected with mCherry - tagged p62 / SQSMT1 and mitochondrially targeted GFP . Note recruitment of p62 to large mitochondrial aggregates ( g , yellow , arrows ) in FCCP - treated cells , which was not observed in control or rotenone - treated cells . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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

figf4(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain.(b,c) Likewise, SH-SY5Y cells did not show beclin 1 cleavage on exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin.(d) Cell lysates from stable PINK1-Flag-expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), rotenone (1 μM) or FCCP (2 μM), and analysed by Flag immunoblot for PINK1-Flag levels.(e) SH-SY5Y cells transfected with HA-parkin were treated with the indicated toxins, immunolabelled for HA (green) and mitochondrial p60 antigen (red) and analysed for HA-parkin redistribution to mitochondria. FCCP elicited translocation of parkin to mitochondria (arrows), which was not seen with the other mitophagy stimuli.(f) Cytosolic and mitochondrial pellets were prepared from SH-SY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or rotenone (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers.(g,h) Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or rotenone-treated cells. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.


figf5 ( a ) Recombinant LC3 incubated with tetraoleoyl - CL ( TOCL ) or dioleoylphosphatidylcholine ( DOPC ) liposomes at the indicated molar ratios was analysed by blue native PAGE ; liposome binding impaired the gel entry of proteins . ( b ) Titration of phospholipids ( for example , TOCL ) to LC3 was performed to evaluate the ratio that prevents 50 % of the LC3 from entering the gels ( IC50 ) as an index of relative affinity . Bottom left inset : representative gel for TOCL / LC3 = 12 ; top right inset : comparison of IC50 values for TOCL versus dioleoyl - phosphatidic acid ( DOPA ) and tetralinoleoyl - CL ( TLCL ) versus monolyso - trilinoleoyl - CL ( lyso - CL ) . The IC50 for DOPG was > 15 . * P0 . 05 versus TOCL ; † P0 . 05 versus TLCL . ( f - j ) The arrowhead in e shows the position of the N - terminal truncation used to create GFP - LC3 deletion mutants , which were analysed for GFP - LC3 puncta formation ( f , g , i ) and participation in mitophagy ( h , j ) in response to the indicated stimuli . Rot , rotenone ; Veh , vehicle . ( e , k ) Dual point mutations were prepared on the basis of predicted CL contact residues ( e ) , and analysed for participation in mitophagy elicited by Rot or 6 - OHDA ( k ) . Inset : expression of GFP - LC3 plasmids ( uncropped blots in Supplementary Fig . S7 ) . UT , untransfected . Mean ± s . d . of n = 3 independent experiments for f - h , and n = 4 independent experiments for i , j ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus respective vehicle . † P0 . 05 versus toxin - treated wild type ( WT ) .

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

figf5(a) Recombinant LC3 incubated with tetraoleoyl-CL (TOCL) or dioleoylphosphatidylcholine (DOPC) liposomes at the indicated molar ratios was analysed by blue native PAGE; liposome binding impaired the gel entry of proteins.(b) Titration of phospholipids (for example, TOCL) to LC3 was performed to evaluate the ratio that prevents 50% of the LC3 from entering the gels (IC50) as an index of relative affinity. Bottom left inset: representative gel for TOCL/LC3 = 12; top right inset: comparison of IC50 values for TOCL versus dioleoyl-phosphatidic acid (DOPA) and tetralinoleoyl-CL (TLCL) versus monolyso-trilinoleoyl-CL (lyso-CL). The IC50 for DOPG was >15. *P0.05 versus TOCL; †P0.05 versus TLCL.(f-j) The arrowhead in e shows the position of the N-terminal truncation used to create GFP-LC3 deletion mutants, which were analysed for GFP-LC3 puncta formation (f,g,i) and participation in mitophagy (h,j) in response to the indicated stimuli. Rot, rotenone; Veh, vehicle.(e,k) Dual point mutations were prepared on the basis of predicted CL contact residues (e), and analysed for participation in mitophagy elicited by Rot or 6-OHDA (k). Inset: expression of GFP-LC3 plasmids (uncropped blots in Supplementary Fig. S7). UT, untransfected. Mean ± s.d. of n = 3 independent experiments for f-h, and n = 4 independent experiments for i,j (see Supplementary Table S4 for statistics source data); *P0.05 versus respective vehicle. †P0.05 versus toxin-treated wild type (WT).


Figure 1D ) HEK293 cells were genetically engineered to knockout RIG - I , MDA5 , or both , and subsequently subjected to retroviral transduction to stably express FLAG - LGP2 or an empty vector ( EV ) . Correct gene editing and intact type I IFN responsiveness was validated by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) . * , non - specific band . E ) Cells generated in ( D ) were transfected with siCtrl or siADAR1 . The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post - transfection as in ( B ) . Data are means ± s . d . from a representative of four biological replicate experiments . F ) Cells generated in ( D ) were transfected with siCtrl ( siC ) or siADAR1 ( siA ) . Protein lysates were prepared 78h post - transfection , followed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) . G ) HEK293 WT cells and FLAG - LGP2 - expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence . Cells were fixed , permeabilized and stained 72h post - transfection with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 450 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of three biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analyses were performed using unpaired two - tailed Mann - Whitney U tests . ns , not significant ; * * * * , p < 0 . 0001 .

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

Figure 1D) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band.E) Cells generated in (D) were transfected with siCtrl or siADAR1. The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post-transfection as in (B). Data are means ± s.d. from a representative of four biological replicate experiments.F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).G) HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained 72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>450 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.


Figure 2A ) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR / Cas9 . Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness , respectively . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) . B ) ADAR1 - knockout HEK293 cells ( clone 1 ) were co - transfected with an empty vector ( EV ) or a FLAG - LGP2 - encoding vector ( LGP2 ) combined with a vector encoding GFP - tagged ADAR1 p110 or p150 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 expression , normalized to ACTB . Data are means ± s . d . from a representative of four biological replicate experiments . C ) ADAR1 - knockout cells ( clone 1 ) were transfected as in ( B ) . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 4 ) .

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

Figure 2A) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR/Cas9. Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness, respectively. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3).B) ADAR1-knockout HEK293 cells (clone 1) were co-transfected with an empty vector (EV) or a FLAG-LGP2-encoding vector (LGP2) combined with a vector encoding GFP-tagged ADAR1 p110 or p150. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 expression, normalized to ACTB. Data are means ± s.d. from a representative of four biological replicate experiments.C) ADAR1-knockout cells (clone 1) were transfected as in (B). Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=4).


Figure 3A ) ADAR1 - knockout HEK293 cells ( clone 1 ) were modified with a lentiviral - based inducible system to express FLAG - LGP2 WT or a FLAG - LGP2 RNA binding mutant ( K138E / R490E / K634E , denoted as " KRK mutant " ) in a doxycycline - regulated manner . Cells were treated 72h with doxycycline ( dox ) . Protein lysates were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 3 ) . iEV = inducible empty vector ; iLGP2 = inducible LGP2 . C ) Cells generated in ( A ) were plated on coverslips and treated with or without doxycycline for 72h . Cells were fixed , permeabilized and stained with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 500 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of two biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analysis was performed using a Kruskal - Wallis test with a Dunn ' s post hoc test for multiple comparisons . ns , not significant ; * * * * , p < 0 . 0001 . E ) MDA5 - knockout HEK293 cells , were transfected with an ADAR1 - targeting siRNA ( siADAR1 ) or a control siRNA ( siCtrl ) and 8h later with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of MDA5 . Cells were harvested 72h post - siRNA transfection and the type I IFN response was monitored by RT - qPCR analysis of IFIT1 and ISG15 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of two biological replicate experiments .

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

Figure 3A) ADAR1-knockout HEK293 cells (clone 1) were modified with a lentiviral-based inducible system to express FLAG-LGP2 WT or a FLAG-LGP2 RNA binding mutant (K138E/R490E/K634E, denoted as "KRK mutant") in a doxycycline-regulated manner. Cells were treated 72h with doxycycline (dox). Protein lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies (n=3). iEV = inducible empty vector; iLGP2 = inducible LGP2.C) Cells generated in (A) were plated on coverslips and treated with or without doxycycline for 72h. Cells were fixed, permeabilized and stained with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>500 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of two biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analysis was performed using a Kruskal-Wallis test with a Dunn's post hoc test for multiple comparisons. ns, not significant; ****, p<0.0001.E) MDA5-knockout HEK293 cells, were transfected with an ADAR1-targeting siRNA (siADAR1) or a control siRNA (siCtrl) and 8h later with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of MDA5. Cells were harvested 72h post-siRNA transfection and the type I IFN response was monitored by RT-qPCR analysis of IFIT1 and ISG15 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of two biological replicate experiments.


Figure 4B ) HEK293 WT or ADAR1 - knockout cells ( clone 1 ) were transiently transfected with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of LGP2 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of three biological replicate experiments . C ) Cells were treated as in ( B ) . Protein lysates were prepared and analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) . The dotted line indicates the juxtaposition of two non - adjacent lanes .

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

Figure 4B) HEK293 WT or ADAR1-knockout cells (clone 1) were transiently transfected with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of LGP2. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of three biological replicate experiments.C) Cells were treated as in (B). Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3). The dotted line indicates the juxtaposition of two non-adjacent lanes.


Figure 5A ) Kaplan - Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer ( BLCA ; n = 407 ) , breast cancer ( BRCA ; n = 1099 ) and sarcoma ( SARC ; n = 263 ) TCGA datasets . Median cut - offs were used for patient stratification and logrank test P values are shown . B ) CAL27 cells transduced with doxycycline - inducible shRNAs targeting ADAR1 or GFP ( negative control ) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 ( siLGP2 # 1 or # 2 ) or a control siRNA ( siCtrl ) . To determine cell confluency at 120h post - transfection , cells were fixed , stained with Crystal Violet , and imaged . Images of a representative experiment are shown ( n = 3 ) . C ) Crystal Violet was extracted from stained cells ( B ) and the dye intensity was quantified using a colorimetric assay ( OD590 ) . OD590 values of doxycycline - treated cells were normalized to the OD 590 values of untreated cells . Quantification of data from three independent experiments is shown as mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 .

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

Figure 5A) Kaplan-Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer (BLCA; n=407), breast cancer (BRCA; n=1099) and sarcoma (SARC; n=263) TCGA datasets. Median cut-offs were used for patient stratification and logrank test P values are shown.B) CAL27 cells transduced with doxycycline-inducible shRNAs targeting ADAR1 or GFP (negative control) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 (siLGP2 #1 or #2) or a control siRNA (siCtrl). To determine cell confluency at 120h post-transfection, cells were fixed, stained with Crystal Violet, and imaged. Images of a representative experiment are shown (n=3). C) Crystal Violet was extracted from stained cells (B) and the dye intensity was quantified using a colorimetric assay (OD590). OD590 values of doxycycline-treated cells were normalized to the OD590 values of untreated cells. Quantification of data from three independent experiments is shown as mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.


Figure 6A ) HT29 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed by RT - qPCR analysis of IFN - β , IFIT1 , and ISG15 transcript expression , normalized to ACTB . B ) LIM1215 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed as in ( A ) . C ) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days . Three days after treatment initiation , cells were transfected with the indicated siRNAs and cultured for an additional 72h . The type I IFN response was monitored as in ( A ) . Data information : Data from three biological independent experiments are shown with mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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Figure 6A) HT29 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed by RT-qPCR analysis of IFN-β, IFIT1, and ISG15 transcript expression, normalized to ACTB. B) LIM1215 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed as in (A). C) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days. Three days after treatment initiation, cells were transfected with the indicated siRNAs and cultured for an additional 72h. The type I IFN response was monitored as in (A). Data information: Data from three biological independent experiments are shown with mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


Figure 1Ribosome occupancy map at the LINC00665 locus . MCF10A were either mock - treated or treated with TGF - β and Ribosome profiling on three biological replicates were shown . Relative levels of CIP2A - BP were determined by polysome profiling and qRT - PCR in MCF - 10A and TNBC cells either mock treated or treated with TGF - β . Relative levels of LINC00665 were determined by qRT - PCR in MCF - 10A and TNBC cells either mock treated or treated with TGF - β . Upper : putative ORFs in LINC00665 . Lower : The ORFs were constructed into pcDNA3 . 1 vector and transfected to MCF - 10A cells for 24h . The ORFs - His fusion proteins were determined by western blot with anti - His antibody . The indicated constructs were transfected into MCF - 10A cells for 24h , then the GFP fluorescence was detected using fluorescence microscope ( F ) and fusion protein levels were determined by western blot with anti - GFP and anti - CIP2A - BP antibodies respectivelyDiagram of the His fusion constructs . The start codon ATG of the LINC00665 ORF is mutated to ATT . The indicated constructs were stably expressed in MDA - MB - 231 cells , micropeptide CIP2A - BP was immuno - stained using anti - His ( I ) and anti - CIP2A - BP antibodies ( K ) respectively ; CIP2A - BP - His fusion protein levels were determined by western blotting with anti - His and CIP2A - BP antibodiesMCF - 10A cells incubated in the absence or presence of puromycin ( 200μM ) were fractionated on sucrose gradients , and distribution of CIP2A - BP transcripts was quantified by qPCR .

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Figure 1Ribosome occupancy map at the LINC00665 locus. MCF10A were either mock-treated or treated with TGF-β and Ribosome profiling on three biological replicates were shown.Relative levels of CIP2A-BP were determined by polysome profiling and qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β. Relative levels of LINC00665 were determined by qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β.Upper: putative ORFs in LINC00665. Lower: The ORFs were constructed into pcDNA3.1 vector and transfected to MCF-10A cells for 24h. The ORFs-His fusion proteins were determined by western blot with anti-His antibody.The indicated constructs were transfected into MCF-10A cells for 24h, then the GFP fluorescence was detected using fluorescence microscope (F) and fusion protein levels were determined by western blot with anti-GFP and anti-CIP2A-BP antibodies respectivelyDiagram of the His fusion constructs. The start codon ATG of the LINC00665 ORF is mutated to ATT. The indicated constructs were stably expressed in MDA-MB-231 cells, micropeptide CIP2A-BP was immuno-stained using anti-His (I) and anti-CIP2A-BP antibodies (K) respectively; CIP2A-BP-His fusion protein levels were determined by western blotting with anti-His and CIP2A-BP antibodiesMCF-10A cells incubated in the absence or presence of puromycin (200μM) were fractionated on sucrose gradients, and distribution of CIP2A-BP transcripts was quantified by qPCR.


Figure 2Micropeptide CIP2A - BP was immune - stained with anti - CIP2A - BP antibody in MCF - 10A and MDA - MB - 231 cells either mock treated or treated with TGF - β . Micropeptide CIP2A - BP levels were determined by western blot with anti - CIP2A - BP antibody in MCF - 10A and TNBC cells either mock treated or treated with TGF - β . MCF - 10A cells were first transfected with anti - LINC00665 translation - blocking antisense oligo , then micropeptide CIP2A - BP was detected . Micropeptide CIP2A - BP levels were detected by western blot with anti - CIP2A - BP antibody in the indicated non - metastatic TNBC tissues ( non - metastasis ) and metastatic TNBC tissues ( metastasis ) . Micropeptide CIP2A - BP expression in MCF - 10A cells was used as the positive control . Kaplan - Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A - BP expression in Suzhou cohort ( discovery set , n = 112 ) and Guangzhou cohort ( validation set , n = 105 ) .

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Figure 2Micropeptide CIP2A-BP was immune-stained with anti-CIP2A-BP antibody in MCF-10A and MDA-MB-231 cells either mock treated or treated with TGF-β.Micropeptide CIP2A-BP levels were determined by western blot with anti-CIP2A-BP antibody in MCF-10A and TNBC cells either mock treated or treated with TGF-β.MCF-10A cells were first transfected with anti-LINC00665 translation-blocking antisense oligo, then micropeptide CIP2A-BP was detected.Micropeptide CIP2A-BP levels were detected by western blot with anti-CIP2A-BP antibody in the indicated non-metastatic TNBC tissues (non-metastasis) and metastatic TNBC tissues (metastasis). Micropeptide CIP2A-BP expression in MCF-10A cells was used as the positive control.Kaplan-Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A-BP expression in Suzhou cohort (discovery set, n = 112) and Guangzhou cohort (validation set, n = 105).


Figure 3Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h . Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel ( upper ) and invaded through Matrigel - coated membrane ( lower ) . Migration and invasion of wild - type and CIP2A - BP knockout TNBC cells and CIP2A - BP re - expressed Hs578TKO and MDA - MB - 231KO cells . Right panel is the quantification of cells that migrated through the membrane without the Matrigel ( upper ) and invaded through Matrigel - coated membrane ( lower ) . Wound - healing assay on Hs578T and MDA - MB - 231 cells transfected with LINC00665 ORF expressing and control lentiviruses . The wound edges were photographed at the indicated time points after wounding . Right panel is the quantification of the relative wound healing area . Wild - type and CIP2A - BP knockout TNBC cells and CIP2A - BP re - expressed Hs578TKO and MDA - MB - 231KO were seeded on cell culture inserts for wound - healing assay . The wound edges were photographed at the indicated time points after wounding . Right panel is the quantification of the relative wound healing area . Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T ( G ) and MDA - MB - 231 cells ( H ) ( 1 . 0 × 105 - 106 cells / mouse ) via tail vein ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

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Figure 3Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h. Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).Migration and invasion of wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO cells. Right panel is the quantification of cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).Wound-healing assay on Hs578T and MDA-MB-231 cells transfected with LINC00665 ORF expressing and control lentiviruses. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.Wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO were seeded on cell culture inserts for wound-healing assay. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T (G) and MDA-MB-231 cells (H) (1.0×105-106 cells/mouse) via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Figure 4Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF - β . Chromatin immunoprecipitation showing Smad4 occupancy at the 4E - BP1 locus in TNBC cells . Co - precipitated DNA was analyzed for amplicons A - E by qPCR . Values represent the enrichment of bound protein fractions relative to input . Luciferase reporter assay was performed in Hs578T ( F ) and MDA - MB - 231 ( G ) cells following co - transfection with wildtype or mutant 4E - BP1 promoter fragment and Smad4 over - expressed , or knocked down and respective controls for 36h and treatment with TGF - β for 24h . The reporter constructs were expressing the luciferase gene under 4E - BP1 promoter segment or 4E - BP1 promoter deleted 0 to + 200 region . Regulation of 4E - BP1 expression and binding to eIF4E by TGF - β . TNBC cells were treated with TGF - β and equal amounts of proteins were subjected to immunoprecipitation and / or examined by immunoblotting analysis . Immunoblotting analysis of expression of micropeptide CIP2A - BP and β - actin in TNBC cells cultured with or without TGF - β . Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF - β . The indicated proteins were determined by Immunoblotting analysis . TNBC cells were pretreated with specific antagonist against Smad3 ( SIS3 , 3μM ) for 1h and then cultured with or without TGF - β . The indicated proteins were determined by immunoblotting analysis . 4E - BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF - β . The indicated proteins were determined by immunoblotting analysis .

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Figure 4Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF-β.Chromatin immunoprecipitation showing Smad4 occupancy at the 4E-BP1 locus in TNBC cells. Co-precipitated DNA was analyzed for amplicons A-E by qPCR. Values represent the enrichment of bound protein fractions relative to input.Luciferase reporter assay was performed in Hs578T (F) and MDA-MB-231 (G) cells following co-transfection with wildtype or mutant 4E-BP1 promoter fragment and Smad4 over-expressed, or knocked down and respective controls for 36h and treatment with TGF-β for 24h. The reporter constructs were expressing the luciferase gene under 4E-BP1 promoter segment or 4E-BP1 promoter deleted 0 to +200 region.Regulation of 4E-BP1 expression and binding to eIF4E by TGF-β. TNBC cells were treated with TGF-β and equal amounts of proteins were subjected to immunoprecipitation and/or examined by immunoblotting analysis.Immunoblotting analysis of expression of micropeptide CIP2A-BP and β-actin in TNBC cells cultured with or without TGF-β.Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by Immunoblotting analysis.TNBC cells were pretreated with specific antagonist against Smad3 (SIS3, 3μM) for 1h and then cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.4E-BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.


Figure 5LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were subjected to co - immunoprecipitation using anti - His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A - BP . LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were subjected to co - immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A - BP antibodies to verify that CIP2A - BP bound to CIP2A . Endogenous CIP2A - BP and CIP2A were co - immunoprecipitated in MCF - 10A cell line . LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were treated with 30 mg / mL RNase A for 30 min , the indicated complexes were co - immunoprecipitated by anti - His or anti - CIP2A antibody , and CIP2A or micropeptide CIP2A - BP were detected . Upper : A diagram of the domain structure of full length CIP2A protein . Red : RIP = Required for Interaction with PPP2R5C ; Orange : RH = Required for Homodimerization ; Blue , CC = Coiled coil . Lower : Co - immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A - BP with CIP2A via the N - terminus domain of CIP2A . Upper : A diagram of the domain structure of CIP2A protein fragment ( 1 - 245 ) . Red : RIP = Required for Interaction with PPP2R5C . Lower : Co - immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A - BP with CIP2A via the N - terminus domain of CIP2A ( amino acid 159 - 245 ) . Co - immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A - BP expression .

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Figure 5LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP.LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A-BP antibodies to verify that CIP2A-BP bound to CIP2A.Endogenous CIP2A-BP and CIP2A were co-immunoprecipitated in MCF-10A cell line.LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were treated with 30 mg/mL RNase A for 30 min, the indicated complexes were co-immunoprecipitated by anti-His or anti-CIP2A antibody, and CIP2A or micropeptide CIP2A-BP were detected.Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A. Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245).Co-immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A-BP expression.