Figure 6Profiles showing average Ca2 + influx induced by Thapsigargin ( TG ) in freshly sorted NKT1 ( Red ) and NKT2 ( Black ) cells . The timing of TG ( 1uM ) , ionomycin ( Iono ) ( 2uM ) and CCCP ( 1uM ) addition , and extracellular [ Ca2 + ] in mM are shown .

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

Figure 6Profiles showing average Ca2+ influx induced by Thapsigargin (TG) in freshly sorted NKT1 (Red) and NKT2 (Black) cells. The timing of TG (1uM), ionomycin (Iono) (2uM) and CCCP (1uM) addition, and extracellular [Ca2+] in mM are shown.


Figure 1 ( B ) Bar graph summarizing the efficiency of single RNAi knockdowns for 28 out of the 30 TRs , and their effects in the reprogramming efficiency in comparison to control cells expressing scramble shRNA . The efficiency of each TR ' s knockdown ( KD ) does not correlate with its effect in reprogramming efficiency . The knockdown of 9 ( Class III ) out of the 28 TRs decreased the efficiency of the reprogramming in a statistically significant manner ( unpaired two - tailed Student ' s t - test , p - value < 0 . 05 ) . Data are shown as mean ± SEM of at least two independent experiments . Class I corresponds to one TR inhibiting reprogramming , whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming . Shown are the effects of 28 out of 30 TRs , because we did not succeed in knocking down Cphx and Phox2a . ( C ) Left : Shown is a line graph depicting normalized mRNA expression levels of the 9 TRs at the indicated time points during the reprogramming of MEFs . The data were plotted after normalization with the endogenous Gapdh using the " ΔCt method " . The grey vertical dashed line depicts day 6 in which the expression levels of nearly all TRs peaks . Notice that the y - axis is broken to accommodate the large spread of expression levels . Right : Shown are western blots using antibodies specific for Irf6 , Tead4 , Taf1c , Rcan1 and Tfap4 . Whole cell extracts were prepared from MEFs at day 6 of reprogramming and run side - by - side with extracts from control samples ( day 0 ) . Gapdh , mTOR and b - Tubulin were used as loading controls . ( D ) Same as in ( B ) except MEFs were co - transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs . Left : Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency ( RE % ) . Shown are the average REs values from at least two independent experiments . The REs of the double KDs were evaluated against the RE of the corresponding single KDs . Right : Alkaline Phosphatase ( AP ) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA - based screens .

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 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 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

Figure 1(B) Bar graph summarizing the efficiency of single RNAi knockdowns for 28 out of the 30 TRs, and their effects in the reprogramming efficiency in comparison to control cells expressing scramble shRNA. The efficiency of each TR's knockdown (KD) does not correlate with its effect in reprogramming efficiency. The knockdown of 9 (Class III) out of the 28 TRs decreased the efficiency of the reprogramming in a statistically significant manner (unpaired two-tailed Student's t-test, p-value <0.05). Data are shown as mean ± SEM of at least two independent experiments. Class I corresponds to one TR inhibiting reprogramming, whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming. Shown are the effects of 28 out of 30 TRs, because we did not succeed in knocking down Cphx and Phox2a.(C) Left: Shown is a line graph depicting normalized mRNA expression levels of the 9 TRs at the indicated time points during the reprogramming of MEFs. The data were plotted after normalization with the endogenous Gapdh using the "ΔCt method". The grey vertical dashed line depicts day 6 in which the expression levels of nearly all TRs peaks. Notice that the y-axis is broken to accommodate the large spread of expression levels. Right: Shown are western blots using antibodies specific for Irf6, Tead4, Taf1c, Rcan1 and Tfap4. Whole cell extracts were prepared from MEFs at day 6 of reprogramming and run side-by-side with extracts from control samples (day 0). Gapdh, mTOR and b-Tubulin were used as loading controls.(D) Same as in (B) except MEFs were co-transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs. Left: Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency (RE%). Shown are the average REs values from at least two independent experiments. The REs of the double KDs were evaluated against the RE of the corresponding single KDs. Right: Alkaline Phosphatase (AP) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA-based screens.


Figure 2 ( A ) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming . The boundaries of the early - iPSCs formations are indicated by a blue dashed line . Scale bars : 100 μm . ( C ) Bar graph showing the percentage of cells isolated from early - iPSCs colonies or outside of the colonies ( rest of population ) expressing each of the 9 TRs . Data are shown as mean ± SEM of at least two independent experiments . 0 . 05 < ( * ) p < 0 . 1 , * p < 0 . 05 , * * p < 0 . 01 by unpaired Student ' s t - test or Welch ' s t - test , when appropriate . ( D ) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs . Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments . Data are shown as mean ± SEM of at least two independent experiments . Anova test p < 0 . 0001 . ( E ) Schematic representation of the statistical analysis estimating the probability of co - expression of the nine TRs per cell within the early - iPSCs colonies . Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs ( shown with different color ) in 282 cells ( right side legend ) . The co - expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure . The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells , while the right part depicts the co - expression of combinations of the nine TRs based on double and triple single - cell experiments .

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

Figure 2(A) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming. The boundaries of the early-iPSCs formations are indicated by a blue dashed line. Scale bars: 100 μm.(C) Bar graph showing the percentage of cells isolated from early-iPSCs colonies or outside of the colonies (rest of population) expressing each of the 9 TRs. Data are shown as mean ± SEM of at least two independent experiments. 0.05 < (*)p < 0.1, *p < 0.05, **p < 0.01 by unpaired Student's t-test or Welch's t-test, when appropriate.(D) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs. Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments. Data are shown as mean ± SEM of at least two independent experiments. Anova test p<0.0001.(E) Schematic representation of the statistical analysis estimating the probability of co-expression of the nine TRs per cell within the early-iPSCs colonies. Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs (shown with different color) in 282 cells (right side legend). The co-expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure. The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells, while the right part depicts the co-expression of combinations of the nine TRs based on double and triple single-cell experiments.


Figure 3 ( A - I ) Shown are ChIP - seq big wig files in the IGV browser depicting the binding of Oct4 ( Red ) , Sox2 ( Blue ) , Klf4 ( Green ) and c - Myc ( Black ) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming ( 18 hrs , D3 and D5 ) and in control MEFs , mESCs and miPSCs . All peaks have been normalized against input DNA . The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated . The side color bar depicts the expression levels of each TR at the indicated time points .

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

Figure 3(A-I) Shown are ChIP-seq big wig files in the IGV browser depicting the binding of Oct4 (Red), Sox2 (Blue), Klf4 (Green) and c-Myc (Black) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming (18 hrs, D3 and D5) and in control MEFs, mESCs and miPSCs. All peaks have been normalized against input DNA. The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated. The side color bar depicts the expression levels of each TR at the indicated time points.


Figure 4 ( C ) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6 ; The white color denotes no change in expression levels . ( D ) Scatter plots depicting the extent of co - expression of either the broadly - or early - iPSCs - specifically expressed TRs in individual cells obtained from the early - iPSCs colonies or from the rest of the population ( left and right panel respectively ) . Each dot depicts the percentage of cells co - expressing a unique combination of two or three TRs . Data are shown as mean ± SEM of at least two independent experiments . ( E ) Heatmap summarizing the most striking effects of double nine TRs KDs on the expression of the other TRs and on the expression of the Epcam and Lin28a pluripotency markers ; the white color indicates no change in expression levels . The genes examined have been ranked in the heatmap according to their order of upregulation during reprogramming ; Lin28a is the latest upregulated gene .

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 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 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

Figure 4(C) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6; The white color denotes no change in expression levels.(D) Scatter plots depicting the extent of co-expression of either the broadly- or early-iPSCs-specifically expressed TRs in individual cells obtained from the early-iPSCs colonies or from the rest of the population (left and right panel respectively). Each dot depicts the percentage of cells co-expressing a unique combination of two or three TRs. Data are shown as mean ± SEM of at least two independent experiments.(E) Heatmap summarizing the most striking effects of double nine TRs KDs on the expression of the other TRs and on the expression of the Epcam and Lin28a pluripotency markers; the white color indicates no change in expression levels. The genes examined have been ranked in the heatmap according to their order of upregulation during reprogramming; Lin28a is the latest upregulated gene.


Figure 5 ( B ) Shown is a bar graph depicting the effect of overexpression of the indicated TRs in reprogramming efficiency . Data are shown as mean ± SEM of at least two independent experiments . The y axis labeled as % reprogramming efficiency refers to the AP + colonies scored as iPSCs . ( C ) Shown is a bar graph depicting the effects of rescue experiments in reprogramming efficiency . MEFs undergoing reprogramming were knocked down using shRNAs for the endogenous Nanog or Irf6 expression and were co - infected with a vector expressing an shRNA - resistant human homologue of Nanog or Irf6 ( hNanog OE , hIrf6 OE ) . Data are shown as mean ± SEM of two independent experiments .

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 O O O O O O O O O

Figure 5(B) Shown is a bar graph depicting the effect of overexpression of the indicated TRs in reprogramming efficiency. Data are shown as mean ± SEM of at least two independent experiments. The y axis labeled as % reprogramming efficiency refers to the AP+ colonies scored as iPSCs.(C) Shown is a bar graph depicting the effects of rescue experiments in reprogramming efficiency. MEFs undergoing reprogramming were knocked down using shRNAs for the endogenous Nanog or Irf6 expression and were co-infected with a vector expressing an shRNA-resistant human homologue of Nanog or Irf6 (hNanog OE, hIrf6 OE). Data are shown as mean ± SEM of two independent experiments.


Figure 1 ( A ) Protein abundance is the normalized signal intensity ( LFQ ) for a protein divided by its molecular weight . Specificity ( enrichment ) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples . The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes . LFQ , Label - Free - Quantification ; M . W . , molecular weight . ( B ) Flp - In T - REx 293 cells with induced MIA40FLAG expression were solubilized , and the affinity purification of MIA40FLAG was performed . Fractions were analyzed by SDS - PAGE and Western blot . Load : 2 . 5 % . Eluate : 100 % . Unbound : 2 . 5 % . ( C ) Flp - In T - REx 293 cells with induced MIA40FLAG expression were solubilized , and the affinity purification of MIA40FLAG was performed . Eluate fractions were solubilized under reducing ( DTT ) or non - reducing ( IAA ) conditions and analyzed by SDS - PAGE and Western blot . DTT , dithiothreitol ; IAA , iodoacetamide . Eluate : 100 % . ( D ) Cellular protein extracts from Flp - In T - REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification . Load and eluate fractions were analyzed by reducing SDS - PAGE and Western blot . Load : 2 . 5 % . Eluate : 100 % .

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 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 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

Figure 1(A) Protein abundance is the normalized signal intensity (LFQ) for a protein divided by its molecular weight. Specificity (enrichment) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples. The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes. LFQ, Label-Free-Quantification; M.W., molecular weight.(B) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Fractions were analyzed by SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %. Unbound: 2.5 %.(C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %.(D) Cellular protein extracts from Flp-In T-REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification. Load and eluate fractions were analyzed by reducing SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %.


Figure 2 ( A ) Schematic representation of the thiol trapping assay . Mitochondria were solubilized in sample buffer with either dithiothreitol ( DTT ) , iodoacetamide ( IAA ) , or 4 - acetamido - 4 - maleimidylstilbene - 2 , 2 - disulfonic acid ( AMS ) . The samples were analyzed by SDS - PAGE and Western blot . ( B ) Indirect thiol trapping assay . Mitochondria were pretreated with IAA as indicated to block free cysteine residues , and disulfide bonds were subsequently reduced by DTT . Mitochondria were solubilized in sample buffer with AMS . ( C ) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria ( 250 mM sucrose ) , mitoplasts ( 5 mM sucrose ) , and mitochondrial lysates ( 1 % Triton X - 100 ) . The samples were analyzed by SDS - PAGE and Western blot . Mitos , mitochondria ; Sup , post - mitochondria supernatant ; OM , outer membrane ; IM , inner membrane ; IMS , intermembrane space . ( D ) N - SIM super - resolution micrographs of one Z - stack ( 0 . 15 μm ) orthogonal section ( XYZ ) of HeLa cells or HeLa cells that stably expressed COA7 - HA transfected with different markers TOMM20 - DsRed ( OM ) , COX8A - DsRed ( IM ) , and matrix targeted photoactivatable GFP ( Matrix target ) labeled with anti - HA , anti - COA7 and Smac / Diablo ( IMS ) antibodies . The picture represents the majority population of cells from three independent experiments . Scale bar = 2 μm , scale bar in the magnified insert = 0 . 5 μm . The panel shows Pearson ' s coefficient in a co - localized volume of different subcompartment combinations with anti - HA or anti - COA7 . The data are expressed as a mean ± SD ( n = 5 ) . * * * p < 0 . 001 [ IMSxEnd vs . IMxEnd p = 0 . 0001 ; IMSxEnd vs . MxEnd p = 0 . 0002 ] , * * * * p < 0 . 0001 [ OMxHA vs . IMSxHA p < 0 . 0001 ; IMSxHA vs . IMxHA p < 0 . 0001 ] ( one - way ANOVA ) . M , matrix ; End , endogenous COA7 ; HA , COA7 - HA .

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

Figure 2(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot.(B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS.(C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space.(D) N-SIM super-resolution micrographs of one Z-stack (0.15 μm) orthogonal section (XYZ) of HeLa cells or HeLa cells that stably expressed COA7-HA transfected with different markers TOMM20-DsRed (OM), COX8A-DsRed (IM), and matrix targeted photoactivatable GFP (Matrix target) labeled with anti-HA, anti-COA7 and Smac/Diablo (IMS) antibodies. The picture represents the majority population of cells from three independent experiments. Scale bar = 2 μm, scale bar in the magnified insert = 0.5 μm. The panel shows Pearson's coefficient in a co-localized volume of different subcompartment combinations with anti-HA or anti-COA7. The data are expressed as a mean ± SD (n=5). ***p<0.001 [IMSxEnd vs. IMxEnd p=0.0001; IMSxEnd vs. MxEnd p=0.0002], ****p<0.0001 [OMxHA vs. IMSxHA p<0.0001; IMSxHA vs. IMxHA p<0.0001] (one-way ANOVA). M, matrix; End, endogenous COA7; HA, COA7-HA.


Figure 3 ( A ) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector . Mitochondria were solubilized and analyzed by reducing SDS - PAGE and Western blot . Mitos , mitochondria . ( B ) Radiolabeled [ 35S ] TIMM8A and [ 35S ] COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 30 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide . ( C ) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing ( DTT ) and non - reducing ( IAA ) conditions and analyzed for levels of MIA40 by Western blot . ( D ) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG - PCMal . Control cells were pretreated with the thiol oxidizing agent diamide ( DAM ) or the reductant DTT . The samples were analyzed by SDS - PAGE and Western blot . ( E ) Flp - In T - REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector . The affinity purification of MIA40FLAG was performed , and eluate fractions were analyzed by SDS - PAGE and Western blot .

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 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 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 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 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

Figure 3(A) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. Mitochondria were solubilized and analyzed by reducing SDS-PAGE and Western blot. Mitos, mitochondria.(B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.(C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot.(D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot.(E) Flp-In T-REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector. The affinity purification of MIA40FLAG was performed, and eluate fractions were analyzed by SDS-PAGE and Western blot.


Figure 4Radiolabeled [ 35S ] COA7 ( A ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express MIA40FLAG . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide . Radiolabeled [ 35S ] TIMM8A ( B ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express MIA40FLAG . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide . ( C ) Mitochondria were isolated from Flp - In T - REx 293 cells induced to express MIA40FLAG and control cells . The samples were analyzed by SDS - PAGE and Western blot . Mitos , mitochondria . Radiolabeled [ 35S ] COA7 ( D ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express ALRFLAG and control cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological experiments were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . Radiolabeled [ 35S ] TIMM8A ( E ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express ALRFLAG and control cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological experiments were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . ( F ) Mitochondria were isolated from Flp - In T - REx 293 cells induced to express ALRFLAG and control cells . The samples were analyzed by SDS - PAGE and Western blot .

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 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 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 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 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 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

Figure 4Radiolabeled [35S]COA7 (A) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.Radiolabeled [ 35S]TIMM8A (B) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.(C) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria.Radiolabeled [35S] COA7 (D) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).Radiolabeled [35S] TIMM8A (E) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).(F) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot.


Figure 5 ( A ) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides . The samples were analyzed by reducing SDS - PAGE and Western blot . ( B ) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides . The samples were analyzed by reducing SDS - PAGE and Western blot . ( D ) Cellular protein extracts were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells . The samples were analyzed by SDS - PAGE and Western blot . ( E ) Cellular protein extracts were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells transfected with a plasmid that encoded MIA40 or an empty vector . The samples were subjected to reducing SDS - PAGE and Western blot . Radiolabeled [ 35S ] COA7 ( F ) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide . Radiolabeled [ [ 35S ] TIMM8A ( G ) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

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 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 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 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 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 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

Figure 5(A) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.(B) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.(D) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by SDS-PAGE and Western blot.(E) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells transfected with a plasmid that encoded MIA40 or an empty vector. The samples were subjected to reducing SDS-PAGE and Western blot.Radiolabeled [35S]COA7 (F) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.Radiolabeled [ [35S]TIMM8A (G) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


Figure 6 ( B ) Cellular protein extracts were isolated from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7 . The samples were analyzed by reducing SDS - PAGE and Western blot . ( C ) Cellular fractions were prepared from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7 . The fractions were analyzed by reducing SDS - PAGE and Western blot . T , total ; C , cytosol ; M , mitochondria . ( D ) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were solubilized , and the affinity purification of COA7HIS was performed . The samples were analyzed by reducing and non - reducing SDS - PAGE and Western blot . Load : 3 % . Eluate : 100 % . ( E ) Equal amounts of wildtype and mutant radiolabeled [ 35S ] COA7 precursors were imported into mitochondria isolated from HEK293 cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to wildtype COA7 at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

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

Figure 6(B) Cellular protein extracts were isolated from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The samples were analyzed by reducing SDS-PAGE and Western blot.(C) Cellular fractions were prepared from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The fractions were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were solubilized, and the affinity purification of COA7HIS was performed. The samples were analyzed by reducing and non-reducing SDS-PAGE and Western blot. Load: 3 %. Eluate: 100 %.(E) Equal amounts of wildtype and mutant radiolabeled [35S]COA7 precursors were imported into mitochondria isolated from HEK293 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to wildtype COA7 at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


Figure 7 ( B ) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times , and protein extracts were isolated . The samples were analyzed by reducing SDS - PAGE and Western blot . ( D ) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and / or MG132 for the indicated times , and protein extracts were isolated . The samples were analyzed by reducing SDS - PAGE and Western blot . CHX , cycloheximide . ( F ) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132 . The samples were analyzed by reducing SDS - PAGE and Western blot .

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

Figure 7(B) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot.(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and/or MG132 for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot. CHX, cycloheximide.(F) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132. The samples were analyzed by reducing SDS-PAGE and Western blot.


Figure 8 ( A ) Cellular protein extracts were isolated from immortalized patient - derived skin fibroblasts that were treated with the indicated concentrations of MG132 , bortezomib , or carfilzomib . The samples were analyzed by reducing SDS - PAGE and Western blot . ( B ) Cellular fractions were prepared from immortalized patient - derived skin fibroblasts that were treated with the indicated concentrations of MG132 , bortezomib , or carfilzomib . The samples were analyzed by reducing SDS - PAGE and Western blot . T , total ; C , cytosol ; M , mitochondria .

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 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

Figure 8(A) Cellular protein extracts were isolated from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot.(B) Cellular fractions were prepared from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.


Figure 9 ( A ) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient - derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified , and normalized to control fibroblasts . Data are expressed as a mean ± SEM ( n = 3 , * p = 0 . 01 ) . ( B ) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient - derived skin fibroblasts treated with MG132 ( 1 μM ) , bortezomib ( 20 nM ) , or carfilzomib ( 50 nM ) for 12 h and recovered for another 6 h . The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM ( n = 3 , * p = 0 . 04 [ DMSO vs MG132 ] , * * p = 0 . 0025 [ DMSO vs Bortezomib ] , * p = 0 . 02 [ DMSO vs Carfilzomib ] ) . ( C ) Immortalized patient - derived skin fibroblasts transiently expressing wildtype or mutant COA7 ( COA7 - Y137C and COA7 - Ex2 ∆ ) for 48 h were treated with bortezomib ( 20 nM ) during the last 12 h . The fibroblasts were harvested , and complex IV activity was measured in digitonized cellular extracts . The results of three biological replicates were quantified and normalized to empty vector - bortezomib treated samples and the data are expressed as a mean ± SEM ( n = 3 , * p = 0 . 01 [ Empty vector vs COA7 - Wt ] , * p = 0 . 005 [ Empty vector vs COA7 - Y137C ] ) . ( D ) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4 - 13 % gel BN - PAGE and Western blot . SC , supercomplexes . ( E ) Immortalized patient - derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7 - Y137CHIS were solubilized in DDM buffer and analyzed by 4 - 13 % gel BN - PAGE and Western blot . ( F ) Mitochondria were isolated from immortalized patient - derived skin fibroblasts treated with bortezomib ( 10 nM ) for 12 h and recovered for another 6 h . Mitochondria were solubilized in digitonin buffer and analysed by 4 - 13 % gel BN - PAGE and Western blot . SC , supercomplexes .

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

Figure 9(A) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient-derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified, and normalized to control fibroblasts. Data are expressed as a mean ± SEM (n=3, *p=0.01).(B) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient-derived skin fibroblasts treated with MG132 (1 μM), bortezomib (20 nM), or carfilzomib (50 nM) for 12 h and recovered for another 6 h. The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.04 [DMSO vs MG132], **p=0.0025 [DMSO vs Bortezomib], *p=0.02 [DMSO vs Carfilzomib]).(C) Immortalized patient-derived skin fibroblasts transiently expressing wildtype or mutant COA7 (COA7-Y137C and COA7-Ex2∆) for 48 h were treated with bortezomib (20 nM) during the last 12 h. The fibroblasts were harvested, and complex IV activity was measured in digitonized cellular extracts. The results of three biological replicates were quantified and normalized to empty vector-bortezomib treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.01 [Empty vector vs COA7- Wt], *p=0.005 [Empty vector vs COA7 - Y137C]).(D) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.(E) Immortalized patient-derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7-Y137CHIS were solubilized in DDM buffer and analyzed by 4-13 % gel BN-PAGE and Western blot.(F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.


Fig . 1 : Three - dimensional structure of 2019 - nCoV Mpro , in two different views . One protomer of the dimer is shown in light blue , the other one in orange . Amino - acid residues of the catalytic site are indicated as yellow and blue spheres , for Cys145 and His41 , respectively . Black spheres indicate the positions of Ala285 of each of the two domains III ( see text ) . Chain termini are labeled N and C for molecule A ( light blue ) and N * and C * for molecule B ( orange ) .

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

Fig. 1: Three-dimensional structure of 2019-nCoV Mpro, in two different views. One protomer of the dimer is shown in light blue, the other one in orange. Amino-acid residues of the catalytic site are indicated as yellow and blue spheres, for Cys145 and His41, respectively. Black spheres indicate the positions of Ala285 of each of the two domains III (see text). Chain termini are labeled N and C for molecule A (light blue) and N* and C* for molecule B (orange).


Fig . 2 : Compound 13b in the substrate - binding cleft located between domains I and II of the Mpro , in the monoclinic crystal form ( space group C2 ) . 2Fo - Fc electron density is shown for the inhibitor ( contouring level : 1σ ) . Carbon atoms of the inhibitor are magenta , oxygens red , nitrogens blue , and sulfur yellow . Note the interaction between the N - terminal residue of chain B , S1 * , and E166 of chain A .

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

Fig. 2: Compound 13b in the substrate-binding cleft located between domains I and II of the Mpro, in the monoclinic crystal form (space group C2). 2Fo-Fc electron density is shown for the inhibitor (contouring level: 1σ). Carbon atoms of the inhibitor are magenta, oxygens red, nitrogens blue, and sulfur yellow. Note the interaction between the N-terminal residue of chain B, S1*, and E166 of chain A.


Figure 1 ( A ) Optically evoked auditory brainstem response in an AAV - CatCh - injected gerbil in response to a 1 ms laser pulse of ~ 35 mW ( mean of 1000 stimulus presentations at 10 Hz repetition rate ) . ( B - D ) Catch - eYFP - expressing SGNs in the apical cochlear turn identified by parvalbumin expression ( B ) and CatCh - eYFP ( C ) . The inlay ( D ´ ) of the merged immunostaining ( D ) indicates the lack of CatCh - EYFP signal in inner hair cells ( dashed white line ) . Scale bar : 20 µm . ( I ) oCI inserted into the cochlea ( dashed black lines ) via a cochleostomy in the basal cochlear turn ( black , solid line ) . The round window niche is highlighted by a dashed white line , the round window by a solid white line . SA : stapedial artery . ( J ) 3D X - ray tomography reconstruction of a 16 - channel oCI implanted in a gerbil cochlea via the round window . Cables and μLEDs are marked in blue , the most apical µLED is indicated by the black arrowhead ; the basilar membrane is marked in green . µLEDs have a size of 60 × 60 µm .

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

Figure 1(A) Optically evoked auditory brainstem response in an AAV-CatCh-injected gerbil in response to a 1 ms laser pulse of ~35 mW (mean of 1000 stimulus presentations at 10 Hz repetition rate). (B-D) Catch-eYFP-expressing SGNs in the apical cochlear turn identified by parvalbumin expression (B) and CatCh-eYFP (C). The inlay (D´) of the merged immunostaining (D) indicates the lack of CatCh-EYFP signal in inner hair cells (dashed white line). Scale bar: 20 µm.  (I) oCI inserted into the cochlea (dashed black lines) via a cochleostomy in the basal cochlear turn (black, solid line). The round window niche is highlighted by a dashed white line, the round window by a solid white line. SA: stapedial artery.  (J) 3D X-ray tomography reconstruction of a 16-channel oCI implanted in a gerbil cochlea via the round window. Cables and μLEDs are marked in blue, the most apical µLED is indicated by the black arrowhead; the basilar membrane is marked in green. µLEDs have a size of 60×60 µm.


Figure 2 ( A - D ) Exemplar STCs in response to SGN illumination with a single μLED ( A ) , a block of four neighboring μLEDs ( B ) and all μLEDs of an oCI inserted via the round window in a CatCh - transduced gerbil ( C ) and with all µLEDs in a wildtype gerbil ( WT ; D ) . μLEDs were spaced by 250 μm . ( E ) Acoustic auditory brainstem responses ( average of 1000 stimuli at a stimulation rate of 10 Hz ) upon click stimulation from 20 - 100 dB SPL before ( left ) and after deafening ( right ; star : threshold ) . ( F ) Optically evoked auditory brainstem responses before ( left ) and after deafening ( right ) , elicited by ~ 35 mW pulses of 1 ms duration , delivered by a laser - coupled fiber ( 1000 stimuli presented at 10 Hz ) , recorded from the same animal . Optogenetic activation of the auditory system was still possible after deafening . Differences in amplitude and waveform before and after deafening are likely due to different positioning of recording electrodes and the optical fiber . ( G - H ) Exemplar STCs in response to SGN illumination with all µLEDs in a CatCh - transduced , kanamycin - deafened gerbil before ( G ) and after ( H ) sacrifice . ( I ) Maximum strength of ICC responses ( mean ± standard deviation ) evoked by oCI stimulation with 1 ( indiv . ; n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( block ; n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , and 16 ( all ; n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) active µLEDs , and with a laser - coupled optical fiber ( ~ 35 mW ; n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 73 . 25 , 4 degrees of freedom , p = 5 . 19 * 10 - 45 ) and post - hoc multiple comparison test ; indiv . vs . block : p = 9 . 9 * 10 - 9 ; block vs . all : p = 0 . 0096 ; all vs . fiber : p = 0 . 0016 ; all CatCh vs . all WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . oCI stimulation with all µLEDs has also been performed in WT animals ( n = 7 / 7 oCIs , N = 2 / 2 hearing / deaf gerbils ) and in sacrificed CatCh - transduced animals ( post - mortem ; n = 5 / 1 oCIs in N = 5 / 1 hearing / deaf gerbils ) . Brown dots indicate data recorded from deafened animals . ( J ) Number of active electrodes ( d ´ > 1 ; mean ± standard deviation ) upon oCI stimulation with 1 ( n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , and 16 µLEDs ( n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) , and with optical fiber stimulation ( n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 30 . 18 , 4 degrees of freedom , p = 1 . 42 * 10 - 21 ) and post - hoc multiple comparison test ; indiv . vs . block : p = 6 . 8 * 10 - 5 ; block vs . all : p = 0 . 006 ; all vs . fiber : p = 0 . 25 ; all CatCh vs . all WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . Responses to oCI stimulation with all µLEDs were also observed in hearing WT animals ( n = 3 oCIs in 2 gerbils ) . No active electrodes were found in response to 4 / 7 oCI stimulations in 2 / 2 hearing / deaf wildtype gerbils ) . ( K ) Thresholds ( d ´ = 1 , measured at the best electrode ( BE ) ) for ICC activation evoked with 16 active µLEDs on an oCI ( n = 24 / 9 in N = 9 / 3 hearing / deaf gerbils ) and with a laser - coupled optical fiber ( n = 9 / 3 in N = 9 / 3 hearing / deaf gerbils ; p = 0 . 012 , Wilcoxon rank sum test ) . Central horizontal line indicates the median , lower , upper horizontal lines indicate the minimum and maximum values , and box edges indicate the 25th and 75th percentile , respectively .

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

Figure 2 (A-D) Exemplar STCs in response to SGN illumination with a single μLED (A), a block of four neighboring μLEDs (B) and all μLEDs of an oCI inserted via the round window in a CatCh-transduced gerbil (C) and with all µLEDs in a wildtype gerbil (WT; D). μLEDs were spaced by 250 μm.  (E) Acoustic auditory brainstem responses (average of 1000 stimuli at a stimulation rate of 10 Hz) upon click stimulation from 20-100 dB SPL before (left) and after deafening (right; star: threshold). (F) Optically evoked auditory brainstem responses before (left) and after deafening (right), elicited by ~35 mW pulses of 1 ms duration, delivered by a laser-coupled fiber (1000 stimuli presented at 10 Hz), recorded from the same animal. Optogenetic activation of the auditory system was still possible after deafening. Differences in amplitude and waveform before and after deafening are likely due to different positioning of recording electrodes and the optical fiber.  (G-H) Exemplar STCs in response to SGN illumination with all µLEDs in a CatCh-transduced, kanamycin-deafened gerbil before (G) and after (H) sacrifice.  (I) Maximum strength of ICC responses (mean ± standard deviation) evoked by oCI stimulation with 1 (indiv.; n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (block; n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 (all; n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils) active µLEDs, and with a laser-coupled optical fiber (~35 mW; n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 73.25, 4 degrees of freedom, p = 5.19*10-45) and post-hoc multiple comparison test; indiv. vs. block: p = 9.9*10-9; block vs. all: p = 0.0096; all vs. fiber: p = 0.0016; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). oCI stimulation with all µLEDs has also been performed in WT animals (n = 7/7 oCIs, N = 2/2 hearing/deaf gerbils) and in sacrificed CatCh-transduced animals (post-mortem; n = 5/1 oCIs in N = 5/1 hearing/deaf gerbils). Brown dots indicate data recorded from deafened animals. (J) Number of active electrodes (d´ > 1; mean ± standard deviation) upon oCI stimulation with 1 (n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 µLEDs (n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils), and with optical fiber stimulation (n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 30.18, 4 degrees of freedom, p = 1.42*10-21) and post-hoc multiple comparison test; indiv. vs. block: p = 6.8*10-5; block vs. all: p = 0.006; all vs. fiber: p = 0.25; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). Responses to oCI stimulation with all µLEDs were also observed in hearing WT animals (n = 3 oCIs in 2 gerbils). No active electrodes were found in response to 4/7 oCI stimulations in 2/2 hearing/deaf wildtype gerbils). (K) Thresholds (d´ = 1, measured at the best electrode (BE)) for ICC activation evoked with 16 active µLEDs on an oCI (n = 24/9 in N = 9/3 hearing/deaf gerbils) and with a laser-coupled optical fiber (n = 9/3 in N = 9/3 hearing/deaf gerbils; p = 0.012, Wilcoxon rank sum test). Central horizontal line indicates the median, lower, upper horizontal lines indicate the minimum and maximum values, and box edges indicate the 25th and 75th percentile, respectively.


Figure 3 ( A ) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window , from apical to basal direction ( A - D ) . White markers define the best electrode ( BE ) , and measurement of the spread of excitation ( SoE ) is indicated in the left panel . In STCs where more than one peak was detected , the dorsal - and ventral - most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE . ( C ) Spectral spread of excitation upon SGN stimulation with µLED - based oCIs ( blocks of 4 µLEDs , solid lines ; mean ± standard error of the mean ) , acoustic ( black dashed line ) , laser - coupled optical fibers ( blue dashed line ) , as well as mono - and bipolar electrical stimulation with a clinical - style eCI ( dashed and dotted red lines , respectively ) . Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al ( Dieter et al , 2019 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 (A) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window, from apical to basal direction (A-D). White markers define the best electrode (BE), and measurement of the spread of excitation (SoE) is indicated in the left panel. In STCs where more than one peak was detected, the dorsal- and ventral-most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE.  (C) Spectral spread of excitation upon SGN stimulation with µLED-based oCIs (blocks of 4 µLEDs, solid lines; mean ± standard error of the mean), acoustic (black dashed line), laser-coupled optical fibers (blue dashed line), as well as mono- and bipolar electrical stimulation with a clinical-style eCI (dashed and dotted red lines, respectively). Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al (Dieter et al, 2019).


f1a , Silver staining of LC3 complexes purified from U2OS cells expressing ZZ - Flag - LC3 in normal medium or Earle ' s balanced salt solution ( EBSS ) for 2 h . Asterisks mark centriolar satellite proteins . b - d , Co - immunoprecipitation ( IP ) of OFD1 with LC3 , LC3 with PCM1 , or OFD1 with PCM1 in HEK293T cells . HA , haemagglutinin . e , Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown ( KD ) HEK293T cells . f , Co - immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells . IP efficiency is calculated as the ratio of immunoprecipitated OFD1 / LC3 . g , h , Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation ( SS ) . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments . g , h , Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation ( SS ) , 50 nM bafilomycin A1 ( Baf ) , 20 µM chloroquine ( CQ ) or 1 µM MG132 . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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 B-PANEL_START 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 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 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

f1a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ-Flag-LC3 in normal medium or Earle's balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins.b-d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin.e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells.f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3.g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


f2a , Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin ( Ac - tubulin ) , or centriole marker γ - tubulin in hTERT - RPE1 cells in normal medium ( Un ) or subjected to 24 hserum starvation ( SS ) . b , Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5 + / + and Atg5 − / − MEFs subjected to 24 hserum starvation . a , b , Data shown represent mean ± s . d . percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples . * * * P 0 . 001 , two - tailed unpaired student ' s t - test . Similar results were observed in three independent experiments .

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

f2a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker γ-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 hserum starvation (SS).b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 hserum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments.


f3a , b , Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation . Data shown represent mean ± s . d . percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well , respectively , in triplicate samples . * * * P 0 . 001 , two - tailed unpaired student ' s t - test . Similar results were observed in three independent experiments . Un , untreated ; SS , serum - starved .

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

f3a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.


f4a , Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 ( C - 19 ) cells , quantified OFD1 level was normalized with alpha - tubulin . b , Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation . Data shown represent mean ± s . d . percentage of cells with primary cilia for 500 cells per well in triplicate samples . * * * P 0 . 001 , two - tailed unpaired student ' s t - test . c , Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation . d , e , Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation . Data shown represent 100 cells per well in triplicate samples . d , e , Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation . Data shown represent 100 cells per well in triplicate samples . f , Scanning electron microscope analysis of primary cilia ( marked by arrows ) formed in C - 19 cells subject to 72 h serum starvation and cycling Atg5 + / + MEFs in normal medium for 24 h . Similar results were observed in three independent experiments .

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

f4a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with alpha-tubulin.b, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test.c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation.d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples.d, e, Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation. Data shown represent 100 cells per well in triplicate samples.f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.


Figure 1E ) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets . Removing all charged residues except for arginine from hnRNPA2 LC ( hnRNPA2 LCCD , R ) allows partitioning of hnRNPF PLD into droplets , indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning . hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions , see Figure S1J for quantification of phase separation of variants . Conditions : 20 µM proteins , 20 mM MES pH 5 . 5 50 mM NaCl , 150 mM urea . Scale bar : 10 µm .

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

Figure 1E) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LCCD,R) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions, see Figure S1J for quantification of phase separation of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.


Figure 2A ) AlexaFluor488 - tagged ( green ) hnRNPA2 LC undergoes LLPS , while AlexaFluor555 - tagged ( red ) TOG D1 does not . However , TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1 : 1 ratio . Conditions : 20 µM indicated protein ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm . hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently . B ) Similar to hnRNPF PLD , AlexaFluor555 - tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488 - tagged FUS LC droplets with both proteins at 300 µM . Conditions : 300 µM proteins ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 150 mM NaCl , 150 mM urea . Scale bar : 10 µm . E ) AlexaFluor555 - tagged hnRNPF PLD or FL and AlexaFluor405 - tagged TOG D1 partition simultaneously into AlexaFluor488 - tagged hnRNPA2 LC droplets . Conditions : 20 µM of each indicated protein ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 20 µm .

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 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

Figure 2A) AlexaFluor488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently. B) Similar to hnRNPF PLD, AlexaFluor555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm. E) AlexaFluor555-tagged hnRNPF PLD or FL and AlexaFluor405-tagged TOG D1 partition simultaneously into AlexaFluor488-tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm.


Figure 3C ) Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets ( rows 1 and 3 ) , they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets ( rows 2 and 4 ) . Conditions : 20 µM proteins ( ~ 1 % fluorescently labeled ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm . D ) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions ( hnRNPA2 LC5E and hnRNPA2 LC12E , respectively ) both allow partitioning of TOG D1 and hnRNPF PLD , even though hnRNPA2 LC12E barely phase separates at these conditions , indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD . See Appendix Figure S7B for quantification of LLPS of hnRNPA2 LC5E and hnRNPA2 LC12E . Conditions : 20 µM proteins ( ~ 1 % fluorescently labeled ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm .

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Figure 3C) Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets (rows 1 and 3), they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets (rows 2 and 4). Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. D) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions (hnRNPA2 LC5E and hnRNPA2 LC12E, respectively) both allow partitioning of TOG D1 and hnRNPF PLD, even though hnRNPA2 LC12E barely phase separates at these conditions, indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD. See Appendix Figure S7B for quantification of LLPS of hnRNPA2 LC5E and hnRNPA2 LC12E. Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.


Figure 4A ) After cleavage of a C - terminal maltose binding protein solubility tag , hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate . In contrast , phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested . Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested . Conditions : 20 µM proteins , 20 mM Tris pH 7 . 4 , 50 mM NaCl . Scale bar : 10

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Figure 4A) After cleavage of a C-terminal maltose binding protein solubility tag, hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate. In contrast, phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested. Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested. Conditions: 20 µM proteins, 20 mM Tris pH 7.4, 50 mM NaCl. Scale bar: 10 µm


Figure 5D ) After 22 hours of paraquat induced oxidative stress , both HRPA - 1mScarlet and HRPA - 1HsLCWTmScarlet assemble more than when unstressed ( ANOVA p < 0 . 0001 ) . No significant differences were found within the no stress ( p = 0 . 8 ) stress groups ( p = 0 . 45 ) by ANOVA , indicating that there is no difference in assembly between HRPA - 1mScarlet and HRPA - 1HsLCWTmScarlet . N = 12 animals per genotype . F ) There is no significant difference in number of spots in HRPA - 1HsLCWTmScarlet lines with or without Fyn , although there seems to be a trend of reduced spots in HRPA - 1HsLCD290VmScarlet lines in animals also expressing Fyn * ; this difference is not statistically significant ( ANOVA ) . N = 12 animals per genotype . G ) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA - 1HsLCWTmScarlet lines with or without Fyn , although there seems to be a trend of reduced spots in HRPA - 1HsLCD290VmScarlet lines in animals also expressing Fyn * ; this difference is not statistically significant ( ANOVA ) . N = 12 animals per genotype .

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Figure 5D) After 22 hours of paraquat induced oxidative stress, both HRPA-1mScarlet and HRPA-1HsLCWTmScarlet assemble more than when unstressed (ANOVA p<0.0001). No significant differences were found within the no stress (p=0.8) stress groups (p=0.45) by ANOVA, indicating that there is no difference in assembly between HRPA-1mScarlet and HRPA-1HsLCWTmScarlet. N = 12 animals per genotype.F) There is no significant difference in number of spots in HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype. G) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype.


Figure 6D ) Loss of C . elegans TDP - 43 ortholog , tdp - 1 , rescues glutamatergic tail / phasmid neurodegeneration caused by hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress , but not hrpa - 1 ( ∆ ) . N = 4 - 12 animals / genotype / trial , significance from two tailed t - test ; tdp - 1 ∆ = tdp - 1 ( tgx58 ) . E ) Loss of C . elegans TDP - 43 ortholog , tdp - 1 , rescues glutamatergic tail / phasmid neurodegeneration caused by hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress , but not hrpa - 1 ( ∆ ) . N = 4 - 12 animals / genotype / trial , significance from two tailed t - test ; tdp - 1 ∆ = tdp - 1 ( ok803 ) . G ) Expression of Fyn * in sensory glutamatergic neurons ( osm - 10 promoter , unc - 54 3 ' UTR ) reduces glutamatergic tail / phasmid neuron degeneration in hrpa - 1HsLCD290V animals after 22 hours paraquat induced oxidative stress . N = 9 - 12 animals / genotype / trial , significance from two tailed t - test . H ) Expression of an empty Fyn control ( Fynempty ) in sensory glutamatergic neurons ( osm - 10 promoter , unc - 54 3 ' UTR ) does not alter glutamatergic phasmid neuron degeneration in hrpa - 1HsLCD290V animals after 22 hours paraquat induced oxidative stress . N = 9 - 12 animals / genotype / trial .

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Figure 6D) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(tgx58). E) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(ok803). G) Expression of Fyn* in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) reduces glutamatergic tail/phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial, significance from two tailed t-test. H) Expression of an empty Fyn control (Fynempty) in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) does not alter glutamatergic phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial.


Figure 1 ( F ) 5 - day - old Col - 0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 30 - 32 biological replicates from 2 pooled experiments ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 001 ) . ( G ) Ethylene production was measured in Col - 0Cas9 WT and CRISPR glv 3 . 5 h after elicitation with 500 nM flg22 or co - treatment with 1 µM GLV2 . Ethylene production was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 11 - 12 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 01 ) . ( H ) Mean cfu of Pto DC3000 COR - 4 days after spray inoculation of Col - 0Cas9 WT and CRISPR glv ; n = 12 biological replicates from 3 pooled experiments - / + SD ( Students T - Test , * p < 0 . 05 ) .

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

Figure 1(F) 5-day-old Col-0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=30-32 biological replicates from 2 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001).(G) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01).(H) Mean cfu of Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).


Figure 2 ( B ) ROS burst in WT after elicitation with 10 nM flg22 , 1 μM GLV2 , co - treatment or pre - treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22 . Shown is the mean of total ROS production ( Total RLU ) over 40 minutes normalized to the flg22 response set as 100 % - / + SD , n = 96 - 120 biological replicates from 15 pooled experiments ( one - way ANOVA , Tukey post - hoc test , a - b / c / d , p < 0 . 001 ; b - c , p < 0 . 05 ; b - d , p < 0 . 001 ( D ) PR1 expression was measured in 10 - day - old WT seedlings treated for 24 h with 100 nM flg22 , 1 μM GLV2 or co - treatment . Expression was normalized to the UBQ house - keeping gene . Shown is the mean of n = 4 . Symbol colors represent different biological

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

Figure 2(B) ROS burst in WT after elicitation with 10 nM flg22, 1 μM GLV2, co-treatment or pre-treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22. Shown is the mean of total ROS production (Total RLU) over 40 minutes normalized to the flg22 response set as 100% -/+ SD, n=96-120 biological replicates from 15 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b/c/d, p<0.001; b-c, p<0.05; b-d, p<0.001(D) PR1 expression was measured in 10-day-old WT seedlings treated for 24 h with 100 nM flg22, 1 μM GLV2 or co-treatment. Expression was normalized to the UBQ house-keeping gene. Shown is the mean of n=4. Symbol colors represent different biological replicates


Figure 3 ( B ) Ethylene production was measured in the indicated genotypes 3 . 5 h after elicitation with mock or 500 nM flg22 . Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 18 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 001 ; a - c , p < 0 . 001 ; b - c , p < 0 . 001 ) . ( E ) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3 : : RGI3 # 2 and # 3 3 . 5 h after elicitation with 500 nM flg22 . Ethylene production was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 8 biological replicates from 2 pooled experiments - / + SD ( one - way ANOVA , Dunnett post - hoc test , a - b p < 0 . 05 ) . ( F ) 5 - day - old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 36 biological replicates from 3 pooled experiments ( Students T - test , * * * p < 0 . 001 ) .

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

Figure 3(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM flg22. Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).(E) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3::RGI3 #2 and #3 3.5 h after elicitation with 500 nM flg22. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05).(F) 5-day-old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36 biological replicates from 3 pooled experiments (Students T-test, ***p<0.001).


Figure 4 ( C ) 5 - day - old seedlings of WT , rgi5x and rgi5x pRGI3 : : RGI3 # 2 and # 3 were treated with 10 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 32 biological replicates pooled from 4 experiments ( Students T - test , * * p < 0 . 01 ) . ( D ) Quantitative analysis of synthetic GLV2 or GLV2 - S peptide binding to RGI3ECD using microscale thermophoresis . Shown is the mean - / + SD , n = 3 technical replicates . ( E ) Co - immunoprecipitation experiment upon co - expression of BAK1 - HA and RGI3 - GFP in N . benthamiana . IP was performed using GFP - TRAP agarose beads . Western blots for protein detection were probed with α - GFP or α - HA antibodies . Similar results were obtained in 3 independent experiments .

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Figure 4(C) 5-day-old seedlings of WT, rgi5x and rgi5x pRGI3::RGI3 #2 and #3 were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=32 biological replicates pooled from 4 experiments (Students T-test, **p<0.01).(D) Quantitative analysis of synthetic GLV2 or GLV2-S peptide binding to RGI3ECD using microscale thermophoresis. Shown is the mean -/+ SD, n=3 technical replicates.(E) Co-immunoprecipitation experiment upon co-expression of BAK1-HA and RGI3-GFP in N. benthamiana. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Similar results were obtained in 3 independent experiments.


Figure 5 ( C ) Co - immunoprecipitation experiment upon co - expression of FLS2 - HA , BAK1 - HA and RGI3 - GFP in N . benthamiana . Protein extraction was performed 30 min after mock , 1 μM flg22 or 1 μM GLV2 treatment . IP was performed using GFP - TRAP agarose beads . Western blots for protein detection were probed with α - GFP or α - HA antibodies . Data information : Similar results were obtained in 3 independent experiments .

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Figure 5(C) Co-immunoprecipitation experiment upon co-expression of FLS2-HA, BAK1-HA and RGI3-GFP in N. benthamiana. Protein extraction was performed 30 min after mock, 1 μM flg22 or 1 μM GLV2 treatment. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Data information: Similar results were obtained in 3 independent experiments.


Figure 6 ( B ) Quantitative real - time PCR of FLS2 transcripts from 12 - day - old seedlings treated with mock or 1 μM GLV2 for the indicated time , n = 3 biological replicates . UBQ was used as a house keeping gene . Students T - test revealed no statistical difference for each of the different timepoints . Data information : Similar results were obtained in 3 independent experiments . ( C ) Protein extraction was performed from 12 - day - old seedlings of the indicated genotypes . Western blot was probed with α - FLS2 antibodies . ( D ) Protein extraction was performed from 12 - day - old seedlings after 1 hour treatment with mock or 50 μM MG132 . Western blots were probed with α - FLS2 antibodies . Data information : Similar results were obtained in 3 independent experiments .

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Figure 6(B) Quantitative real-time PCR of FLS2 transcripts from 12-day-old seedlings treated with mock or 1 μM GLV2 for the indicated time, n=3 biological replicates. UBQ was used as a house keeping gene. Students T-test revealed no statistical difference for each of the different timepoints. Data information: Similar results were obtained in 3 independent experiments.(C) Protein extraction was performed from 12-day-old seedlings of the indicated genotypes. Western blot was probed with α-FLS2 antibodies. (D) Protein extraction was performed from 12-day-old seedlings after 1 hour treatment with mock or 50 μM MG132. Western blots were probed with α-FLS2 antibodies. Data information: Similar results were obtained in 3 independent experiments.


Figure 1A ) H1299 cell line was transfected with increasing concentrations of MDM2 , and the RB + 5 ´ UTR exogenous ( left panel ) and endogenous ( right panel ) levels were evaluated . B ) Evaluation of the RB expression as in ( A ) but inducing DNA damage with doxorubicin 1 µM during 16 h . C ) Evaluation of the RB levels in an MDM2 dose - dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line . D ) Evaluation of the RB levels in a MDM2 dose - dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line . E ) Evaluation of the endogenous RB expression using H1299 cell line , under normal conditions , transfecting the cells with an increasing amount of the phosphomimetic mutant MDM2 ( S395D ) . F ) Evaluation of the endogenous RB as in ( E ) but using C33 cell line .

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Figure 1A) H1299 cell line was transfected with increasing concentrations of MDM2, and the RB+5´UTR exogenous (left panel) and endogenous (right panel) levels were evaluated. B) Evaluation of the RB expression as in (A) but inducing DNA damage with doxorubicin 1 µM during 16 h. C) Evaluation of the RB levels in an MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line. D) Evaluation of the RB levels in a MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line. E) Evaluation of the endogenous RB expression using H1299 cell line, under normal conditions, transfecting the cells with an increasing amount of the phosphomimetic mutant MDM2(S395D). F) Evaluation of the endogenous RB as in (E) but using C33 cell line.