E . F . Bar plots of EMSA - derived cooperativity factors for Oct - Oct homodimers on MORE ( E ) or Sox2 - Oct heterodimers on SoxOct element ( F ) are shown for a panel of six POU . mean is shown with standard deviation as error bars ( n ≥ 3 ) Tukey multiple comparison of means was used for assessment of statistical significances ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) . N . A . : not assessed .

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E. and F. Bar plots of EMSA-derived cooperativity factors for Oct-Oct homodimers on the MORE (E) or Sox2-Oct heterodimers on the SoxOct element (F) are shown for a panel of six POU proteins. The mean is shown with standard deviation as error bars (n≥3) and Tukey multiple comparison of means was used for assessment of statistical significances (*** p < 0.001, ** p < 0.01, * p < 0.05). N.A.: not assessed.


G . Properties of Oct4 Oct6 homodimers from molecular dynamics ( MD ) simulations . structural snapshots illustrate Oct6 ( first monomer in orange , second in blue ) 3 parameters used : drmsd = root mean square deviation of interatomic distances measured for backbone of structured domains ( excluding linker ) ; θ = angle describing kink in helix α4 ( data for both monomers 1 2 are shown ) ; d = between centers of mass of 2 helices α5 ; w = minor groove width in region between 2 monomers .

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G. Properties of Oct4 and Oct6 homodimers from molecular dynamics (MD) simulations. The structural snapshots illustrate Oct6 (first monomer in orange, second in dark blue) and the 3 parameters used: drmsd = the root mean square deviation of interatomic distances measured for the backbone of the structured domains (excluding the linker); θ = the angle describing the kink in helix α4 (data for both monomers 1 and 2 are shown); d = the distance between the centers of mass of the 2 helices α5; w = the DNA minor groove width in the region between the 2 monomers.


A . Zoom - in view of Oct6 - Oct6 homodimer interface in molecular models on crystal structures of two POU homodimers on MORE . Oct6 POUS , orange ; POUHD of other monomer , blue . Important residues are labelled Met ( Oct6 ) in position 151 which was identified to guide - binding preference is marked with a star . helixes of POU domains are numbered . Colour representation as in Fig . 1B , 1C . For clarity , second monomer of Oct4 / Oct6 is shown in blue .

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A. Zoom-in view of the Oct6-Oct6 homodimer interface in the molecular models based on crystal structures of two POU homodimers on MORE DNA. Oct6 POUS, orange; POUHD of the other monomer, blue. Important residues are labelled and Met (Oct6) in position 151 which was identified to guide the DNA-binding preference is marked with a star. Individual helixes of POU domains are numbered. Colour representation as in Fig. 1B, 1C. For clarity, the second monomer of Oct4/Oct6 is shown in dark blue.


B . C . Network of hydrophobic interactions in Oct6 ( B ) Oct4 ( C ) . numbers show minimal interatomic distances ( excluding hydrogens ) between residues involved calculated from simulations . In blue are those distances that change significantly between Oct4 Oct6 in red those unchanged . shown standard deviations represent a measure for variability during simulations not for uncertainty in calculations .

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B. and C. Network of hydrophobic interactions in Oct6 (B) and Oct4 (C). The numbers show the minimal interatomic distances (excluding hydrogens) between the residues involved calculated from the simulations. In blue are those distances that change significantly between Oct4 and Oct6 and in red those unchanged. The shown standard deviations represent a measure for the variability during the simulations and not for the uncertainty in the calculations.


D . EMSA showing a differential binding of WT Oct4 Oct6 POU domains as well as mutated Oct4151M Oct6151S POU domains on MORE ( OctOct ) element . free bound by monomer or dimer are indicated .

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D. EMSA showing a differential binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains on MORE (OctOct) element. The free DNA and DNA bound by monomer or dimer are indicated.


E . EMSA showing binding of WT Oct4 Oct6 POU domains as well as mutated Oct4151M Oct6151S POU domains to SoxOct element in absence or presence of Sox2 HMG . free bound by monomers or dimer are indicated . Quantifications of cooperativity measurements are shown in Fig . EV3C .

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E. EMSA showing binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains to SoxOct element in the absence or presence of the Sox2 HMG. The free DNA and DNA bound by monomers or dimer are indicated. Quantifications of cooperativity measurements are shown in Fig. EV3C.


F . Bar plot on single tube EMSAs using cy5 - SoxOct FAM - MORE elements showing difference in - binding of WT Oct4 , WT Oct6 , Oct4151M Oct6151S POU domains . mean log2 of cy5 ( heterodimer ) FAM ( homodimer ) band intensities are depicted with standard deviation ( n = 9 ) . data points are shown as gray jitter plots . Tukey multiple comparisons of means was performed to assess significance ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , p > 0 . 05 n . s . ) . Compare to Fig . EV1D EV3D .

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F. Bar plot based on single tube EMSAs using cy5-SoxOct and FAM-MORE DNA elements showing the difference in DNA-binding of WT Oct4, WT Oct6, Oct4151M and Oct6151S POU domains. The mean log2 ratio of cy5 (heterodimer) and FAM (homodimer) band intensities are depicted with standard deviation (n=9). Individual data points are shown as gray jitter plots. Tukey multiple comparisons of means was performed to assess significance (*** p < 0.001, ** p < 0.01, * p < 0.05, p > 0.05 n.s.). Compare to Fig. EV1D and EV3D.


A B . Molecular models show position of mutated residues in structures of two dimers on : Sox2 - Oct4 heterodimer ( A ) Oct4 - Oct4 homodimer ( B ) . Ser in position 151 ( asterisk ) was mutated to Met in order to shift preference of Oct4 for homodimerization . helixes of POU domains are numbered .

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A and B. Molecular models show the position of mutated residues in the structures of two dimers on DNA: Sox2-Oct4 heterodimer (A) and Oct4-Oct4 homodimer (B). Ser in position 151 (asterisk) was mutated to Met in order to shift the preference of Oct4 for homodimerization. The individual helixes of the POU domains are numbered.


D . GFP - positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2 , Klf4 , c - Myc . Colonies were imaged 16 days after second viral infection , using a microscope . Scale bars : 250 μm ; objective 10x .

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D. GFP-positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2, Klf4, and c-Myc. Colonies were imaged 16 days after second viral infection, using a fluorescence microscope. Scale bars: 250 μm; objective 10x.


B . Oct4 - GFP - positive colonies of two O6SKM iPSC lines , expanded from single colonies of pluripotent that were generated by contribution of synthetic Oct6 molecule ( Oct67K , 22T , LinkO4 , 151S ) . Scale bars : 250 μm .

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B. Oct4-GFP-positive colonies of two O6SKM iPSC lines, expanded from single colonies of pluripotent cells that were generated by the contribution of the synthetic Oct6 molecule (Oct67K,22T,LinkO4,151S). Scale bars: 250 μm.


C . Genotyping of two O6SKM iPSC lines . Two stable lines of iPSCs generated with Oct6 , Sox2 , Klf4 , c - Myc retroviral set are positive for these four transgenes but negative for Oct4 transgene . OSKM iPSCs OG2 - MEFs were used as PCR controls .

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C. Genotyping of two O6SKM iPSC lines. Two stable lines of iPSCs generated with the Oct6, Sox2, Klf4, and c-Myc retroviral set are positive for these four transgenes but negative for the Oct4 transgene. OSKM iPSCs and OG2-MEFs were used as PCR controls.


D . Expression of viral transgenes in two O6SKM iPSC lines done by qRT - PCR using specific primers . OG2 - MEFs were harvested 5 days after infection used for comparison as a positive control .

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D. Expression analysis of viral transgenes in two O6SKM iPSC lines done by qRT-PCR using specific primers. OG2-MEFs were harvested 5 days after infection and used for comparison as a positive control.


E . Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 Nanog . Oct4 expression was confirmed by Oct4 - GFP transgene . stained by DAPI . Scale bars : 100 μm .

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E. Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog. Oct4 expression was confirmed by the Oct4-GFP transgene. DNA stained by DAPI. Scale bars: 100 μm.


F . Bisulfite sequencing of genomic Oct4 , Nanog , Col1a1 promoter regions of O6SKM iPSCs , OSKM iPSCs , OG2 - MEFs . black circles represent unmethylated methylated CpG sites , respectively .

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F. Bisulfite sequencing of genomic Oct4, Nanog, and Col1a1 promoter regions of O6SKM iPSCs, OSKM iPSCs, and OG2-MEFs. White and black circles represent unmethylated and methylated CpG sites, respectively.


G . Pairwise scatter plots comparing global gene expression profile of O6SKM iPSCs1 with OG2 ESCs ( left ) O4SK iPSCs ( right ) . Black lines represent a two - fold change in gene expression between paired lines . On right side of plots , color bar indicates scattering density . Red green dots represent up - downregulated genes , respectively . Positions of selected pluripotency - related genes are highlighted as orange points .

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G. Pairwise scatter plots comparing the global gene expression profile of O6SKM iPSCs1 with OG2 ESCs (left) and O4SK iPSCs (right). Black lines represent a two-fold change in gene expression levels between the paired cell lines. On the right side of the plots, the color bar indicates scattering density. Red and green dots represent up- and downregulated genes, respectively. Positions of selected pluripotency-related genes are highlighted as orange points.


H . Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs O4SK iPSCs , illustrating close relationship between their global gene expression profiles .

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H. Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs and O4SK iPSCs, illustrating the close relationship between their global gene expression profiles.


A . In vitro differentiation of O6SKM iPSC lines into of three germ layers as shown by immunochemistry : endoderm ( α - fetoprotein , AFP ) , mesoderm ( α - smooth muscle actin , SMA ) , ectoderm ( α - tubulin , TUJ1 ) . Nuclei ( ) were stained by Hoechst ( blue ) . Scale bars : 200 μm .

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A. In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry: endoderm (α-fetoprotein, AFP), mesoderm (α-smooth muscle actin, SMA), and ectoderm (α-tubulin, TUJ1). Nuclei (DNA) were stained by Hoechst (blue). Scale bars: 200 μm.


B . F1 offspring of chimera mice with contribution from O6SKM iPSCs CD1 mice .

B. F1 offspring of chimera male mice with contribution from O6SKM iPSCs and CD1 female mice.


C . Pictures demonstrating germline transmission ; endogenous Oct4 - GFP signal was detected in gonads of F1 pups . Scale bars : 200 μm .

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C. Pictures demonstrating germline transmission; endogenous Oct4-GFP signal was detected in the gonads of F1 pups. Scale bars: 200 μm.


( A ) Top panel displays structure of each CDK4 / 6 inhibitor tested . Bottom panel shows dose response curves with these inhibitors displaying of G1 - arrested RPE1 - FUCCI . To obtain dose response curves , number of mKO - Cdt1 positive ( G1 - arrested ) were calculated following 24hr drug addition ( blue solid lines ) or 24h after subsequent drug washout ( blue dotted lines ) . Cmax values observed in ( taken from ( He et al . , 2017 ; Klein et al . , 2018 ) ) are represented on each graph with red dotted lines . Graphs display mean data - / + from 3 experiments , with at least 500 counted per condition per experiment . ( B ) of G1 - arrested RPE1 - FUCCI , calculated as in panel A , but using a fixed of CDK4 / 6 inhibitor for different durations of time , as indicated . Note , this is a fixed that quantifies of G1 - arrested prior to , or 24 hrs following , CDK4 / 6 inhibitor washout . Each bar displays mean data + from 3 experiments , with at least 500 counted per condition per experiment . (

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(A) Top panel displays structure of each CDK4/6 inhibitor tested. Bottom panel shows dose response curves with these inhibitors displaying percentage of G1-arrested RPE1-FUCCI cells. To obtain dose response curves, the number of mKO-Cdt1 positive (G1-arrested) cells were calculated following 24hr drug addition (dark blue solid lines) or 24h after subsequent drug washout (light blue dotted lines). Cmax values observed in patients (taken from (He et al., 2017; Klein et al., 2018)) are represented on each graph with red dotted lines. Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (B) Percentage of G1-arrested RPE1-FUCCI cells, calculated as in panel A, but using a fixed concentration of CDK4/6 inhibitor for different durations of time, as indicated. Note, this is a fixed assay that quantifies the percentage of G1-arrested cells prior to, or 24 hrs following, CDK4/6 inhibitor washout. Each bar displays mean data + SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (


( A ) Dose response curves displaying of G1 - arrested p53 - WT ( blue ) or KO ( green ) RPE1 - FUCCI following 24hr incubation with palbociclib ( solid lines ) or 24h after subsequent washout ( dotted lines ) . Graphs display mean data - / + from 3 experiments , with at least 500 counted per condition per experiment .

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(A) Dose response curves displaying the percentage of G1-arrested p53-WT (blue) or KO (green) RPE1-FUCCI cells following 24hr incubation with palbociclib (dark solid lines) or 24h after subsequent washout (light dotted lines). Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment.


( C ) Representative quantifications of colony forming assays in p53 - WT or KO RPE1 treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days then grown at low density without inhibitor for 10 days . Each bar displays mean data + from 3 experiments .

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(C) Representative images and quantifications of colony forming assays in p53-WT or KO RPE1 cells treated with palbociclib (1.25μM) for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Each bar displays mean data + SEM from 3 experiments.


( E ) of cycle profiles from treated as in D . Graph shows mean of + SD that divide at each round of division , with 150 analysed in total from 3 experimental repeats .

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(E) Quantification of cell cycle profiles from cells treated as in D. Graph shows the mean percentage of cells + SD that divide at each round of division, with 150 cells analysed in total from 3 experimental repeats.


( A ) Representative immunofluorescence of p21 in p53 - WT or KO RPE1 , 48 hours after release from 1 , 4 or 7 days palbociclib ( 1 . 25μM ) treatment . Zoom inserts are 3x magnification of indicated regions . Scale bars = 250 μM . ( B ) of p21 intensities in treated as in panel A . At least 100 were analysed per experiment graph shows data from 3 experimental repeats . Violin plots display variation in intensities between . Horizontal lines display median , error bars show 95 % confidence intervals . (

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(A) Representative immunofluorescence images of p21 levels in p53-WT or KO RPE1 cells, 48 hours after release from 1, 4 or 7 days palbociclib (1.25μM) treatment. Zoom inserts are 3x magnification of the indicated regions. Scale bars = 250 μM. (B) Quantification of p21 intensities in cells treated as in panel A. At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats. Violin plots display the variation in intensities between individual cells. Horizontal lines display the median, and error bars show 95% confidence intervals. (


( D - E ) of morphologies ( D ) γH2AX - positive damage foci ( E ) following palbociclib ( 1 . 25μM ) treatment in p53 WT KO RPE1 . were treated for 1 , 4 or 7 days then analysed before or after drug washout for 48 hours . 100 ( ) or 50 ( γH2AX foci ) were scored per condition per experiment , bar graphs represent mean data + from 6 experiments .

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(D-E) Quantification of nuclear morphologies (D) and γH2AX-positive DNA damage foci (E) following palbociclib (1.25μM) treatment in p53 WT and KO RPE1 cells. Cells were treated for 1, 4 or 7 days and then analysed before or after drug washout for 48 hours. 100 cells (nuclear morphology) or 50 cells (γH2AX foci) were scored per condition per experiment, and bar graphs represent mean data +SEM from 6 experiments.


( J - K ) Representative immunofluorescence ( J ) quantifications ( K ) of mitotic replication assays ( MiDAS ) in p53 - KO RPE1 released from 7 days of palbociclib ( 1 . 25μM ) treatment or following 0 . 4uM aphidicolin treatment for 40Hrs . EdU foci were quantified in nocodazole - arrested . Scale bar = 5 μM , zoom inserts = 3x magnification of highlighted areas . 10 were analysed per experiment bar chart shows mean + from 3 experimental repeats .

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(J-K) Representative immunofluorescence images (J) and quantifications (K) of mitotic DNA replication assays (MiDAS) in p53-KO RPE1 cells released from 7 days of palbociclib (1.25μM) treatment or following 0.4uM aphidicolin treatment for 40Hrs. EdU foci were quantified in nocodazole-arrested cells. Scale bar= 5 μM, zoom inserts = 3x magnification of highlighted areas. 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats.


( A ) Volcano plot of up or downregulated following prolonged palbociclib ( 1 . 25μM ) treatment in RPE1 . top 10 significantly upregulated downregulated are shown in blue red , respectively .

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(A) Volcano plot of proteins up or downregulated following prolonged palbociclib (1.25μM) treatment in RPE1 cells. The top 10 significantly upregulated and downregulated proteins are shown in blue and red, respectively.


( D ) Representative western blots of whole lysates from RPE1 - WT treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days , or treated identically , then washed out for indicated times to reflect when majority of are in S - phase ( see Figure 1C ) .

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(D) Representative western blots of whole cell lysates from RPE1-WT cells treated with palbociclib (1.25μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times to reflect when the majority of cells are in S-phase (see Figure 1C).


( F - G ) Representative ( F ) quantifications ( G ) of colony forming assays with RPE1 treated with palbociclib ( 1 . 25μM ) for indicated times , then grown at low density without palbociclib for 10 days . DMSO ( control ) or indicated genotoxic drugs were applied for first 24 h after palbociclib washout . Each bar displays mean data + SD from 4 experiments .

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(F-G) Representative images (F) and quantifications (G) of colony forming assays with RPE1 cells treated with palbociclib (1.25μM) for indicated times, and then grown at low density without palbociclib for 10 days. DMSO (control) or indicated genotoxic drugs were applied for the first 24 h after palbociclib washout. Each bar displays mean data + SD from 4 experiments.


( A ) Representative western blots of whole lysates from MCF7 treated with palbociclib ( 1 μM ) for 1 , 4 or 7 days , or treated identically , then washed out for indicated times ( see Figure 1C ) . ( B ) of adjusted relative density from 3 independent western blot experiments . Bars display mean values - / + SD . Significance determined by unpaired Student ' s T comparing treated target to asynchronous target control . ( * < 0 . 01 , * * < 0 . 001 , * * * < 0 . 0001 ) .

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(A) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib (1 μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times (see Figure 1C). (B) Analysis of adjusted relative density from 3 independent western blot experiments. Bars display mean values -/+ SD. Significance determined by unpaired Student's T test comparing treated target protein to asynchronous target control. (* < 0.01, ** < 0.001, *** < 0.0001).


( C , D ) of morphologies ( C ) or γH2AX - positive damage foci ( D ) from MCF7 , MCF7 p53 KO or T47D that were treated with palbociclib ( 1μM ) for 0 , 1 , 4 , or 7 days then analysed 48hr after drug washout . Either 100 ( ) or 50 ( γH2AX ) were scored per condition per experiment bar graphs represent mean data + from 3 experiments .

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(C,D) Quantification of the nuclear morphologies (C) or γH2AX-positive DNA damage foci (D) from MCF7, MCF7 p53 KO or T47D cells that were treated with palbociclib (1μM) for 0, 1, 4, or 7 days and then analysed 48hr after drug washout. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( F ) of colony forming assays of MCF7 , MCF7 p53 KO T47D treated with CDK4 / 6 inhibitor for 0 , 1 , 4 or 7 days then grown at a low density without palbociclib for 14 - 21 days . Bar graphs displays mean data + from 4 - 5 experiments .

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(F) Quantification of colony forming assays of MCF7, MCF7 p53 KO and T47D cells treated with CDK4/6 inhibitor for 0, 1, 4 or 7 days and then grown at a low density without palbociclib for 14-21 days. Bar graphs displays mean data + SEM from 4-5 experiments.


( B , C ) of morphologies ( B ) or γH2AX - positive damage foci ( C ) from different cancer lines , as indicated , treated continuously with palbociclib ( 1μM ) for 0 , 1 , 2 , or 3 . Either 100 ( ) or 50 ( γH2AX ) were scored per condition per experiment bar graphs represent mean data + from 3 experiments .

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(B,C) Quantification of the nuclear morphologies (B) or γH2AX-positive DNA damage foci (C) from different cancer lines, as indicated, treated continuously with palbociclib (1μM) for 0, 1, 2, or 3 weeks. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( D ) of weekly proliferation rate in treated as in B . A total of 60 , 000 of each were cultured in 10cm dishes with 1uM palbociclib for a total of 3 . Every 7 days , were trypsinised total were determined . 0 - week timepoint shows 7 - day fold increase of untreated . data points show fold increase in over each 7 - day period , bars represent mean from 2 experiments .

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(D) Quantification of weekly proliferation rate in cells treated as in B. A total of 60,000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks. Every 7 days, cells were trypsinised and total cell counts were determined. The 0-week timepoint shows the 7-day fold increase of untreated cells. The data points show fold increase in cell count over each 7-day period, and bars represent the mean from 2 experiments.


( A ) RPE1 were transfected with either scramble or TMEM135 siRNAs immunostained for LAMP1 as a lysosome marker ( red ) PMP70 as a peroxisome marker ( green ) . Scale bar , 10 μm . ( B ) of overlap signal between lysosomes peroxisomes shown in ( A ) . Data represent mean ± SD ( n = 3 experiments ) , 50 were scored per condition per experiment . * P < 0 . 05 , Student ' s t - .

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(A) RPE1 cells were transfected with either scramble or TMEM135 siRNAs and immunostained for LAMP1 as a lysosome marker (red) and PMP70 as a peroxisome marker (green). Scale bar, 10 μm. (B) Quantification of overlap signal between lysosomes and peroxisomes shown in (A). Data represent mean ± SD (n=3 experiments), 50 cells were scored per condition per experiment. *P < 0.05, Student's t-test.


( C ) Subcellular fractions of a discontinuous sucrose gradient collected from top to bottom , separated by SDS - PAGE , immunoblotted for LAMP1 , EEA1 , Rab11 , Rab8 , PMP70 , GM130 , Na + / K + ATPase .

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(C) Subcellular fractions of a discontinuous sucrose gradient collected from top to bottom, separated by SDS-PAGE, and immunoblotted for LAMP1, EEA1, Rab11, Rab8, PMP70, GM130, and Na+/K+ ATPase.


( D ) of total cholesterol in subcellular fractions Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - .

(D) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.


( E ) transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage for precursor of SREBP2 SREBP2 .

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(E) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2.


( F ) transfected with TMEM135 siRNA were subjected to qPCR . Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - .

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(F) Cells transfected with TMEM135 siRNA were subjected to qPCR. Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.


( A ) RPE1 were transfected with siRNAs as indicated , followed by serum starvation for 24 h , immunostained for ARL13B ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( B ) of of ciliated shown in ( A ) . Data represent mean ± SD ( n = 3 experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by serum starvation for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (B) Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) were transfected with siRNAs as indicated incubated with HPβCD in serum - starvation media followed by filipin . Scale bar , 20 μm .

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(C) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm.


( D ) were transfected with siRNAs shown in ( C ) immunostained for ARL13B ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) of of ciliated shown in ( D ) . Data represent mean ± SD ( n = 3 experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(D) Cells were transfected with siRNAs shown in (C) and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of ciliated cells shown in (D). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( F ) were transfected with siRNAs as indicated , incubated with cholesterol - MβCD in serum - starvation media , immunostained for ARL13B ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( G ) of of ciliated shown in ( F ) . Data represent mean ± SD ( n = 3experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(F) Cells were transfected with siRNAs as indicated, incubated with cholesterol-MβCD complex in serum-starvation media, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of ciliated cells shown in (F). Data represent mean ± SD (n=3experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) RPE1 were transfected with siRNA as indicated , immunostained for Rab8 ( red ) γ - tubulin ( green ) in either 10 % FBS medium ( upper panel ) or serum - starvation medium ( lower panel ) . Scale bar , 10 μm . Arrowhead indicates Rab8 localized to centrioles .

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(A) RPE1 cells were transfected with siRNA as indicated, and immunostained for Rab8 (red) and γ-tubulin (green) in either 10% FBS medium (upper panel) or serum-starvation medium (lower panel). Scale bar, 10 μm. Arrowhead indicates Rab8 localized to the centrioles.


( B ) of of with Rab8 localized to centriole Data represent mean ± SD ( n = 3 experiments ) , 200 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) were transfected with siRNA immunostained for IFT20 ( red ) γ - tubulin ( green ) . Scale bar , 10 μm .

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(C) Cells were transfected with siRNA and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.


( D ) of of IFT20 at centrioles Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(D) Quantification of the percentage of IFT20 intensity at the centrioles Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) RPE1 were transfected with siRNAs as indicated , followed by transfection with wild - type pGFP - Rab8a ( WT - Rab8 ) , constitutively active pGFP - Rab8a ( Q67L ) ( CA - Rab8 ) , or DN Rab8 dominant - negative pGFP Rab8a ( T22N ) , incubated in serum - starved media for 12 h , immunostained for ARL13B ( Red ) , GFP - Rab8 ( green ) , DAPI ( blue ) . Scale bar , 10 μm .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild-type pGFP-Rab8a (WT-Rab8), constitutively active pGFP-Rab8a (Q67L) (CA-Rab8), or DN Rab8 dominant-negative pGFP Rab8a (T22N), incubated in serum-starved media for 12 h, and immunostained for ARL13B (Red), GFP-Rab8 (green), and DAPI (blue). Scale bar, 10 μm.


( B ) of of ciliated Data represent mean ± SD ( n = 3 experiments ) , 200 GFP - positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(B) Quantification of the percentage of ciliated cells Data represent mean ± SD (n=3 experiments), 200 GFP-positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) ( Upper panel ) were transfected as indicated lysates were incubated with purified , including GST or GST - JCF1 ( RBD ) . amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by western blot with Rab8 antibody . ( Lower panel ) of bands was quantified by Image J software . amount of GTP - Rab8 were normalized to control level . Bar graph represents mean ± SD ( n = 3 experiments ) . * P < 0 . 05 , Student ' s t - .

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(C) (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST-JCF1 (RBD). The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. Bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.


( D ) were transfected lysates were incubated with purified GST - JCF1 ( RBD ) fusion . amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody . ( Lower panel ) were transfected lysates were incubated with purified GST - . amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody .

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(D) Cells were transfected and cell lysates were incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected and cell lysates were incubated with purified GST- protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody.


( A ) RPE1 were transfected with siRNAs as indicated , followed by 24 h incubation in serum - starved media . were then subjected to fractionation , Western blot for IFT20 , Golgi marker GM130 , marker UBXD8 , marker CREB .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by 24 h incubation in serum-starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB.


( B ) Efficiency of IFT20 knockdown by western blot .

(B) Efficiency of IFT20 knockdown by western blot.


( C ) were transfected with siRNAs as indicated , followed by incubation in serum - starved media for 24 h , immunostained for ARL13B ( red ) . Scale bar , 10 μm . bar graph represents of of ciliated . Data represent mean ± SD ( n = 3 experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(C) Cells were transfected with siRNAs as indicated, followed by incubation in serum-starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( D ) were transfected immunostained for Rab8 γ - tubulin , followed by of of with Rab8 localized to centriole . Data represent average ( n = 2 experiments ) .

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(D) Cells were transfected and immunostained for Rab8 and γ-tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average (n=2 experiments).


( E ) were transfected with siRNAs as indicated , followed by transfection with Flag - IFT20 , incubation in serum - starvation media for 12 h , immunostained for ARL13B . Representative of Flag - IFT20 ( green ) , ARL13B ( red ) , DAPI ( blue ) are shown . Scale bar , 10 μm . ( F ) of of ciliated with both Flag - IFT20 ARL13B localized in cilium . Data represent mean ± SD ( n = 3 experiments ) , 150 Flag positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(E) Cells were transfected with siRNAs as indicated, followed by transfection with Flag-IFT20, incubation in serum-starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag-IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. (F) Quantification of the percentage of ciliated cells with both the Flag-IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n=3 experiments), 150 Flag positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( G ) were transfected with siRNAs as indicated , followed by further transfection with CA - Rab8 , incubation in serum - starvation media for 12 h , immunostained for acetylated - tubulin . Representative of GFP - Rab8 Q67L ( green ) , acetylated - tubulin ( red ) , ( blue ) are shown . Scale bar , 10 μm . ( H ) of of GFP - positive ciliated ( only those cilia having both GFP - Rab8 acetylated - tubulin on cilium were considered for ) . Data represent mean ± SD ( n = 3 experiments ) , 200 GFP positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(G) Cells were transfected with siRNAs as indicated, followed by further transfection with CA-Rab8, incubation in serum-starvation media for 12 h, and immunostained for acetylated-tubulin. Representative fluorescent images of GFP-Rab8 Q67L (green), acetylated-tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. (H) Quantification of the percentage of GFP -positive ciliated cells (only those cilia having both GFP-Rab8 and acetylated-tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), 200 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 .

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(A) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells.


( B ) RPE1 were transfected by siRNAs as indicated , followed by incubation in serum - starvation media for 24 h , immunostained for ARL13B ( red ) γ - tubulin ( green ) . Scale bar , 10 μm .

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(B) RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm.


( C ) of of ciliated Data represent mean ± SD ( n = 3 experiments ) , 250 GFP positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(C) Quantification of the percentage of ciliated cells Data represent mean ± SD (n = 3 experiments), 250 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( D ) were transfected by siRNa as indicated followed by incubation in serum - starvation media for 24 h , immunostained for EHD1 ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) of of with EHD1 in cilium or in distal end of basal as shown in ( D ) . Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(D) Cells were transfected by siRNa as indicated followed by incubation in serum-starvation media for 24 h, and immunostained for EHD1 (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( F ) were transfected by siRNAs as indicated , followed by incubation in serum - starvation media for 24 h , immunostained for IFT20 ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( G ) of of IFT20 at centriole shown in ( F ) . Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(F) Cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD (n=3 experiments), 150 were scored per condition per experiment, *P < 0.05, Student's t-test.


( H , I , J ) were transfected by siRNAs as indicated , followed by transfection with GFP - Rab8 WT , GFP - Rab8 Q67L , or GFP - Rab8 T22N , further incubated in serum - starvation media for 12 h . lysate was subjected to immunoprecipitation with anti - GFP antibody , followed by Western blot with antibody against GFP .

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(H, I, J) Cells were transfected by siRNAs as indicated, followed by transfection with GFP-Rab8 WT, GFP-Rab8 Q67L, or GFP-Rab8 T22N, and further incubated in serum-starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti-GFP antibody, followed by Western blot with antibody against GFP.


( A ) RPE1 were transfected as indicated , followed by incubation in serum - starvation media in presence or absence of cholesterol / MCD for 24 h , then immunostained for Rab8 ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . Representative magnified are shown from labeled with asterisks .

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(A) RPE1 cells were transfected as indicated, followed by incubation in serum-starvation media in the presence or absence of cholesterol/MCD for 24 h, and then immunostained for Rab8 (red) and γ-tubulin (green). Scale bar, 10 μm. Representative magnified images are shown from cells labeled with white asterisks.


( B ) of of with Rab8 localized to centriole Data represent mean ± SD ( n = 3 experiments ) , 200 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) ( Upper panel ) were transfected lysate was incubated with purified GST - JCF1 ( RBD ) fusion . amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by western blot with Rab8 antibody . ( Lower panel ) of bands was quantified by Image J software . amount of GTP - Rab8 were normalized to control level . bar graph represents mean ± SD ( n = 3 experiments ) . * P < 0 . 05 , Student ' s t - .

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(C) (Upper panel) Cells were transfected and cell lysate was incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) The intensity of bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. The bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.


( D ) were transfected immunostained for IFT20 ( red ) γ - tubulin ( green ) . Scale bar , 10 μm .

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(D) Cells were transfected and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.


( E ) of IFT20 at centriole Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(E) Quantification of IFT20 intensity at the centriole Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


of wild - type ( Tefm + / + ) Tefm homozygous knockout ( Tefm - / - ) at embryonic day 8 . 5 ( analyzed , n = 43 ) . Scale bar , 500 μm .

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Morphology of wild-type (Tefm+/+) and Tefm homozygous knockout (Tefm-/-) embryos at embryonic day 8.5 (analyzed embryos, n= 43). Scale bar, 500 μm.


Western blot analyses of TEFM level in heart mitochondrial extracts from 8 - week - old control ( L / L ) tissue - specific Tefm knockout ( Tefm knockout ; L / L , cre ) mice ( n = 12 mice for each group ) . VDAC was used as loading control .

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Western blot analyses of the TEFM protein level in heart mitochondrial extracts from 8-week-old control (L/L) and tissue-specific Tefm knockout (Tefm knockout; L/L, cre) mice (n = 12 mice for each group). VDAC was used as loading control.


Survival curve for control ( L / L ; n = 60 ) Tefm knockout ( L / L , cre ; n = 22 ) mice .

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Survival curve for control (L/L; n= 60) and Tefm knockout (L/L, cre; n= 22) mice.


Heart to in 4 - 8 - week - old control ( L / L ) Tefm knockout mice ( L / L , cre ) . At 4 : L / L n = 22 , L / L , cre n = 15 ; 8 : L / L n = 41 , L / L , cre n = 39 .

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Heart weight to body weight ratio in 4- and 8-week-old control (L/L) and Tefm knockout mice (L/L, cre). At 4 weeks: L/L n= 22, L/L, cre n= 15; 8 weeks: L/L n= 41, L/L, cre n= 39.


Transmission electron of heart tissue in 8 - week - old control Tefm knockout mice ( n = 5 mice for each group ) . Scale bar , 2 μm ( upper panel ) 1 μm ( lower panel ) .

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Transmission electron micrographs of heart tissue in 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Scale bar, 2 μm (upper panel) and 1 μm (lower panel).


Southern blot analyses of mtDNA in control Tefm knockout hearts at of 4 8 ( Upper 2 blots ; n = 6 mice at each time - point for each group ) . 18S rDNA was used as loading control . isolated from Lrpprc Mterf4 knockout hearts used as controls . Western blot analyses of VDAC Histone H3 in control Tefm knockout hearts at of 4 8 ( Bottom 2 blots ; ; n = 6 mice at each time - point for each group ) . VDAC represents mitochondria loading Histone H3 was used as a loading control for nucleus .

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Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Upper 2 blots; n= 6 mice at each time-point for each group). 18S rDNA was used as loading control. DNA isolated from Lrpprc and Mterf4 knockout hearts used as controls. Western blot analyses of VDAC and Histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Bottom 2 blots; ; n= 6 mice at each time-point for each group). VDAC represents mitochondria loading and Histone H3 was used as a loading control for nucleus DNA.


Respiratory chain activities were measured spectrophotometrically normalized to citrate synthase activity in heart mitochondria from 8 - week - old control Tefm knockout mice ( n = 5 mice for each group ) . analyzed enzyme activities are NADH coenzyme Q reductase ( I ) , NADH cytochrome c reductase ( I / III ) , succinate dehydrogenase ( II ) cytochrome c oxidase ( IV ) .

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Respiratory chain complex activities were measured spectrophotometrically and normalized to citrate synthase activity in heart mitochondria from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The analyzed enzyme activities are NADH coenzyme Q reductase (complex I), NADH cytochrome c reductase (complex I/III), succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV).


Barplot of significantly changed ( Benjamini - Hochberg adjusted P < 0 . 05 ) in Tefm knockout mice compared to control mice ( n = 5 mice for each group ) , organized by OXPHOS complexes . are organized in alphabetic order on gene name . Bars represent mean log2 [ L / L , cre / L / L ] ± 95 % confidence interval .

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Barplot of the significantly changed proteins (Benjamini-Hochberg adjusted P <0.05) in Tefm knockout mice compared to control mice (n= 5 mice for each group), organized by individual OXPHOS complexes. Proteins are organized in alphabetic order based on gene name. Bars represent mean log2 [L/L, cre / L/L] ± 95% confidence interval.


Boxplots of ( LFQ ) of TEFM ( top ) POLRMT ( bottom ) . Red dot : mean ; blue dots : value for each biological replicate ( n = 5 mice for each group ) .

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Boxplots of the protein intensity (LFQ) of TEFM (top) and POLRMT (bottom). Red dot: mean; blue dots: intensity value for each biological replicate (n= 5 mice for each group).


Western blot analyses of POLRMT in control Tefm knockout heart mitochondria at of 8 ( n = 12 mice for each group ) . VDAC was used as loading control .

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Western blot analyses of POLRMT protein levels in control and Tefm knockout heart mitochondria at the age of 8 weeks (n= 12 mice for each group). VDAC was used as loading control.


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Northern blot analyses and relative quantification of mtRNA transcripts in control and Tefm knockout mouse hearts at the age of 4 and 8 weeks (n= 12 mice at each time-point for each group). 18S rRNA and 5.8S rRNA were used as loading controls, respectively. HSP transcripts: 12S rRNA 4 weeks P= 0.005966044, 8 weeks P= 0.0806819; 16S rRNA 4 weeks P= 0.000223004, 8 weeks P= 0.00243443; Nd1 4 weeks P= 0.0352088, 8 weeks P= 0.000344158; Cox1 4 weeks P= 0.000353895, 8 weeks P= 0.00145773; Cox2 4 weeks P= 0.0145193, 8 weeks P= 0.00578484; Cox3 4 weeks P= 0.00834317, 8 weeks P= 0.000392364; Nd5/Cyb 4 weeks P= 0.0010185, 8 weeks P= 0.00153049; Nd5 4 weeks P= 0.024621, 8 weeks P= 7.38074E-05; Cyb 4 weeks P= 3.46175E-06, 8 weeks P= 0.000991942; LSP transcript: Nd6 4 weeks P= 0.000162704, 8 weeks P= 0.000187855. (E) HSP transcripts: Tf 4 weeks P= 0.0297685, 8 weeks P= 0. 000212544; Tv 4 weeks P= 0.842023, 8 weeks P= 0.269231; Tl1 4 weeks P= 0.00921072, 8 weeks P= 0.000430068; Tm 4 weeks P= 0.0084742, 8 weeks P= 0.000142524; Tk 4 weeks P= 0.00297015, 8 weeks P= 0.00010425; Ts2 4 weeks P= 0.0128829, 8 weeks P= 2.27321E-05; Tl2 4 weeks P= 0.00701324, 8 weeks P= 0.000411549; Tt 4 weeks P= 0.00572118, 8 weeks P= 0.000807943; LSP transcript: Tp 4 weeks P= 0.0427191, 8 weeks P= 0.0483328; Te 4 weeks P= 0.464031, 8 weeks P= 0.13412; Ts1 4 weeks P= 0.0382215, 8 weeks P= 0.000478237; Tc 4 weeks P= 0.00418795, 8 weeks P= 0.000287525; Tn 4 weeks P= 0.00531668, 8 weeks P= 0.000374367; Ta 4 weeks P= 0.00247257, 8 weeks P= 0.000564597; Tq 4 weeks P= 0.00385524, 8 weeks P= 0.000727981.


In organello transcription analyses using heart mitochondria isolated from 8 - week - old control Tefm knockout mice ( n = 5 mice for each group ) . steady - state of 12S rRNA tRNAV were used as endogenous controls , due to their unchanged . VDAC was used as loading control for equal input of mitochondrial extracts . This experiment has performed five times with either two or three replicates .

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In organello transcription analyses using heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The steady-state levels of 12S rRNA and tRNAV were used as endogenous controls, due to their unchanged levels. VDAC was used as loading control for the equal input of mitochondrial extracts. This experiment has performed five times with either two or three replicates.


Northern blot analyses of 7S level in 4 - 8 - week - old control Tefm knockout mouse hearts ( n = 12 mice at each time - point for each group ) . 18S rRNA was used as loading control . from tissue - specific Mterf4 knockout mouse hearts tissue - specific Polrmt knockout mouse hearts were loaded as positive negative control , respectively .

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Northern blot analyses of 7S RNA level in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice at each time-point for each group). 18S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively.


Southern blot analyses of mtDNA to obtain 7S in 8 - week - old control Tefm knockout mice . Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA . Equal amounts of mtDNA shown on top of gel , were loaded as endogenous control . This experiment has performed three times .

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Southern blot analyses of mtDNA to obtain 7S DNA in 8-week-old control and Tefm knockout mice. Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA. Equal amounts of mtDNA shown on the top of the gel, were loaded as the endogenous control. This experiment has performed three times.


In organello replication assays were performed on heart mitochondria isolated from 8 - week - old control Tefm knockout mice ( n = 5 mice for each group ) . VDAC was used as loading control .

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In organello replication assays were performed on heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). VDAC was used as loading control.


Northern blot analyses of 7S in 4 - 8 - week - old control Tefm knockout mouse hearts ( n = 12 mice for each group ) . 5S rRNA was used as loading control . from tissue - specific Mterf4 knockout mouse hearts tissue - specific Polrmt knockout mouse hearts were loaded as positive negative control , respectively .

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Northern blot analyses of the 7S RNA levels in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice for each group). 5S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively.


Strand - specific RT - qPCR detection of mitochondrial precursor transcripts containing antisense of mt - tRNAA antisense of mt - tRNAC in control Tefm knockout mice ( n = 12 mice for each group ) . mt - mRNAs are shown in blue mt - tRNAs in grey . Arrows show binding position of RT - qPCR primers . Actin transcript was used as endogenous control .

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Strand-specific RT-qPCR detection of the mitochondrial precursor transcripts containing the antisense of mt-tRNAA and antisense of mt-tRNAC in control and Tefm knockout mice (n= 12 mice for each group). The mt-mRNAs are shown in light blue and mt-tRNAs in grey. Arrows show the binding position of RT-qPCR primers. Actin transcript was used as endogenous control.


Strand - specific RT - qPCR detection of mitochondrial precursor transcripts spanning junction of Nd5 antisense of Nd6 in control Tefm knockout mice ( n = 25 mice for each group ) . mt - mRNAs are shown in blue . Arrows show binding position of RT - qPCR primers . Actin transcript was used as endogenous control .

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Strand-specific RT-qPCR detection of the mitochondrial precursor transcripts spanning the junction of Nd5 and antisense of Nd6 in control and Tefm knockout mice (n= 25 mice for each group). The mt-mRNAs are shown in light blue. Arrows show the binding position of RT-qPCR primers. Actin transcript was used as endogenous control.


Northern blot analyses of precursor transcripts containing antisense of mt - tRNAA in 4 - 8 - week - old control Tefm knockout mouse hearts ( n = 6 mice for each group ) .

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Northern blot analyses of the precursor transcripts containing the antisense of mt-tRNAA in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 6 mice for each group).


Volcano plot showing TEFM - BioID . TEFM - BirA * - HA was stably expressed in HEK 293 , followed by streptavidin affinity purification mass spectrometry to detect associated . Mitochondrial targeted BirA * ( MTS - BirA * ) was used as a control . significantly enriched are presented above horizontal dashed with 5 % false discovery rate ( FDR ) significance . blue dots highlight involved in mtDNA transcription green dots represent involved in metabolism . other are in grey .

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Volcano plot showing TEFM-BioID. TEFM-BirA*-HA was stably expressed in HEK 293 cells, followed by streptavidin affinity purification and mass spectrometry analysis to detect the associated proteins. Mitochondrial targeted BirA* (MTS-BirA*) was used as a control. The significantly enriched proteins are presented above the horizontal dashed line with 5% false discovery rate (FDR) significance. The blue dots highlight proteins involved in mtDNA transcription and the green dots represent proteins involved in RNA metabolism. All other proteins are in grey.


( A - C ) Individuals in neuropathology cohort were divided in groups of no significant pathology ( N = 31 ) , AD ( N = 36 ) , PD ( N = 13 ) , others ( N = 8 ) , analyzed together with N = 9 additional subjects with non - AD tauopathies ( PSP / CBD ) . Data is shown for plasma P - tau217 ( panel A ) , plaque density ( panel B ) , tangle density ( panel C ) . Plasma P - tau217 was not elevated in PSP / CBD compared to subjects without significant pathology . AD , Alzheimer ' s disease ; CBD , corticobasal degeneration ; PSP , progressive supranuclear palsy ; PD , Parkinson ' s disease . central band of boxes are medians boxes show inter - quartile ranges . whiskers are defined as smallest ( largest ) observation greater ( less ) than or equal to first ( third ) quartile minus ( plus ) 1 . 5 times inter - quartile range .

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(A-C) Individuals in the neuropathology cohort were divided in groups of no significant primary pathology (N=31), AD (N=36), PD (N=13), and others (N=8), and analyzed together with N=9 additional subjects with primary non-AD tauopathies (PSP/CBD). Data is shown for plasma P-tau217 (panel A), plaque density (panel B), and tangle density (panel C). Plasma P-tau217 was not elevated in PSP/CBD compared to subjects without significant primary pathology. AD, Alzheimer's disease; CBD, corticobasal degeneration; PSP, progressive supranuclear palsy; PD, Parkinson's disease. The central band of the boxes are medians and the boxes show inter-quartile ranges. The whiskers are defined as the smallest (largest) observation greater (less) than or equal to the first (third) quartile minus (plus) 1.5 times the inter-quartile range.


Panel B shows associations between plasma P - tau217 Aβ PET stratified by tertiles ( T ) of tau PET ( T1 : SUVR ≤ 1 . 05 , T2 : 1 . 05 < SUVR ≤ 1 . 11 , T3 : 1 . 11 < SUVR ≤ 3 . 03 ) . Panel C shows associations between plasma P - tau217 tau PET stratified by tertiles ( T ) of Aβ PET ( T1 : SUVR ≤ 0 . 468 , T2 : 0 . 468 < SUVR ≤ 0 . 579 , T3 : 0 . 579 < SUVR ≤ 1 . 08 ) .

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Panel B shows associations between plasma P-tau217 and Aβ PET stratified by tertiles (T) of tau PET (T1: SUVR ≤ 1.05, T2: 1.05 < SUVR ≤ 1.11, T3: 1.11 < SUVR ≤ 3.03).   Panel C shows associations between plasma P-tau217 and tau PET stratified by tertiles (T) of Aβ PET (T1: SUVR ≤ 0.468, T2: 0.468 < SUVR ≤ 0.579, T3: 0.579 < SUVR ≤ 1.08).


G . Purified recombinant FLAG - FAM111A were incubated at indicated temperatures for 4 h , FAM111A auto - proteolytic activity was analyzed by immunoblotting with FLAG antibody . H . As in ( G ) , using recombinant human FLAG - FAM111B .

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G. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting with FLAG antibody. H. As in (G), using recombinant human FLAG-FAM111B proteins.


I . U2OS lines conditionally expressing indicated GFP - FAM111A alleles were fixed at indicated times after treatment with Doxycycline ( DOX ) to induce expression of transgenes stained with crystal violet .

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I. U2OS cell lines conditionally expressing indicated GFP-FAM111A alleles were fixed at the indicated times after treatment with Doxycycline (DOX) to induce expression of the transgenes and stained with crystal violet.


A . Immunoblot of stable U2OS lines left untreated or incubated in presence of DOX to induce expression of WT or mutant forms of GFP - FAM111A .

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A. Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP-FAM111A.


B . replication rates in U2OS / GFP - FAM111A treated with DOX for indicated times , - labeled with EdU stained with DAPI were analyzed by quantifying EdU signal in S phase using quantitative image - cytometry ( QIBC ) ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 per condition ) . See also Appendix Figure S6A .

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B. DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse-labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image-based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6A.


C . treated as in ( B ) were pre - extracted , fixed stained with PCNA or MCM2 antibody , subsequently analyzed by QIBC ( n > 2000 per condition ) .

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C. Cells treated as in (B) were pre-extracted, fixed and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC (n>2000 cells per condition).


D - F . of data in ( C ) ( red bars , mean ) . in S phase were identified on EdU positivity . See also Appendix Figure S6B - D .

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D-F. Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Figure S6B-D.


G . of FAM111A interactors . U2OS / GFP - FAM111A WT were treated or not with DOX for 4 h , subjected to GFP immunoprecipitation ( IP ) analyzed by mass spectrometry . Volcano plot shows enrichment of ( + DOX / - DOX ) plotted against P value . Dashed lines indicate significance thresholds ( FDR < 0 . 05 ; s0 = 1 ) .

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G. Analysis of FAM111A interactors. U2OS/GFP-FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP) and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/-DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).


H . U2OS or U2OS / ∆ FAM111A were subjected to IP with IgG ( control ) or RFC1 antibody followed by immunoblotting with indicated antibodies .

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H. U2OS or U2OS/∆FAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies.


I . U2OS transfected with empty vector ( EV ) or indicated RFC subunit expression plasmids were subjected to FLAG IP immunoblotted with indicated antibodies .

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I. U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies.


J . As in ( C ) , except that were stained with RFC1 antibody ( n > 2000 per condition ) .

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J. As in (C), except that cells were stained with RFC1 antibody (n>2000 cells per condition).


K . of data in ( J ) for S phase ( EdU - positive ) ( red bars , mean ) .

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K. Quantification of data in (J) for S phase (EdU-positive) cells (red bars, mean).


A . Immunoblot of U2OS / GFP - FAM111A WT treated with DOX for indicated times .

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A. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated with DOX for the indicated times.


B . Representative of U2OS / GFP - FAM111A lines that were treated or not with DOX , fixed stained with γ - H2AX antibody DAPI .

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B. Representative images of U2OS/GFP-FAM111A cell lines that were treated or not with DOX, fixed and stained with γ-H2AX antibody and DAPI.


C . in ( B ) were subjected to QIBC of γ - H2AX signal ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 per condition ) .

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C. Cells in (B) were subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


D . Immunoblot of U2OS / GFP - FAM111A WT treated or not with DOX pan - Caspase inhibitor Z - VAD - FMK as indicated .

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D. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated or not with DOX and the pan-Caspase inhibitor Z-VAD-FMK as indicated.


Transcriptional activity in U2OS / GFP - FAM111A treated or not with DOX for indicated times were analyzed by QIBC ( red bars , mean ; n > 2000 per condition ) .

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Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX for the indicated times were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


Transcriptional activity in U2OS / GFP - FAM111A treated or not with DOX - labeled with EU were analyzed by QIBC ( red bars , mean ; n > 2000 per condition ) .

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Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


Transcriptional activity in U2OS / GFP - FAM111A treated or not with DOX - labeled with EU stained with DAPI were analyzed by QIBC ( red bars , mean ; n > 2000 per condition ) .

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Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU and stained with DAPI were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


H . U2OS / GFP - FAM111A lines treated or not with DOX were pre - extracted , fixed stained with RPB1 antibody , analyzed by QIBC ( n > 2000 per condition ) . I . of data in ( H ) ( red bars , mean ) .

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H. U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, and analyzed by QIBC (n>2000 cells per condition). I. Quantification of data in (H) (red bars, mean).


U2OS / GFP - FAM111A lines treated or not with DOX were pre - extracted , fixed stained with RPB1 antibody , J . Representative from experiment in ( H ) . Scale bars , 10 μm .

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U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, J. Representative images from the experiment in (H). Scale bars, 10 μm.


B . Immunoblot of U2OS lines left untreated or incubated in presence of DOX to induce expression of indicated GFP - FAM111A alleles . U2OS / GFP - FAM111A WT ( low ) express transgene at a lower level than U2OS / GFP - FAM111A WT used in Figure 1 2 ( see Figure EV4A ) .

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B. Immunoblot analysis of U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of the indicated GFP-FAM111A alleles. U2OS/GFP-FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP-FAM111A WT cells used in Figure 1 and 2 (see Figure EV4A).


C . U2OS / GFP - FAM111A lines treated or not with DOX , - labeled with EdU stained with DAPI were analyzed for DAPI EdU signal using QIBC .

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C. U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC.


U2OS / GFP - FAM111A lines treated or not with DOX , - labeled with EdU stained with DAPI were analyzed for DAPI EdU signal using QIBC . D . of data in ( C ) for S phase ( EdU - positive ) ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 per condition ) .

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U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. D. Quantification of data in (C) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


U2OS / GFP - FAM111A lines treated or not with DOX , stained with DAPI were analyzed for DAPI signal using QIBC . were - labeled with EU .

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U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. cells were pulse-labeled with EU.


U2OS / GFP - FAM111A lines treated or not with DOX , stained with DAPI were analyzed for DAPI signal using QIBC . of EU incorporation in in ( E ) ( red bars , mean ; n > 2000 per condition ) .

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U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. Quantification of EU incorporation in cells in (E) (red bars, mean; n>2000 cells per condition).


G . U2OS / GFP - FAM111A lines that were treated or not with DOX were stained with RFC1 antibody , pre - extracted fixed , stained with DAPI . RFC1 DAPI signal intensities were analyzed by QIBC .

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G. U2OS/GFP-FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre-extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC.


U2OS / GFP - FAM111A lines that were treated or not with DOX were pre - extracted fixed , stained with DAPI . DAPI signal intensities were analyzed by QIBC . H . were stained with RPB1 antibody .

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U2OS/GFP-FAM111A cell lines that were treated or not with DOX were pre-extracted and fixed, and stained with DAPI. DAPI signal intensities were analyzed by QIBC. H. cells were stained with RPB1 antibody.


I . Immunoblot of U2OS / GFP - FAM111A lines treated or not with DOX in absence or presence of Z - VAD - FMK .

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I. Immunoblot analysis of U2OS/GFP-FAM111A cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


Immunoblot of lines treated or not with DOX in absence or presence of Z - VAD - FMK . J . using U2OS lines conditionally expressing ectopic untagged FAM111A alleles .

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Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK. J. using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles.


K . Purified recombinant FLAG - FAM111A were incubated at indicated temperatures for 4 h , FAM111A auto - proteolytic activity was analyzed by immunoblotting .

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K. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting.


L . GFP IPs from U2OS lines expressing GFP - FAM111A WT or D528G mutant were analyzed by mass spectrometry . Volcano plot shows enrichment of ( WT / D528G ) plotted against P value . Dashed lines indicate significance thresholds ( FDR < 0 . 05 ; s0 = 1 ) .

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L. GFP IPs from U2OS cell lines expressing GFP-FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).


B . U2OS / GFP - FAM111A WT treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies . C . As in ( B ) , using U2OS / GFP - FAM111B WT .

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B. U2OS/GFP-FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies. C. As in (B), using U2OS/GFP-FAM111B WT cells.


D . Purified recombinant FLAG - FAM111A FLAG - FAM111B were incubated separately or mixed , subjected to IP with FAM111A antibody immunoblotted with FLAG antibody .

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D. Purified recombinant FLAG-FAM111A and FLAG-FAM111B proteins were incubated separately or mixed, subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody.


E . Immunoblot of U2OS / GFP - FAM111B lines treated or not with DOX in absence or presence of Z - VAD - FMK .

E. Immunoblot analysis of U2OS/GFP-FAM111B cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


F . in ( E ) were - labeled with EdU , stained with DAPI analyzed for DAPI EdU signal using QIBC .

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F. Cells in (E) were pulse-labeled with EdU, stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC.


analyzed using QIBC . G . of data in ( F ) for S phase ( EdU - positive ) ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 per condition ) .

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Cells analyzed using QIBC. G. Quantification of data in (F) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


stained with DAPI analyzed for DAPI signal using QIBC . H . were - labeled with EU .

Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC. H. cells were pulse-labeled with EU.


analyzed using QIBC . I . of EU incorporation in in ( H ) ( red bars , mean ; n > 2000 per condition ) .

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Cells analyzed using QIBC. I. Quantification of EU incorporation in cells in (H) (red bars, mean; n>2000 cells per condition).


J . U2OS / GFP - FAM111B lines ( Figure 5E ) were fixed at indicated times after DOX treatment stained with crystal violet .

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J. U2OS/GFP-FAM111B cell lines (Figure 5E) were fixed at the indicated times after DOX treatment and stained with crystal violet.


K . in ( E ) were fixed , immunostained with γ - H2AX antibody subjected to QIBC of γ - H2AX signal ( red bars , mean ; n > 2000 per condition ) .

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K. Cells in (E) were fixed, immunostained with γ-H2AX antibody and subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean; n>2000 cells per condition).


L . Immunoblot of purified recombinant FLAG - FAM111B their auto - cleavage products .

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L. Immunoblot analysis of purified recombinant FLAG-FAM111B proteins and their auto-cleavage products.


C . Aliquots of supernatants of indicated HEK293T transfectants ( expressing λ chains ) were resolved under non - reducing conditions blotted with anti - µ antibodies . Wild type µs chains are secreted only as pentamers hexamers , whilst mutants lacking C575 release mostly µs2L2 subunits ( indicated with a red arrow ) as does double C414A - C575A . L566A releases instead mainly µs2L2 subunits of faster migration ( blue arrow ) . C414 mutants release no hexamers , but smaller assemblies , including fast migrating µs2L2 subunits .

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C. Aliquots of the supernatants of the indicated HEK293T transfectants (all expressing λ chains) were resolved under non-reducing conditions and blotted with anti-µ antibodies. Wild type µs chains are secreted only as pentamers and hexamers, whilst mutants lacking C575 release mostly µs2L2 subunits (indicated with a red arrow) as does the double C414A-C575A. L566A releases instead mainly µs2L2 subunits of faster migration (blue arrow). C414 mutants release no hexamers, but smaller assemblies, including fast migrating µs2L2 subunits.


E . Aliquots of supernatants were treated with or without Endo - H electrophoresed under reducing conditions . blot was decorated with anti - µ antibodies . N563 - glycans acquire Endo - H resistance in µsL566A µsC575A , but remain largely sensitive in wild type µs .

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E. Aliquots of the supernatants were treated with or without Endo-H and electrophoresed under reducing conditions. The blot was decorated with anti-µ antibodies. N563-glycans acquire Endo-H resistance in µsL566A and µsC575A, but remain largely sensitive in wild type µs.


A . Aliquots of lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non - reducing conditions before or after removal of N - glycans by PNGaseF . Blots were decorated with anti - µ antibodies . Blue red arrows point to closed ( oxidized ) open µs2L2 species respectively .

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A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively.


Intracellular µs2L2 species purified from murine hybridoma B1 . 8 were subjected to sequential alkylation analyzed by mass spectrometry In MS / MS spectrum Y B ions are shown in blue red , respectively .

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Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS/MS spectrum Y and B ions are shown in blue and red, respectively.


A . I . 29µ + B lymphoma were treated with LPS to induce differentiation towards IgM secretion . At indicated times , were lysed in TritonX114 to separate soluble integral ( aqueous detergent phases , respectively ) . Aliquots from two phases were resolved electrophoretically blot was decorated by anti - µ antibodies . blue arrow points at a band likely corresponding to µs2L2C575OX , of which increases upon LPS stimulation .

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A. I.29µ+ B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion. At the indicated times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to µs2L2C575OX, the intensity of which increases upon LPS stimulation.


C . µ - chains with oxidized C575 bind AMS after reduction migrate more slowly than those which were reduced at time of lysis hence alkylated by NEM .

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C. µ-chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM.


D . bar graphs show of C575 C414 oxidation , expressed as between IAA NEM modified peptides .

D. The bar graphs show the levels of C575 and C414 oxidation, expressed as the ratio between IAA and NEM modified peptides.


ERp44KO HeLa expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . A . Aliquots from spent media ( Extracellular ) were blotted under non - reducing conditions decorated with anti - µ antibodies . blue arrow points at faster migrating µs2L2 released by expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575 - dependent retention , wild type µs are secreted only as polymers ( black arrow ) . Traces of covalent HaloERp44 - µ2L2 complexes ( black asterisks ) are detected in media of expressing HaloERp44 ∆ RDEL µs or L566A . slightly faster mobility of µs2L2 µsL complexes released by expressing ERp44 ∆ RDEL suggest that latter inhibits processing of N563 glycan , likely hindering it to Golgi enzymes upon non - covalent binding . Data information : membranes were decorated with anti - μ antibodies . Black roundish spots seen in left panels are artifacts that formed during blotting procedures signal acquisition .

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ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


ERp44KO HeLa expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . B . corresponding lysates are shown in this panel . short black arrow points at polymers formed in ERp44KO HeLa upon expression of wild type HaloERp44 . black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2 . Traces of intracellular µs2L2 - ERp44 ∆ RDEL complexes are visible in µs L566A but absent from C575A expressing . Data information : membranes were decorated with anti - μ antibodies . Black roundish spots seen in left panels are artifacts that formed during blotting procedures signal acquisition .

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ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. B. The corresponding cell lysates are shown in this panel. The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44. The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2. Traces of intracellular µs2L2-ERp44∆RDEL complexes are visible in µs and L566A but absent from C575A expressing cells. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


C . Lysates of ERp44KO transfectants were precipitated with NP - sepharose , resolved under non reducing conditions blot decorated sequentially with anti - µ , anti - ERp44 or anti - Halo . two black asterisks point at bands corresponding to covalent complexes containing µs2L2 endogenous or Halo - tagged ERp44 .

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C. Lysates of the ERp44KO transfectants were precipitated with NP-sepharose, resolved under non reducing conditions and the blot decorated sequentially with anti-µ, anti-ERp44 or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.