Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx - SGTA and recovery on HisPur Cobalt resin the eluted fractions were subjected to RNaseA treatment ( C ) - tRNA " and " + tRNA " indicate tRNA - lacking and tRNA - bound protein species , 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

Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin the eluted fractions were subjected to RNaseA treatment (C) -tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively.


D Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx - SGTA and recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide ( CTAB ) ( D ) ; - tRNA " and " + tRNA " indicate tRNA - lacking and tRNA - bound protein species , 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

D Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide (CTAB) (D) ;-tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively.


B Variants of PPLssKO - C99 were translated in the presence of 2 μM HisTrx - SGTA using templates lacking a stop codon and terminating either before ( " - TMD " ) or at indicated amino acid distance after the TMD . Pull - down with HisPur Cobalt resin was carried out as described in Materials and Methods , eluted material resolved by SDS - PAGE and results visualised by phosphorimaging . tRNA - bound species were verified by treating PPLssKO - C99 variant terminating immediately after the TMD ( TMD + 0 ) with RNaseA ( lane 9 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Variants of PPLssKO-C99 were translated in the presence of 2 μM HisTrx-SGTA using templates lacking a stop codon and terminating either before ("-TMD") or at indicated amino acid distance after the TMD. Pull-down with HisPur Cobalt resin was carried out as described in Materials and Methods, eluted material resolved by SDS-PAGE and results visualised by phosphorimaging. tRNA-bound species were verified by treating PPLssKO-C99 variant terminating immediately after the TMD (TMD + 0) with RNaseA (lane 9).


translation reactions were carried out using RNAs coding for the indicated TA - proteins and lacking a stop codon . Sec61β 3R was used as a control protein without a functional TMD . Filled circles indicate tRNA - bound TA - protein species recovered with HisTrx - SGTA .

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

translation reactions were carried out using RNAs coding for the indicated TA-proteins and lacking a stop codon. Sec61β 3R was used as a control protein without a functional TMD. Filled circles indicate tRNA-bound TA-protein species recovered with HisTrx-SGTA.


D Rabbit reticulocyte lysate was left untreated ( " untreat . " ) , incubated with control immobilised antibodies ( " contr . depl . " ) or immunodepleted of Hbs1L , a factor that together with Pelota and ABCE1 [ 4 ] , mediates splitting of ribosomes stalled on truncated mRNAs ( see diagram ) . These lysates were used to translate FLAG - tagged Sec61β from an mRNA lacking a stop codon in the presence of 2 μM HisTrx or HisTrx - SGTA and samples were processed as described for ( B ) .

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

D Rabbit reticulocyte lysate was left untreated ("untreat."), incubated with control immobilised antibodies ("contr. depl.") or immunodepleted of Hbs1L, a factor that together with Pelota and ABCE1 [4], mediates splitting of ribosomes stalled on truncated mRNAs (see diagram). These lysates were used to translate FLAG-tagged Sec61β from an mRNA lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and samples were processed as described for (B).


E tRNA - bound species of FLAG - tagged Sec61β from experiment shown in panel ( D ) were quantified and expressed relative to the value obtained for the control - depleted RRL . Shown is the mean with standard error of mean for n = 4 biological replicates . Statistical significance was calculated using unpaired t test with Welch ' s correction . ns - not significant .

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

E tRNA-bound species of FLAG-tagged Sec61β from experiment shown in panel (D) were quantified and expressed relative to the value obtained for the control-depleted RRL. Shown is the mean with standard error of mean for n=4 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.


Sec61β wild - type ( WT ) and the 3R variant tagged at their N - terminus with the FLAG epitope were translated in RRL from RNAs lacking a stop codon . Ribosome - nascent chain complexes ( RNCs ) were recovered using anti - FLAG affinity resin , beads were washed , bound proteins eluted with 3xFLAG peptide and RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS - PAGE and immunoblotted for endogenous rabbit SGTA , FLAG - tagged translation products , proteins of the 40S ( Rps3 ) and 60S ( Rpl17 ) ribosomal subunits or analysed by phosphorimaging ( 35S ) . The 3xFLAG peptide eluted material ( lanes 4 and 5 ) corresponds to ~ 10 % of the sedimented RNCs ( lanes 7 and 8 ) .

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

Sec61β wild-type (WT) and the 3R variant tagged at their N-terminus with the FLAG epitope were translated in RRL from RNAs lacking a stop codon. Ribosome-nascent chain complexes (RNCs) were recovered using anti-FLAG affinity resin, beads were washed, bound proteins eluted with 3xFLAG peptide and RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS-PAGE and immunoblotted for endogenous rabbit SGTA, FLAG-tagged translation products, proteins of the 40S (Rps3) and 60S (Rpl17) ribosomal subunits or analysed by phosphorimaging (35S). The 3xFLAG peptide eluted material (lanes 4 and 5) corresponds to ~10% of the sedimented RNCs (lanes 7 and 8).


A , B PPL - C99 - TMD ( A ) and PPL - C99FL ( B ) were in vitro translated in RRL from mRNAs lacking a stop codon in the presence of 2 μM HisTrx or HisTrx - SGTA and ε - TDBA - Lys - tRNA analogue . Reactions were irradiated with the UV light to induce photo - cross - linking , RNCs isolated and adducts immunoprecipitated with mouse anti - SRP54 , mouse anti - SGTA or a control antibody as described in Materials and Methods . Samples were resolved by SDS - PAGE and results visualised by phosphorimaging . C - terminal 40 amino acid residues located in the ribosomal exit tunnel are indicated . " x SRP54 " and " x HisTrx - SGTA " indicate cross - linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx - SGTA , respectively . Arrows indicate unmodified translation products .

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

A, B PPL-C99-TMD (A) and PPL-C99FL (B) were in vitro translated in RRL from mRNAs lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and ε-TDBA-Lys-tRNA analogue. Reactions were irradiated with the UV light to induce photo-cross-linking, RNCs isolated and adducts immunoprecipitated with mouse anti-SRP54, mouse anti-SGTA or a control antibody as described in Materials and Methods. Samples were resolved by SDS-PAGE and results visualised by phosphorimaging. C-terminal 40 amino acid residues located in the ribosomal exit tunnel are indicated. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively. Arrows indicate unmodified translation products.


C PPL - C99FL variants lacking a stop codon and carrying the indicated Lys to Arg substitutions were in vitro translated in the presence of 2 μM HisTrx - SGTA . Reactions were processed using chicken anti - SGTA antibody for immunoprecipitation of SGTA adducts . D Cross - linking adducts between ribosome - stalled PPL - C99FL lysine mutants and recombinant HisTrx - SGTA shown in panel ( C ) were quantified , any differences in translation efficiency accounted for and cross - linking efficiency expressed relative to the wild - type ( WT ) protein . Shown is the mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t test with Welch ' s correction .

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

C PPL-C99FL variants lacking a stop codon and carrying the indicated Lys to Arg substitutions were in vitro translated in the presence of 2 μM HisTrx-SGTA. Reactions were processed using chicken anti-SGTA antibody for immunoprecipitation of SGTA adducts. D Cross-linking adducts between ribosome-stalled PPL-C99FL lysine mutants and recombinant HisTrx-SGTA shown in panel (C) were quantified, any differences in translation efficiency accounted for and cross-linking efficiency expressed relative to the wild-type (WT) protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.


A , B PPL - C99FL lacking a stop codon was in vitro translated in RRL in the presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε - TDBA - Lys - tRNA analogue . Reactions were carried out in the presence ( A ) or absence ( B ) of 2 μM HisTrx - SGTA . Samples were subjected to UV light - induced photo - cross - linking and processed as described for Fig 5 . " x SRP54 " and " x HisTrx - SGTA " indicate cross - linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx - SGTA , respectively , whilst " endo . SGTA " indicates cross - linking adducts with endogenous SGTA . Cross - linking products between stalled PPL - C99FL and SGTA are also indicated with arrowheads , whilst arrows indicate unmodified translation products .

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

A, B PPL-C99FL lacking a stop codon was in vitro translated in RRL in the presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε-TDBA-Lys-tRNA analogue. Reactions were carried out in the presence (A) or absence (B) of 2 μM HisTrx-SGTA. Samples were subjected to UV light-induced photo-cross-linking and processed as described for Fig 5. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively, whilst "endo. SGTA" indicates cross-linking adducts with endogenous SGTA. Cross-linking products between stalled PPL-C99FL and SGTA are also indicated with arrowheads, whilst arrows indicate unmodified translation products.


C , D chemical cross - linking using DSS reagent was carried out . E DSS - mediated HisTrx - SGTA cross - linking efficiency to PPL - C99FL in the absence or presence of ER - derived microsomes was quantified . For each repeat , intensity of the cross - linked SGTA adduct in the absence of microsomes was set as 100 % . Shown is the mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t test with Welch ' s correction .

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

C, D chemical cross-linking using DSS reagent was carried out. E DSS-mediated HisTrx-SGTA cross-linking efficiency to PPL-C99FL in the absence or presence of ER-derived microsomes was quantified. For each repeat, intensity of the cross-linked SGTA adduct in the absence of microsomes was set as 100%. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.


A Indicated precursor proteins were translated in vitro using RRL supplemented with 20 μM HA - tagged ubiquitin and 2 μM of either HisTrx or HisTrx - SGTA . RNCs were isolated , ubiquitinated nascent chains recovered using anti - HA agarose , samples resolved by SDS - PAGE and analysed by phosphorimaging . Ubiquitinated precursor protein species were assigned based on SDS - PAGE electrophoretic mobility and are indicated in red . A schematic diagram of the proteins used is shown and the ~ 40 residues of the nascent chain located in the ribosomal exit tunnel are indicated . " ss " - signal sequence , " TMD " - transmembrane domain .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Indicated precursor proteins were translated in vitro using RRL supplemented with 20 μM HA-tagged ubiquitin and 2 μM of either HisTrx or HisTrx-SGTA. RNCs were isolated, ubiquitinated nascent chains recovered using anti-HA agarose, samples resolved by SDS-PAGE and analysed by phosphorimaging. Ubiquitinated precursor protein species were assigned based on SDS-PAGE electrophoretic mobility and are indicated in red. A schematic diagram of the proteins used is shown and the ~40 residues of the nascent chain located in the ribosomal exit tunnel are indicated. "ss" - signal sequence, "TMD" - transmembrane domain.


B Individual , clearly resolved ubiquitinated species of the indicated proteins ( labelled 1 to 4 ) were quantified and their intensity in HisTrx - SGTA containing samples shown relative to samples supplemented with HisTrx control protein . Shown is the mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t test with Welch ' s correction . ns - not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Individual, clearly resolved ubiquitinated species of the indicated proteins (labelled 1 to 4) were quantified and their intensity in HisTrx-SGTA containing samples shown relative to samples supplemented with HisTrx control protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.


In panels ( A ) violin plots show the variability in continuous leaf 16 , ear phenotypes among the 60 individual plants used in downstream analyses . Data information : In all violin plots , the median is indicated by the white circle . The black box extends from the 25th to the 75th percentile , and black whiskers extend from each end of the box to the most extreme values within 1 . 5 times the interquartile range from the respective end . Data points beyond this range are shown as black dots . The red open circle indicates the mean of the distribution , with red whiskers extending to 1 standard deviation above and below the mean .

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

In panels (A) violin plots show the variability in continuous leaf 16, ear phenotypes among the 60 individual plants used in downstream analyses. Data information: In all violin plots, the median is indicated by the white circle. The black box extends from the 25th to the 75th percentile, and black whiskers extend from each end of the box to the most extreme values within 1.5 times the interquartile range from the respective end. Data points beyond this range are shown as black dots. The red open circle indicates the mean of the distribution, with red whiskers extending to 1 standard deviation above and below the mean.


In panels ( E ) , violin plots show the variability in plant height phenotypes among the 60 individual plants used in downstream analyses . Data information : In all violin plots , the median is indicated by the white circle . The black box extends from the 25th to the 75th percentile , and black whiskers extend from each end of the box to the most extreme values within 1 . 5 times the interquartile range from the respective end . Data points beyond this range are shown as black dots . The red open circle indicates the mean of the distribution , with red whiskers extending to 1 standard deviation above and below the mean .

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

In panels (E), violin plots show the variability in plant height phenotypes among the 60 individual plants used in downstream analyses. Data information: In all violin plots, the median is indicated by the white circle. The black box extends from the 25th to the 75th percentile, and black whiskers extend from each end of the box to the most extreme values within 1.5 times the interquartile range from the respective end. Data points beyond this range are shown as black dots. The red open circle indicates the mean of the distribution, with red whiskers extending to 1 standard deviation above and below the mean.


Boxplots showing differences of the APTT time ( left ) and ME1 - enriched protein expressions ( right ) between mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Mann - Whitney - Wilcoxon test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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

Boxplots showing differences of the APTT time (left) and ME1-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Boxplots showing differences of IL - 6 level , IL - 10 level ( left ) and ME15 - enriched protein expressions ( right ) between mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Mann - Whitney - Wilcoxon test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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

Boxplots showing differences of IL-6 level, IL-10 level (left) and ME15-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Rose plots indicating the number of all detected and differently expressed tissues - enhanced proteins in mild ( left ) and severe patients ( right ) compared with healthy controls . Tissues labeled in orange represented tissues with the largest proportions ( 100 % of all detected tissue - enhanced proteins ) of altered tissues - enhanced proteins .

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

Rose plots indicating the number of all detected and differently expressed tissues-enhanced proteins in mild (left) and severe patients (right) compared with healthy controls. Tissues labeled in orange represented tissues with the largest proportions (100% of all detected tissue-enhanced proteins) of altered tissues-enhanced proteins.


Boxplots indicating the expression level of known brain dysfunctional biomarkers in control ( n = 12 ) , mild ( n = 13 ) and severe ( n = 18 ) patients . Differences between groups were estimated using Kruskal - Wallis test . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

Boxplots indicating the expression level of known brain dysfunctional biomarkers in control (n=12), mild (n=13) and severe (n=18) patients. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Boxplot showing the selected cell type specific proteins among control ( n = 12 ) , mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Kruskal - Wallis test . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

Boxplot showing the selected cell type specific proteins among control (n=12), mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Heatmap of IFNG , GZMB and PRF1 gene expression in COVID - 19 patients . Average expression values were centered and scaled . Red indicates a higher expression and blue indicates a lower expression .

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

Heatmap of IFNG, GZMB and PRF1 gene expression in COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.


Heatmap of the expression of genes enriched in IL - 17 signaling pathway between healthy control and COVID - 19 patients . Average expression values were centered and scaled . Red indicates a higher expression and blue indicates a lower expression .

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

Heatmap of the expression of genes enriched in IL-17 signaling pathway between healthy control and COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.


COVID - 19 severity is associated with significant changes in lipoprotein subclasses including high - density lipoprotein subclass - 1 ( HDL1 ) , HDL4 , low - density lipoprotein subclasses ( LDL1 , LDL4 , LDL5 ) , very low - density lipoprotein subclass - 5 ( VLDL5 ) and their compositional components ( ApoA1 , triglycerides , cholesterol ) . TG : triglycerides ; FC : free cholesterol ; CE : cholesteryl esters ; CH : total cholesterol ( i . e . , FC + CE ) ; PL : total phospholipids ; A1 : ApoA1 ; A2 : ApoA2 ; L1TG : TG in LDL1 ; L1TG % : percentages of L1TG in total lipids of LDL1 ; L1 % : percentage of LDL1 in all LDL ; L - TG % : percentages of L - TG ( TG in LDL ) in total lipids of LDL ; V5FC % , V5CE % : percentages of V5FC ( FC in VLDL5 ) and V5CE ( CE in VLDL5 ) in total lipids of VLDL5 ; L5CE % , L5CH % : percentages of L5CE ( CE in LDL5 ) and L5CH ( CH in LDL5 ) in total lipids of LDL5 ; H1FC % : percentages of H1FC ( FC in HDL1 ) in total lipids of HDL1 ; H - A2 : ApoA2 in both HDL and nascent HDL ; H4A1 , H4A2 , H4CE , H4CH , H4FC , H4PL : ApoA1 , ApoA2 , CE , CH , FC and PL in HDL4 ;

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

COVID-19 severity is associated with significant changes in lipoprotein subclasses including high-density lipoprotein subclass-1 (HDL1), HDL4, low-density lipoprotein subclasses (LDL1, LDL4, LDL5), very low-density lipoprotein subclass-5 (VLDL5) and their compositional components (ApoA1, triglycerides, cholesterol). TG: triglycerides; FC: free cholesterol; CE: cholesteryl esters; CH: total cholesterol (i.e., FC + CE); PL: total phospholipids; A1: ApoA1; A2: ApoA2; L1TG: TG in LDL1; L1TG%: percentages of L1TG in total lipids of LDL1; L1%: percentage of LDL1 in all LDL; L-TG%: percentages of L-TG(TG in LDL) in total lipids of LDL; V5FC%, V5CE%: percentages of V5FC(FC in VLDL5) and V5CE(CE in VLDL5) in total lipids of VLDL5; L5CE%, L5CH%: percentages of L5CE(CE in LDL5) and L5CH(CH in LDL5) in total lipids of LDL5; H1FC%: percentages of H1FC (FC in HDL1) in total lipids of HDL1; H-A2: ApoA2 in both HDL and nascent HDL; H4A1, H4A2, H4CE, H4CH, H4FC, H4PL: ApoA1, ApoA2, CE, CH, FC and PL in HDL4;


Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID - 19 severity . Data were represented as means ± SD and differences between groups were estimated using a Student ' s t test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Control , n = 12 ; mild , n = 29 ; severe , n = 17 ; discharge , n = 16 ; sLDLR : soluble low - density lipoprotein receptor ; LCAT : lecithin - cholesterol acyltransferase ; CEPT : cholesteryl - ester transfer protein .

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

Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID-19 severity. Data were represented as means ± SD and differences between groups were estimated using a Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Control, n=12; mild, n=29; severe, n=17; discharge, n=16; sLDLR: soluble low-density lipoprotein receptor; LCAT: lecithin-cholesterol acyltransferase; CEPT: cholesteryl-ester transfer protein.


A . EGFR ( pY1068 , left ) , Akt ( pS473 , middle ) and Erk ( pT202 and pY204 , right ) phosphorylation response in WT ( red ) compared to p22phox - KO ( green ) MCF7 cells as function of EGF concentration ( ng / ml ; nM ) upon 5 ' stimulation with different doses of EGF - Alexa647 quantified from Western blot analysis . N = 4 biological replicates with mean ± SD , P : unpaired two - tailed t - test . B . Same as ( A ) comparing WT ( red ) to RPTPγ - KO ( blue ) MCF7 cells . N = 3 biological replicates with mean ± SD , P : unpaired two - tailed t - test . C . Quantitative Western blot analysis as in ( A ) comparing WT ( red ) and RPTPγ - KO ( blue ) MCF7 cells after EGF stimulus ( 20 , 80 , 160 ng / ml from ( B ) , left column : w / o gef . ) to the cells from the corresponding cell line treated with 10 μM of the EGFR - inhibitor Gefitinib for 1 h and the indicated EGF concentration for the last 5 ' ( ng / ml ) . N = 3 biological replicates with mean ± SD , P : unpaired two - tailed t - test . D .

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

A. EGFR (pY1068, left), Akt (pS473, middle) and Erk (pT202 and pY204, right) phosphorylation response in WT (red) compared to p22phox-KO (green) MCF7 cells as function of EGF concentration (ng/ml; nM) upon 5' stimulation with different doses of EGF-Alexa647 quantified from Western blot analysis. N=4 biological replicates with mean±SD, P:unpaired two-tailed t-test. B. Same as (A) comparing WT (red) to RPTPγ-KO (blue) MCF7 cells. N=3 biological replicates with mean±SD, P: unpaired two-tailed t-test. C. Quantitative Western blot analysis as in (A) comparing WT (red) and RPTPγ-KO (blue) MCF7 cells after EGF stimulus (20, 80, 160 ng/ml from (B), left column: w/o gef.) to the cells from the corresponding cell line treated with 10 μM of the EGFR-inhibitor Gefitinib for 1 h and the indicated EGF concentration for the last 5' (ng/ml). N=3 biological replicates with mean±SD, P: unpaired two-tailed t-test. D.


E . Comparison of normalized EGFR - ( pY1068 - ) phosphorylation as a function of EGF concentration ( N = 10 , from Figs 1A , 1B , EV1E , red ) to EGF - Alexa647 bound to WT MCF7 cells at the corresponding , indicated concentrations normalized to the 160 ng / mL , measured by fluorescence microscopy . N = 5 biological replicates , n = 16 - 19 fields of view , mean ± SD , P : One - way ANOVA with Tukey ' s multiple comparison test .

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

E. Comparison of normalized EGFR- (pY1068-) phosphorylation as a function of EGF concentration (N=10, from Figs 1A, 1B, EV1E, red) to EGF-Alexa647 bound to WT MCF7 cells at the corresponding, indicated concentrations normalized to the 160 ng/mL, measured by fluorescence microscopy. N=5 biological replicates, n=16-19 fields of view, mean±SD, P: One-way ANOVA with Tukey's multiple comparison test.


A . Left panel : comparison of normalized EGF - Alexa647 ( 160 ng / ml ; 5 ' ) fluorescence intensity bound to individual endogenous EGFR expressing MCF10A ( yellow ) , to exogenous EGFR - mCitrine expressing EmCit _ MCF7 cells ( black ) and WT MCF7 cells ( red ) ; Right panel : normalized EGF - Alexa647 fluorescence plotted against normalized EGFR - mCitrine fluorescence intensity in WT ( red ) and EmCit _ MCF7 ( black , with 2nd order polynomal fit : grey line ) cells . N = 3 biological replicates , n > 75 cells , mean ± SD .

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

A. Left panel: comparison of normalized EGF-Alexa647 (160 ng/ml; 5') fluorescence intensity bound to individual endogenous EGFR expressing MCF10A (yellow), to exogenous EGFR-mCitrine expressing EmCit_MCF7 cells (black) and WT MCF7 cells (red); Right panel: normalized EGF-Alexa647 fluorescence plotted against normalized EGFR-mCitrine fluorescence intensity in WT (red) and EmCit_MCF7 (black, with 2nd order polynomal fit: grey line) cells. N=3 biological replicates, n>75 cells, mean±SD.


D . Gray : αL upon each administered dose for individual EmCit _ MCF7 cells to cumulative doses of EGF - Alexa647 ( 2 . 5 - 640 ng / ml ) . N = 3 biological replicates , n = 13 cells . Black : EGF - Alexa647 bound to WT MCF7 cells at the indicated concentrations normalized to the mean fluorescence intensity at 160 ng / ml EGF - Alexa647 ( Fig 1E ; mean ± SD , N = 5 biological replicates , n = 16 - 19 fields of view ) .

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

D. Gray: αL upon each administered dose for individual EmCit_MCF7 cells to cumulative doses of EGF-Alexa647 (2.5-640 ng/ml). N=3 biological replicates, n=13 cells. Black: EGF-Alexa647 bound to WT MCF7 cells at the indicated concentrations normalized to the mean fluorescence intensity at 160 ng/ml EGF-Alexa647 (Fig 1E; mean±SD, N=5 biological replicates, n=16-19 fields of view).


F . Left : EGFR - ( pY1068 - ) phosphorylation response in WT MCF7 cells obtained from western blots normalized to maximal phosphorylation obtained by inhibiting all phosphatases by 0 . 33 mM pervanadate ( N = 6 ; red symbols with mean ± SD and fit to the hill equation ( solid line ) ) at 0 ( plotted as 0 . 001 to fit the logarithmic x - axis ) , 0 . 5 , 1 , 2 , 5 , 10 , 20 , 40 and 80 ng / ml plotted against corresponding αL ( obtained from in cell dose response experiments in EmCit _ MCF7 cells ( D ) ) . Correspondig molecular RPTPγ / EGFR - ratio ( see G , H , Fig EV2H ) is depicted above the graphs ; Inserted into each graph are the values of Hill coefficient ( HC ) and EC50 of the fitted hill equation ( 95 % confidence interval ) . 2nd graph : Same as left graph with αp plotted vs αL both obtained from in cell dose response experiments in EmCit _ MCF7 cells . 3rd - 5th graph : Same as 2nd graph for EmCit _ MCF7 RPTPγ - KO with RPTPγ - mTFP ectopic expression clustered by RPTPγ / EGFR - expression ratio and HC ( Fig EV2G ) .

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

F. Left: EGFR- (pY1068-) phosphorylation response in WT MCF7 cells obtained from western blots normalized to maximal phosphorylation obtained by inhibiting all phosphatases by 0.33 mM pervanadate (N=6; red symbols with mean±SD and fit to the hill equation (solid line)) at 0 (plotted as 0.001 to fit the logarithmic x-axis), 0.5, 1, 2, 5, 10, 20, 40 and 80 ng/ml plotted against corresponding αL (obtained from in cell dose response experiments in EmCit_MCF7 cells (D)). Correspondig molecular RPTPγ/EGFR-ratio (see G, H, Fig EV2H) is depicted above the graphs; Inserted into each graph are the values of Hill coefficient (HC) and EC50 of the fitted hill equation (95% confidence interval). 2nd graph: Same as left graph with αp plotted vs αL both obtained from in cell dose response experiments in EmCit_MCF7 cells. 3rd-5th graph: Same as 2nd graph for EmCit_MCF7 RPTPγ-KO with RPTPγ-mTFP ectopic expression clustered by RPTPγ/EGFR-expression ratio and HC (Fig EV2G).


G . Number of molecules obtained by normalizing background - subtracted fluorescence intensities of individual transfected cells against the mean background intensity of untransfected cells , yielding relative expressions levels independent on the fluorophore ( methods ) . This value was then set into proportion to the known mean number of EGFR per MCF10A cell to yield absolute molecule count / cell . 1st column : EGFR - mCitrine in EmCit _ MCF7 cells ( N = 3 biological replicates , n = 102 cells ) ; 2nd column EGFR - mCitrine expressed in EmCit _ MCF7 RPTPγ - KO expressing RPTPγ - mTFP ( N = 3 biological replicates , n = 26 cells ) ; 3rd column : RPTPγ - mCitrine in MCF7 cells expressing additionally EGFR - mCherry ( N = 3 biological replicates , n = 253 cells ) ; 4th column : RPTPγ - mTFP expressed in EmCit _ MCF7 RPTPγ - KO ( N = 3 biological replicates , n = 26 cells ) ( individual cells with mean + SD ) . Color code in 2nd and 4th column is attribution to respective cluster ( H and EV2G ) .

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

G. Number of molecules obtained by normalizing background-subtracted fluorescence intensities of individual transfected cells against the mean background intensity of untransfected cells, yielding relative expressions levels independent on the fluorophore (methods). This value was then set into proportion to the known mean number of EGFR per MCF10A cell to yield absolute molecule count/cell. 1st column: EGFR-mCitrine in EmCit_MCF7 cells (N=3 biological replicates, n=102 cells); 2nd column EGFR-mCitrine expressed in EmCit_MCF7 RPTPγ-KO expressing RPTPγ-mTFP (N=3 biological replicates, n=26 cells); 3rd column: RPTPγ-mCitrine in MCF7 cells expressing additionally EGFR-mCherry (N=3 biological replicates, n=253 cells); 4th column: RPTPγ-mTFP expressed in EmCit_MCF7 RPTPγ-KO (N=3 biological replicates, n=26 cells) (individual cells with mean+SD). Color code in 2nd and 4th column is attribution to respective cluster (H and EV2G).


C - E ( C ) in cell EGF - dose response imaging for RPTPγ - mCitrine oxidation . Left panel : Representative confocal micrographs of RPTPγ - mCitrine in EmTFP _ MCF7 cells ( top row ) together with its oxidized fraction estimated using DyTo - FLIM ( αox , bottom row ) , upon 10 ' stimulation with EGF - Alexa647 ( 0 - 160 ng / ml ) including 5 ' together with 0 . 5 mM DyTo . Scale bar : 10 μm . Right panel : Quantification depicting the PM - proximal ( orange ) and PM - distal ( blue ) oxidized fractions as functions of receptor occupancy ( αL ) and corresponding EGF - Alexa647 , or H2O2 concentration in EmTFP MCF7 cells expressing RPTPγ - mCitrine ( WT ) or RPTPγC1060S - mCitrine ( C1060S ) as well as WT cells treated with 0 . 5 mM atto590 instead of DyTo ( atto590 ) . Mean of individual cells ( symbols ) with mean ± SD ( black lines ) , N = 3 biological replicates , n = 13 - 15 cells per EGF dose . P : unpaired two - tailed t - test , between PM ( serpentine peripheral structures ) and endosomal ( vesicular structures ) fractions . ( D ) Same as in ( C ) , for RPTPγ - mCitrine oxidation in p22phox - KO cells . N = 3 biological replicates , n = 14 - 26 cells per EGF dose . ( E ) Same as in ( C ) , for TCPTP - mCitrine or TCPTPC216S - mCitrine ( C216S ) oxidation in EmTFP _ MCF7 cells . N = 3 biological replicates , n = 18 - 21 cells per EGF dose .

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

C-E (C) in cell EGF-dose response imaging for RPTPγ-mCitrine oxidation. Left panel: Representative confocal micrographs of RPTPγ-mCitrine in EmTFP_MCF7 cells (top row) together with its oxidized fraction estimated using DyTo-FLIM (αox, bottom row), upon 10' stimulation with EGF-Alexa647 (0-160 ng/ml) including 5' together with 0.5 mM DyTo. Scale bar: 10 μm. Right panel: Quantification depicting the PM-proximal (orange) and PM-distal (blue) oxidized fractions as functions of receptor occupancy (αL) and corresponding EGF-Alexa647, or H2O2 concentration in EmTFP MCF7 cells expressing RPTPγ-mCitrine (WT) or RPTPγC1060S-mCitrine (C1060S) as well as WT cells treated with 0.5 mM atto590 instead of DyTo (atto590). Mean of individual cells (symbols) with mean±SD (black lines), N=3 biological replicates, n=13-15 cells per EGF dose. P: unpaired two-tailed t-test, between PM (serpentine peripheral structures) and endosomal (vesicular structures) fractions. (D) Same as in (C), for RPTPγ-mCitrine oxidation in p22phox-KO cells. N=3 biological replicates, n=14-26 cells per EGF dose. (E) Same as in (C), for TCPTP-mCitrine or TCPTPC216S-mCitrine (C216S) oxidation in EmTFP_MCF7 cells. N=3 biological replicates, n=18-21 cells per EGF dose.


A . Representative confocal micrographs of MCF7 WT cells showing the co - localization of RPTPγ - mCitrine ( 1st column : green ; 3rd column : blue ) and EGFR - mCherry ( 2nd column : green ; 3rd column : yellow ) with recycling - endosome defined by immunostaining against Rab11a ( magenta ) , without ( top row ) or after 30 ' EGF - DyLight405 stimulus ( 160 ng / ml ; bottom row ) . Scale bar : 10 μm .

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

A. Representative confocal micrographs of MCF7 WT cells showing the co-localization of RPTPγ-mCitrine (1st column: green; 3rd column: blue) and EGFR-mCherry (2nd column: green; 3rd column: yellow) with recycling-endosome defined by immunostaining against Rab11a (magenta), without (top row) or after 30' EGF-DyLight405 stimulus (160 ng/ml; bottom row). Scale bar: 10 μm.


B . Fraction of RPTPγ - mCitrine ( cyan ) or EGFR - mCherry ( green ) that spatially overlaps with Rab11a ( top left , N = 3 , n = 23 - 26 cells per timepoint ) , PM ( bottom left , N = 3 biological replicates , n = 15 - 17 cells ) , EEA1 - positive early endosomes ( top right , N = 3 biological replicates , n = 25 - 28 cells ) or Rab7 - positive late endosomes ( bottom right , N = 3 biological replicates , n = 23 - 27 cells ) in MCF7 cells as function of time after 160 ng / ml EGF - stimulus . Orange symbols / dotted line : same for EGFR - mCitrine in RPTPγ - KO cells ( N = 3 biological replicates , n = 17 - 21 cells ) . P : unpaired two - tailed t - test ; colored P values compare to the respective species before stimulation , black in between species .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fraction of RPTPγ-mCitrine (cyan) or EGFR-mCherry (green) that spatially overlaps with Rab11a (top left, N=3, n=23-26 cells per timepoint), PM (bottom left, N=3 biological replicates, n=15-17 cells), EEA1-positive early endosomes (top right, N=3 biological replicates, n=25-28 cells) or Rab7-positive late endosomes (bottom right, N=3 biological replicates, n=23-27 cells) in MCF7 cells as function of time after 160 ng/ml EGF-stimulus. Orange symbols/dotted line: same for EGFR-mCitrine in RPTPγ-KO cells (N=3 biological replicates, n=17-21 cells). P: unpaired two-tailed t-test; colored P values compare to the respective species before stimulation, black in between species.


C . Left : Representative confocal micrographs of MCF7 cells depicting the steady state localization of expressed RPTPγ - mCitrine ( cyan ) , without ( top ) or with co - expression of BFP - Rab11a ( yellow , bottom ) . Top right : Quantification of PM - localized fraction of RPTPγ - mCitrine without ( WT ) and with co - expression of BFP - Rab11a . Bottom right : Fraction of RPTPγ - mCitrine localized to the PM in individual cells as a function of BFP - Rab11a expression level , measured by mean BFP - fluorescence intensity . Scale bar : 10 μm . N = 2 biological replicates , n > 40 per condition , mean ± SD , ; P : unpaired two - tailed t - test .

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

C. Left: Representative confocal micrographs of MCF7 cells depicting the steady state localization of expressed RPTPγ-mCitrine (cyan), without (top) or with co-expression of BFP-Rab11a (yellow, bottom). Top right: Quantification of PM-localized fraction of RPTPγ-mCitrine without (WT) and with co-expression of BFP-Rab11a. Bottom right: Fraction of RPTPγ-mCitrine localized to the PM in individual cells as a function of BFP-Rab11a expression level, measured by mean BFP-fluorescence intensity. Scale bar: 10 μm. N=2 biological replicates, n>40 per condition, mean±SD,; P: unpaired two-tailed t-test.


E . Upper panel : Dual - color widefield images ( 1st column ) , SRRF reconstructions ( 2nd column ) with magnifications of boxed areas ( 3rd column ) of Alexa647 - SNAP - EGFR ( green ) and RPTPγ - mCitrine ( magenta ) of cryo - arrested MCF7 cells , unstimulated ( top row ) or stimulated with 100 ng / ml EGF ( bottom row ) for 15 ' . Scale bar : 10 μm . Lower panel : corresponding Manders colocalization coefficients for Alexa647 - Snap - EGFR / RPTPγ - mCitrine from SRRF reconstructions on intracellular compartments or PM area for unstimulated ( n = 12 - 18 ) and 15 ' EGF - stimulated ( n = 13 - 14 ) cells . mean ± SD , P : unpaired two - tailed t - test .

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

E. Upper panel: Dual-color widefield images (1st column), SRRF reconstructions (2nd column) with magnifications of boxed areas (3rd column) of Alexa647-SNAP-EGFR (green) and RPTPγ-mCitrine (magenta) of cryo-arrested MCF7 cells, unstimulated (top row) or stimulated with 100 ng/ml EGF (bottom row) for 15'. Scale bar: 10 μm. Lower panel: corresponding Manders colocalization coefficients for Alexa647-Snap-EGFR/RPTPγ-mCitrine from SRRF reconstructions on intracellular compartments or PM area for unstimulated (n=12-18) and 15' EGF-stimulated (n=13-14) cells. mean±SD, P: unpaired two-tailed t-test.


F . Left : Representative IP - western blot showing co - IP of EGFR ( 2nd row ) upon RPTPγ - mCitrine ( 1st row : lanes 1 - 6 ) or RPTPγC1060S - mCitrine ( lane 7 ) pull - down by anti - GFP antibody from lysates of MCF7 cells co - transfected with EGFR and RPTPγ - mCitrine or RPTPγC1060S - mCitrine : without stimulus ( 0 ng / ml ) , upon 10 ' stimulus with EGF - Alexa647 ( 5 - 320 ng / ml , also displayed as corresponding receptor - occupancy αL , Fig 2D ) or 8 mM of H2O2 . 3rd and 4th row : total protein concentrations of RPTPγ - mCitrine and EGFR in the lysate measured by western blot as input control for the Co - IP . Right : corresponding ratiometric quantification of co - immunoprecipitated EGFR over pulled down RPTPg - mCitrine or RPTPγC1060S - mCitrine protein bands ( mean ± SD , N = 4 biological replicates , P : unpaired two - tailed t - test )

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

F. Left: Representative IP-western blot showing co-IP of EGFR (2nd row) upon RPTPγ-mCitrine (1st row: lanes 1-6) or RPTPγC1060S-mCitrine (lane 7) pull-down by anti-GFP antibody from lysates of MCF7 cells co-transfected with EGFR and RPTPγ-mCitrine or RPTPγC1060S-mCitrine: without stimulus (0 ng/ml), upon 10' stimulus with EGF-Alexa647 (5-320 ng/ml, also displayed as corresponding receptor-occupancy αL, Fig 2D) or 8 mM of H2O2. 3rd and 4th row: total protein concentrations of RPTPγ-mCitrine and EGFR in the lysate measured by western blot as input control for the Co-IP. Right: corresponding ratiometric quantification of co-immunoprecipitated EGFR over pulled down RPTPg-mCitrine or RPTPγC1060S-mCitrine protein bands (mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test)


. C . Catalytic ( ε1 − ε4 ) and autocatalytic ( α1 − α4 ) rate constants obtained from iterative global fitting the ODEs in ( B ) solved for steady state ( dEGFR p / T / dt = dEGF - EGFRp / T / dt = dRPTPγA / T / dt = 0 ) to EGF - dose response ( aL − aP ) data from all EGFR and PTP expression conditions ( Fig 2E , F , I ) . EGFRp , EGF - EGFRp : product of the corresponding

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

. C. Catalytic (ε1 − ε4) and autocatalytic (α1 − α4) rate constants obtained from iterative global fitting the ODEs in (B) solved for steady state (dEGFR p/T/dt = dEGF-EGFRp/T/dt = dRPTPγA/T/dt = 0) to EGF-dose response (aL − aP) data from all EGFR and PTP expression conditions (Fig 2E, F, I). EGFRp, EGF-EGFRp: product of the corresponding reaction


. F . Change of the free parameter groups Γ1 = γ1RPTPγ / EGFRT and Β = β EGFRT / k1 in EmCit _ MCF7 RPTPγ - KO ( blue ) , EmCit _ MCF7 ( red ) , EmCit _ MCF7 RPTPγ - KO expressing RPTPγ - mTFP splitted in 3 clusters with increasing RPTPγ − mTFP / EGFR - mCitrine ratio ( yellow , green , purple ; Fig 2H , EV2G ) and WT MCF7 cells (

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

. F. Change of the free parameter groups Γ1 = γ1RPTPγ/EGFRT and Β = β EGFRT/k1 in EmCit_MCF7 RPTPγ-KO (blue), EmCit_MCF7 (red), EmCit_MCF7 RPTPγ-KO expressing RPTPγ-mTFP splitted in 3 clusters with increasing RPTPγ−mTFP/EGFR-mCitrine ratio (yellow, green, purple; Fig 2H, EV2G) and WT MCF7 cells (black


C . Top : Representative Western blot against Erk and phosphorylated Erk ( pT202 and pY204 ) in WT ( red ) compared to p22phox - KO ( green ) MCF7 cells after the indicated times of sustained stimulation with 20 ng / ml EGF - Alexa647 . Bottom : Corresponding quantification of the fraction of phosphorylated ERK as a function of stimulation time . Mean ± SD , N = 4 biological replicates , P : unpaired two - tailed t - test .

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

C. Top: Representative Western blot against Erk and phosphorylated Erk (pT202 and pY204) in WT (red) compared to p22phox-KO (green) MCF7 cells after the indicated times of sustained stimulation with 20 ng/ml EGF-Alexa647. Bottom: Corresponding quantification of the fraction of phosphorylated ERK as a function of stimulation time. Mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test.


D . Quantification of cell proliferation using retinoblastoma ( Rb ) protein phosphorylation detected by immunofluorescence , for WT ( red ) , RPTPγ - KO ( blue ) and p22phox - KO ( green ) MCF7 cells without or post 24 h of EGF - Alexa647 treatment ( 1 , 20 , 160 ng / ml ) . Mean ± SEM , N = 3 biological replicates , n > 2000 cells per EGF stimulus per cell line , P : two - way ANOVA with Tukey multiple comparisons .

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

D. Quantification of cell proliferation using retinoblastoma (Rb) protein phosphorylation detected by immunofluorescence, for WT (red), RPTPγ-KO (blue) and p22phox-KO (green) MCF7 cells without or post 24 h of EGF-Alexa647 treatment (1, 20, 160 ng/ml). Mean±SEM, N=3 biological replicates, n>2000 cells per EGF stimulus per cell line, P: two-way ANOVA with Tukey multiple comparisons.


E . Quantification of the culture - well area ( % ) occupied by proliferating cell - colonies , obtained from clonogenic assays of WT , RPTPγ - KO and p22phox - KO MCF7 cells , plated either in medium containing 20 ng / ml EGF and 0 . 5 % FCS ( left : orange bars , N = 3 - 4 biological replicates , 11 - 12 wells each ) or complete serum growth medium containing 10 % FCS ( right : pink bars , N = 4 biological replicates , 12 wells each ) . Mean ± SEM , P : unpaired two - tailed t - test with Welch ' s correction .

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

E. Quantification of the culture-well area (%) occupied by proliferating cell-colonies, obtained from clonogenic assays of WT, RPTPγ-KO and p22phox-KO MCF7 cells, plated either in medium containing 20 ng/ml EGF and 0.5% FCS (left: orange bars, N=3-4 biological replicates, 11-12 wells each) or complete serum growth medium containing 10% FCS (right: pink bars, N=4 biological replicates, 12 wells each). Mean±SEM, P: unpaired two-tailed t-test with Welch's correction.


( a ) Relative quantification ( RQ ) of CG7630 transcript after RNAi with a 120 - bp inverted - repeat sequence ( act5c - gal4 > UAS - CG7630RNAi ) compared to control ( act5c - gal4 > + ) .

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

(a) Relative quantification (RQ) of CG7630 transcript after RNAi with a 120-bp inverted-repeat sequence (act5c-gal4>UAS-CG7630RNAi) compared to control (act5c-gal4>+).


( b ) Relative percentage of egg to adult viability of CG7630 RNAi cross ( act5c - gal4 / CyO . GFP x UAS - CG7630RNAi ) and control cross ( act5c - gal4 / CyO . GFP x w1118 ) , calculated at three developmental stages ( eggs , pupae and adults ) , ( n > 250 , Chi - square test 9 . 920 df ( 2 ) , * * p = 0 . 007 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative percentage of egg to adult viability of CG7630 RNAi cross (act5c-gal4/CyO.GFP x UAS-CG7630RNAi) and control cross (act5c-gal4/CyO.GFP x w1118), calculated at three developmental stages (eggs, pupae and adults), (n > 250, Chi-square test 9.920 df(2), **p = 0.007).


( d ) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase ( CS ) activity .

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

(d) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase (CS) activity.


( e ) In gel - activity assays for MRC complexes II and IV in DDM - solubilized mitochondria from RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control larvae ( act5c - gal4 > + ) .

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

(e) In gel-activity assays for MRC complexes II and IV in DDM-solubilized mitochondria from RNAi (act5c-gal4>UAS-CG7630RNAi) and control larvae (act5c-gal4>+).


( f ) BN - PAGE , Western blot immunodetection of MRC complexes from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control ( act5c - gal4 > + ) larvae using antibodies against specific subunits : anti - UQCRC2 ( complex III ) , anti - NDUFS3 ( complex I ) , anti - COX4 ( complex IV ) and anti - SDHA ( complex II ) .

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

(f) BN-PAGE, Western blot immunodetection of MRC complexes from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II).


( g ) Western blot analysis of steady state levels ( SDS - PAGE ) of MRC subunits COX4 ( complex IV ) , SDHA ( complex II ) and UQCR - C2 ( complex III ) in whole lysates from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control ( act5c - gal4 > + ) larvae .

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

(g) Western blot analysis of steady state levels (SDS-PAGE) of MRC subunits COX4 (complex IV), SDHA (complex II) and UQCR-C2 (complex III) in whole lysates from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae.


( h ) Western blot analysis of anti - HA affinity purified samples from S2R + D . melanogaster cells . EV = samples from cells carrying the empty expression vector . CG7630 - HA = samples from cells expressing HA - tagged CG7630 . Tot = total mitochondrial - enriched fractions ; unb = unbound material ; IP : HA = immunoprecipitated material . Samples were probed with antibodies against MRC subunits of complexes I ( NDUFS3 ) , II ( SDHA ) , III ( UQCR - C2 ) , IV ( COX4 ) and V ( ATP5A ) .

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

(h) Western blot analysis of anti-HA affinity purified samples from S2R+ D. melanogaster cells. EV = samples from cells carrying the empty expression vector. CG7630-HA = samples from cells expressing HA-tagged CG7630. Tot = total mitochondrial-enriched fractions; unb = unbound material; IP:HA = immunoprecipitated material. Samples were probed with antibodies against MRC subunits of complexes I (NDUFS3), II (SDHA), III (UQCR-C2), IV (COX4) and V (ATP5A).


( a ) Relative quantification ( RQ ) of CG7630 transcript in wild - type D . melanogaster S2R + cells treated with a dsRNA targeting CG7630 ( CG7630 KD ) and a mock control dsRNA ( mock ) .

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

(a) Relative quantification (RQ) of CG7630 transcript in wild-type D. melanogaster S2R+ cells treated with a dsRNA targeting CG7630 (CG7630 KD) and a mock control dsRNA (mock).


( b ) Relative quantification ( RQ ) of CG7630 and COX7B transcripts in stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . Cells were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) and a mock control dsRNA ( solid bars , mock ) . N / D = not detected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative quantification (RQ) of CG7630 and COX7B transcripts in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). N/D = not detected.


( c ) Immunoblot analysis of stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . The CG7630 transcript was knocked - down ( KD ) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA ( mock ) . Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control .

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

(c) Immunoblot analysis of stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). The CG7630 transcript was knocked-down (KD) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA (mock). Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control.


( d ) Kinetic enzyme activity of COX normalized by citrate synthase activity ( CS ) in stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . Cells were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) and a mock control dsRNA ( solid bars , mock ) .

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

(d) Kinetic enzyme activity of COX normalized by citrate synthase activity (CS) in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock).


( e ) Steady - state levels of MRC subunits in samples from rescue experiment performed as in ( d ) .

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

(e) Steady-state levels of MRC subunits in samples from rescue experiment performed as in (d).


( B ) Representative immunoblot analysis of CALHM6 expression in BMDM and BMDC stimulated for 6 or 24 h with Poly ( I : C ) ( 50 μg / ml ) or LPS ( 100 ng / ml ) ( one representative experiment of two is shown , n = 2 mice ) .

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

(B) Representative immunoblot analysis of CALHM6 expression in BMDM and BMDC stimulated for 6 or 24 h with Poly(I:C) (50 μg/ml) or LPS (100 ng/ml) (one representative experiment of two is shown, n = 2 mice).


( G and H ) Calhm6 mRNA expression measured by qPCR in naïve or IFN - γ ( 10 ng / ml ) primed ( 6 h ) BMDM stimulated with ( G ) Poly ( I : C ) ( 50 μg / ml ) ( one representative experiment of two is shown , n = 2 mice , biological replicates ) , or ( H ) Zymosan ( 100 μg / ml ) for 2 to 24h ( one representative experiment is shown , n = 3 mice , biological replicates ) .

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

(G and H) Calhm6 mRNA expression measured by qPCR in naïve or IFN-γ (10 ng/ml) primed (6 h) BMDM stimulated with (G) Poly(I:C) (50 μg/ml) (one representative experiment of two is shown, n = 2 mice, biological replicates), or (H) Zymosan (100 μg/ml) for 2 to 24h (one representative experiment is shown, n = 3 mice, biological replicates).


CFU / g of spleen are shown ( B ) after 24 h ( pooled results from two independent experiments , WT mice = 9 , Calhm6 - / - mice = 12 , Man Whitney test ) , ( C ) 72 h ( pooled results from two independent experiments , WT mice = 16 , Calhm6 - / - mice = 17 , Man Whitney test )

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

CFU/g of spleen are shown (B) after 24 h (pooled results from two independent experiments, WT mice = 9, Calhm6-/- mice = 12, Man Whitney test), (C) 72 h (pooled results from two independent experiments, WT mice = 16, Calhm6-/- mice = 17, Man Whitney test)


Serum collected from infected mice at peak infection ( day 3 ) and serum cytokines quantified using LEGENDplex Mouse Inflammation Panel assay to determine IFN - γ ( F ) , IL - 6 ( G ) and IL - 10 ( H ) concentration ( pooled results from two independent experiments , WT mice = 16 , Calhm6 - / - mice = 17 , Mann Whitney test ) .

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

Serum collected from infected mice at peak infection (day 3) and serum cytokines quantified using LEGENDplex Mouse Inflammation Panel assay to determine IFN-γ (F), IL-6 (G) and IL-10 (H) concentration (pooled results from two independent experiments, WT mice = 16, Calhm6-/- mice = 17, Mann Whitney test).


( F - J ) WT and Calhm6 - / - mice were injected i . p . with Poly ( I : C ) ( 200 μg / mouse ) or PBS for 3 h ( F - H ) or 12 h ( I - J ) , spleens were collected and processed as in ( A - E ) . Representative contour plots ( F , I ) , pooled frequency of IFN - γ + in CD3 - NK1 . 1 + cells of WT vs Calhm6 - / - mice ( G , J ) and ratio of IFN - γ + cells that are NK1 . 1 + or CD3 + normalised as in C ( H ) ( For Poly ( I : C ) 3 h : pooled results from two independent experiments , Poly ( I : C ) WT mice = 10 , Calhm6 - / - mice = 10 , control WT mice = 2 , Calhm6 - / - mice = 2 , One way ANOVA with multiple comparisons . For Poly ( I : C ) 12 h : results from one experiment , Poly ( I : C ) WT mice = 6 , Calhm6 - / - mice = 5 , control WT mice = 1 , Calhm6 - / - mice = 1 mice , One - way ANOVA with multiple comparisons ) .

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

(F-J) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS for 3 h (F-H) or 12 h (I-J), spleens were collected and processed as in (A-E). Representative contour plots (F, I), pooled frequency of IFN-γ+ in CD3-NK1.1+ cells of WT vs Calhm6-/- mice (G,J) and ratio of IFN-γ+ cells that are NK1.1+ or CD3+ normalised as in C (H) (For Poly(I:C) 3 h: pooled results from two independent experiments, Poly(I:C) WT mice = 10, Calhm6-/- mice = 10, control WT mice = 2, Calhm6-/- mice = 2, One way ANOVA with multiple comparisons. For Poly(I:C) 12 h: results from one experiment, Poly(I:C) WT mice = 6, Calhm6-/- mice = 5, control WT mice = 1, Calhm6-/- mice = 1 mice, One-way ANOVA with multiple comparisons).


( A - B ) WT and Calhm6 - / - mice were injected i . p . with Poly ( I : C ) ( 200 μg / mouse ) or PBS . On day 3 spleens were collected , stained with antibodies against NK cell - maturation markers and analysed by flow cytometry . Representative contour plots ( A ) and pooled flow cytometry results ( B ) are shown ( results from one experiment , Poly ( I : C ) WT mice = 5 , Calhm6 - / - mice = 5 , control WT mice = 2 , Calhm6 - / - mice = 2 mice , One - way ANOVA with multiple comparisons ) .

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

(A-B) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS. On day 3 spleens were collected, stained with antibodies against NK cell-maturation markers and analysed by flow cytometry. Representative contour plots (A) and pooled flow cytometry results (B) are shown (results from one experiment, Poly(I:C) WT mice = 5, Calhm6-/- mice = 5, control WT mice = 2, Calhm6-/- mice = 2 mice, One-way ANOVA with multiple comparisons).


( F ) NO production in WT and Calhm6 - / - BMDM treated overnight with LPS or Poly ( I : C ) quantified with a Griess test on cell supernatant ( results from one experiment , WT mice = 3 , Calhm6 - / - mice = 3 , Kruskal Wallis test with multiple comparisons ) .

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

(F) NO production in WT and Calhm6-/- BMDM treated overnight with LPS or Poly(I:C) quantified with a Griess test on cell supernatant (results from one experiment, WT mice = 3, Calhm6-/- mice = 3, Kruskal Wallis test with multiple comparisons).


( G ) BMDM with or without IFN − γ priming were grown overnight in antibiotic free medium . Cells were infected with L . monocytogenes ( MOI 1 ) for 30 min . Gentamicin ( 5 ng / ml ) was then added to the medium to kill extracellular bacteria . At 0 , 4 and 8 h cells were harvested , lysed with 0 . 1 % Triton X - 100 and the lysate was plated on antibiotic free BHI plates for 24 h , and CFU were quantified ( pooled results from three independent experiments , WT mice = 3 , Calhm6 - / - mice = 3 , Two - way ANOVA test ) .

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

(G) BMDM with or without IFN−γ priming were grown overnight in antibiotic free medium. Cells were infected with L. monocytogenes (MOI 1) for 30 min. Gentamicin (5 ng/ml) was then added to the medium to kill extracellular bacteria. At 0, 4 and 8 h cells were harvested, lysed with 0.1% Triton X-100 and the lysate was plated on antibiotic free BHI plates for 24 h, and CFU were quantified (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-way ANOVA test).


( D - G ) BMDM were transduced with a retroviral vector expressing CALHM6 - eGFP . BMDM ( 3x105 ) were plated on coverslips and pre - treated with IFN - γ and LPS for 6 h . ( D ) Freshly isolated primary CD3 - NK1 . 1 + cells isolated from mouse spleens were added for 0 , 10 , 20 , or 40 min on top of plated BMDMs that were generated from bone marrow cells transduced with a retroviral vector expressing CALHM6 - eGFP . The interacting cells were then fixed and stained with phalloidin to identify actin and DAPI to identify nucleus . Synaptic interaction between BMDM and NK cell is indicated by white arrowheads ; entire collapsed imaging Z - stack is shown . ( E ) Images were blinded and CALHM6 - eGFP polarisation towards CD3 - NK1 . 1 + cells manually scored only in Z - slices where BMDM - NK interaction was identified ( p - value calculated by two - way ANOVA of polarisation and time points ) . Numbers on top of each bar indicate number of synapses scored for that time point . ( F ) IgG2a - opsonised sRBC labelled with PHK26 were added on top of plated CALHM - eGFP - expressing BMDM . One single Z - slice containing BMDM - sRBC interaction plane is shown . Synaptic interaction between BMDM and sRBC cell is indicated by white arrowheads ( G ) Quantified results of experiment in F . Numbers on top of each bar indicate number of synapses scored for that time point . Longer time points were not possible to score for opsonised sRBC because of the speed of the phagocytosis as sRBC were quickly internalised . Images taken with 60x objective ( p - value calculated by two - way ANOVA of polarisation and time points ) .

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

(D-G) BMDM were transduced with a retroviral vector expressing CALHM6-eGFP. BMDM (3x105) were plated on coverslips and pre-treated with IFN-γ and LPS for 6 h. (D) Freshly isolated primary CD3-NK1.1+ cells isolated from mouse spleens were added for 0, 10, 20, or 40 min on top of plated BMDMs that were generated from bone marrow cells transduced with a retroviral vector expressing CALHM6-eGFP. The interacting cells were then fixed and stained with phalloidin to identify actin and DAPI to identify nucleus. Synaptic interaction between BMDM and NK cell is indicated by white arrowheads; entire collapsed imaging Z-stack is shown. (E) Images were blinded and CALHM6-eGFP polarisation towards CD3-NK1.1+ cells manually scored only in Z-slices where BMDM-NK interaction was identified (p-value calculated by two-way ANOVA of polarisation and time points). Numbers on top of each bar indicate number of synapses scored for that time point. (F) IgG2a-opsonised sRBC labelled with PHK26 were added on top of plated CALHM-eGFP-expressing BMDM. One single Z-slice containing BMDM-sRBC interaction plane is shown. Synaptic interaction between BMDM and sRBC cell is indicated by white arrowheads (G) Quantified results of experiment in F. Numbers on top of each bar indicate number of synapses scored for that time point. Longer time points were not possible to score for opsonised sRBC because of the speed of the phagocytosis as sRBC were quickly internalised. Images taken with 60x objective (p-value calculated by two-way ANOVA of polarisation and time points).


( B ) WT or Calhm6 - / - BMDM stimulated with IFN - γ ( 10 ng / ml ) for 24 h were lysed with 1 % Triton X100 - containing buffer with cOmplete protease inhibitor cocktail and treated with increasing concentrations of DTT . The samples , without boiling , were resolved by SDS - PAGE gel ( one representative experiment of two is shown , n = 4 mice ) . Lack of DTT influences solubilisation of GADPH monomer , and its detection for immunoblotting .

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

(B) WT or Calhm6-/- BMDM stimulated with IFN-γ (10 ng/ml) for 24 h were lysed with 1% Triton X100-containing buffer with cOmplete protease inhibitor cocktail and treated with increasing concentrations of DTT. The samples, without boiling, were resolved by SDS-PAGE gel (one representative experiment of two is shown, n = 4 mice). Lack of DTT influences solubilisation of GADPH monomer, and its detection for immunoblotting.


( G ) Current - voltage ( I - V ) relations for WT - mCALHM6 , E119R - mCALHM6 and control ( ASO ) in 2 - mM Ba2 + bath solution ( pooled individual whole - cell patches , WT - mCALHM6 n = 13 , E119R - mCALHM6 n = 4 , control = 4 , SEM error bars ) .

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

(G) Current-voltage (I-V) relations for WT-mCALHM6, E119R-mCALHM6 and control (ASO) in 2-mM Ba2+ bath solution (pooled individual whole-cell patches, WT-mCALHM6 n = 13, E119R-mCALHM6 n = 4, control = 4, SEM error bars).


( A ) Fura - 2 fluorescence ratio ( F340 / 380 ) recorded in N2a cells expressing human CALHM1 ( hCALHM1 ) , mouse CALHM6 ( mCALHM6 ) or control ( vector ) in response to removal and subsequent add - back of extracellular Ca2 + . Traces representative of four independent experiments for each , normalized to baseline ratio . Right : Resting F340 / 380 ratio in N2a cells expressing hCALHM1 , mCALHM6 or vector in 2 mM Ca2 + ( the error bars represent SEM ) .

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

(A) Fura-2 fluorescence ratio (F340/380) recorded in N2a cells expressing human CALHM1 (hCALHM1), mouse CALHM6 (mCALHM6) or control (vector) in response to removal and subsequent add-back of extracellular Ca2+. Traces representative of four independent experiments for each, normalized to baseline ratio. Right: Resting F340/380 ratio in N2a cells expressing hCALHM1, mCALHM6 or vector in 2 mM Ca2+ (the error bars represent SEM).


( E ) Steady - state ATP release from activated macrophages : BMDMs were plated at 150 , 000 cells / well in a 48 - well plate , and incubated for 3 h with Poly ( I : C ) ( 50 μg / ml ) , LPS ( 100 ng / ml ) and ATPase inhibitor ARL67156 ( 1 mM ) in culture medium . Control cells were incubated with 1 mM ARL67156 only . ATP release ( nM ) measured using Sigma ATP Bioluminescent Assay : WT without stimulation : 9 . 12 ± 0 . 76 ; WT with stimulation : 22 . 16 ± 1 . 83 ; Calhm6 - / - cells without stimulation : 8 . 76 ± 0 . 55 ; KO cells with stimulation : 12 . 19 ± . 75 . ( pooled results from three independent experiments , WT mice = 3 , Calhm6 - / - mice = 3 , Two - tailed unpaired Student ' s T test , error bars represent SEM ) .

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

(E) Steady-state ATP release from activated macrophages: BMDMs were plated at 150,000 cells/well in a 48-well plate, and incubated for 3 h with Poly(I:C) (50 μg/ml), LPS (100 ng/ml) and ATPase inhibitor ARL67156 (1 mM) in culture medium. Control cells were incubated with 1 mM ARL67156 only. ATP release (nM) measured using Sigma ATP Bioluminescent Assay: WT without stimulation: 9.12 ± 0.76; WT with stimulation: 22.16 ± 1.83; Calhm6-/- cells without stimulation: 8.76 ± 0.55; KO cells with stimulation: 12.19 ± .75. (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-tailed unpaired Student's T test, error bars represent SEM).


( left ) Luciferase expression in HEK293 cells driven by deletion constructs of POLG proximal promoter . ( right ) Luciferase expression with mutated CAAT - boxes in POLG promoter . The error bars indicate standard deviation in 3 biological replicates . AU ; arbitrary units .

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

(left) Luciferase expression in HEK293 cells driven by deletion constructs of POLG proximal promoter. (right) Luciferase expression with mutated CAAT-boxes in POLG promoter. The error bars indicate standard deviation in 3 biological replicates. AU; arbitrary units.


Expression pattern driven by 500 bp Polg proximal promoter in E12 . 5 mouse embryo . LacZ - positive cell populations in the developing midbrain ( black arrows ) , dorsal root ganglia ( grey arrows ) and motoneuron progenitors ( arrowhead ) of the neural tube . Somites show some expression ( white arrow ) . Sectioning planes indicated by red lines . Scale bars 100 µm .

Expression pattern driven by 500 bp Polg proximal promoter in E12.5 mouse embryo. LacZ-positive cell populations in the developing midbrain (black arrows), dorsal root ganglia (grey arrows) and motoneuron progenitors (arrowhead) of the neural tube. Somites show some expression (white arrow). Sectioning planes indicated by red lines. Scale bars 100 µm.


- ( G ) POLG enhancer elements are functional in vivo and drive expression in E12 . 5 transgenic mice . Sectioning planes indicated by red lines . Black line in ( E ) marks the expression in the dorsal neural tube , whereas rostral and caudal regions lack dorsal expression ( black arrows ) .

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

- (G) POLG enhancer elements are functional in vivo and drive expression in E12.5 transgenic mice. Sectioning planes indicated by red lines. Black line in (E) marks the expression in the dorsal neural tube, whereas rostral and caudal regions lack dorsal expression (black arrows).


( H ) - ( J ) Neural tube lacZ expression driven by ( H ) EE1 : immature neuronal precursors ( grey arrow ) , ( I ) EE2 : dorsal neural tube ( grey arrow ) and dorsal root ganglia ( black arrow ) , and ( J ) EE3 : dorsal neural tube ( grey arrow ) . Scale bars 100 µm .

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

(H)- (J) Neural tube lacZ expression driven by (H) EE1: immature neuronal precursors (grey arrow), (I) EE2: dorsal neural tube (grey arrow) and dorsal root ganglia (black arrow), and (J) EE3: dorsal neural tube (grey arrow). Scale bars 100 µm.


K ) - ( M ) Midbrain lacZ expression , driven by ( K ) EE1 , ( L ) EE2 and ( M ) EE3 . Black arrow indicates neuronal population from EE1 embryo stained in ( N ) . Scale bars 100 µm .

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

K)- (M) Midbrain lacZ expression, driven by (K) EE1, (L) EE2 and (M) EE3. Black arrow indicates neuronal population from EE1 embryo stained in (N). Scale bars 100 µm.


( N ) EE1 drives expression in oculomotor complex ; immunofluorescent co - staining with antibodies against ISL1 / 2 ( motoneurons of oculomotor complex ; red ) and β - Gal ( green ) . LacZ - staining of the region in ( k ) ; black arrow . Scale bars 50 µm .

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

(N) EE1 drives expression in oculomotor complex; immunofluorescent co-staining with antibodies against ISL1/2 (motoneurons of oculomotor complex; red) and β-Gal (green). LacZ-staining of the region in (k); black arrow. Scale bars 50 µm.


Sagittal section of adult mouse brain showing lacZ expression driven by EE2 . HC , hippocampus .

Sagittal section of adult mouse brain showing lacZ expression driven by EE2. HC, hippocampus.


EE2 - driven expression in rostral migratory stream ( black arrow ) and in subventricular zone ( white arrow ) . Scale bar 760 µm .

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

EE2-driven expression in rostral migratory stream (black arrow) and in subventricular zone (white arrow). Scale bar 760 µm.


Adult mouse spinal cord , EE2 and ( D ) EE3 expression pattern . Dorsal horns , laminae I - III ( black arrows ) and central canal ( arrowheads ) . LacZ staining . Dashed lines in ( C ) indicates the region of dorsal horn shown in ( D ) , ( F ) and ( G ) . Inlets in ( C ) and ( E ) show POLG immunohistochemistry of dorsal horn and central canal ( grey arrow ) , respectively . Scale bars : 100 µm if not indicated otherwise .

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

Adult mouse spinal cord, EE2 and (D) EE3 expression pattern. Dorsal horns, laminae I-III (black arrows) and central canal (arrowheads). LacZ staining. Dashed lines in (C) indicates the region of dorsal horn shown in (D), (F) and (G). Inlets in (C) and (E) show POLG immunohistochemistry of dorsal horn and central canal (grey arrow), respectively. Scale bars: 100 µm if not indicated otherwise.


( F ) - ( G ) Calbindin ( red ) and β - Gal ( green ) expression in interneurons of the dorsal horn laminae I - III of ( F ) EE2 and ( G ) EE3 transgenic adult mice . Immunofluorescence . Scale bars 20 µm .

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

(F)-(G) Calbindin (red) and β-Gal (green) expression in interneurons of the dorsal horn laminae I-III of (F) EE2 and (G) EE3 transgenic adult mice. Immunofluorescence. Scale bars 20 µm.


The number of distal DNAse I hypersensitive sites ( DHSs ) of mtDNA maintenance genes and genes in the genomic POLG locus correlating > 0 . 85 with target promoter DHS across 125 cell lines . Abbreviations : TWNK , Twinkle protein ; POLG , DNA Polymerase gamma , catalytic subunit ; POLG2 , DNA Polymerase gamma , accessory subunit ; SSBP1 , Single stranded DNA binding protein 1 ; TFAM , mitochondrial transcription factor A ; FANCI , Fanconi anemia group I protein ; RHCG , Ammonium transporter Rh type C ; TICRR , Treslin .

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

The number of distal DNAse I hypersensitive sites (DHSs) of mtDNA maintenance genes and genes in the genomic POLG locus correlating >0.85 with target promoter DHS across 125 cell lines. Abbreviations: TWNK, Twinkle protein; POLG, DNA Polymerase gamma, catalytic subunit; POLG2, DNA Polymerase gamma, accessory subunit; SSBP1, Single stranded DNA binding protein 1; TFAM, mitochondrial transcription factor A; FANCI, Fanconi anemia group I protein; RHCG, Ammonium transporter Rh type C; TICRR, Treslin.


Distribution histogram of DHSs for mtDNA maintenance genes .

Distribution histogram of DHSs for mtDNA maintenance genes.


Genomic distribution of POLG DHSs . Black arrow shows a cluster upstream from POLG coding region .

Genomic distribution of POLG DHSs. Black arrow shows a cluster upstream from POLG coding region.


Expression of LINC00925 and POLG in different human tissues and cell types . Quantitative PCR amplification of cDNA . Abbreviations : iPS , induced pluripotent stem cell ; SH5Y , neuroblastoma line ; HepG2 , liver hepatocellular carcinoma line ; U2OS bone osteosarcoma line . Error bars indicate standard deviation in 3 technical replicates . AU ; arbitrary units .

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

Expression of LINC00925 and POLG in different human tissues and cell types. Quantitative PCR amplification of cDNA. Abbreviations: iPS, induced pluripotent stem cell; SH5Y, neuroblastoma line; HepG2, liver hepatocellular carcinoma line; U2OS bone osteosarcoma line. Error bars indicate standard deviation in 3 technical replicates. AU; arbitrary units.


Polg and long - non - coding RNA Ai854517 ( mouse homologue of human LINC00925 ) correlate tightly in mouse cerebellar development . Time points : E18 , postnatal days 0 , 3 , 6 , 9 ; three mice per time point . Expression calculated as cap analysis of gene expression ( CAGE ) hits in the transcription start site ( CTSS ) .

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

Polg and long-non-coding RNA Ai854517 (mouse homologue of human LINC00925) correlate tightly in mouse cerebellar development. Time points: E18, postnatal days 0, 3, 6, 9; three mice per time point. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).


Ai854517 and Polg transcripts colocalize in adult mouse brain ; in situ hybridization . HC , hippocampus ; cerebellar molecular layer ( M ) , Purkinje cell layer ( PC ) , granular cell layer ( GC ) . Scale bars : hippocampus 760 µm , cerebellum 300 µm .

Ai854517 and Polg transcripts colocalize in adult mouse brain; in situ hybridization. HC, hippocampus; cerebellar molecular layer (M), Purkinje cell layer (PC), granular cell layer (GC). Scale bars: hippocampus 760 µm, cerebellum 300 µm.


Expression levels of MIR9 - 3 and Ai854517 correlate in mouse cerebellar development . Time points : E18 , postnatal day 0 , 3 , 6 , 9 ; three mice per time point . . Expression calculated as cap analysis of gene expression ( CAGE ) hits in the transcription start site ( CTSS ) .

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

Expression levels of MIR9-3 and Ai854517 correlate in mouse cerebellar development. Time points: E18, postnatal day 0, 3, 6, 9; three mice per time point.. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).


RNA expressions of MIR9 , NR2E2 , LDLDRAP1 and MTHFD2 in HEK293 untransfected controls and in cells transfected with pre - MIR9 or scrambled RNA . Shown is mean with standard error of the mean . Statistical testing was performed using 1 - way ANOVA with Dunnet ' s correction for multiple comparisons . Abbreviations : AU ; arbitrary units .

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

RNA expressions of MIR9, NR2E2, LDLDRAP1 and MTHFD2 in HEK293 untransfected controls and in cells transfected with pre-MIR9 or scrambled RNA. Shown is mean with standard error of the mean. Statistical testing was performed using 1-way ANOVA with Dunnet's correction for multiple comparisons. Abbreviations: AU; arbitrary units.


Spinal cord ( C8 - level ) of MIRAS patient , neurofilament staining . Dorsal columns , especially the gracile , show pallor ( star ) ; motoneurons in the anterior horns are preserved ( black arrow ) . Scale bar 1 mm .

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

Spinal cord (C8-level) of MIRAS patient, neurofilament staining. Dorsal columns, especially the gracile, show pallor (star); motoneurons in the anterior horns are preserved (black arrow). Scale bar 1 mm.


Oculomotor complex of POLG patient with progressive ophthalmoplegia ; midbrain level . Hematoxylin - eosin staining . The whole area shows spongiotic degeneration . Scale bar 50 µm .

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

Oculomotor complex of POLG patient with progressive ophthalmoplegia; midbrain level. Hematoxylin-eosin staining. The whole area shows spongiotic degeneration. Scale bar 50 µm.


Seedling phenotype under GA treatment . After germination , Five - day - old seedlings with the same growth stage were transferred to 0 . 5 MS liquid media without or with different concentrations of GA3 . PA from soybean was added to the media at a final concentration of 20 μM . Pictures were taken 7 days after transfer . The horizontal red line separates different plants and the vertical red scale bar represents 2 cm .

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

Seedling phenotype under GA treatment. After germination, Five-day-old seedlings with the same growth stage were transferred to 0.5 MS liquid media without or with different concentrations of GA3. PA from soybean was added to the media at a final concentration of 20 μM. Pictures were taken 7 days after transfer. The horizontal red line separates different plants and the vertical red scale bar represents 2 cm.


Immunoblotting of His - tagged PLDα6 ( arrow ) expressed in E . coli as separated on an 10 % SDS - PAGE and blotted to a membrane .

Immunoblotting of His-tagged PLDα6 (arrow) expressed in E. coli as separated on an 10% SDS-PAGE and blotted to a membrane.


Lipid hydrolyzing activity assayed in the presence of different phospholipids using purified PLDα6 from E . coli . Solid bars are activities assayed using purified PLDα6 whereas open bars were empty vector control that used an equal volume of eluents from bacteria containing the empty vector that was identically processed as those expressing PLDα6 . Values are means ± SD ( n = 3 biological replicates ) .

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

Lipid hydrolyzing activity assayed in the presence of different phospholipids using purified PLDα6 from E. coli. Solid bars are activities assayed using purified PLDα6 whereas open bars were empty vector control that used an equal volume of eluents from bacteria containing the empty vector that was identically processed as those expressing PLDα6. Values are means ± SD (n = 3 biological replicates).


Total lipid levels in WT , pldα6 and COM without and with 10 μM GA3 . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected and lipids were extracted and profiled using ESI - tandem mass spectrometry . Values are means ± SD ( n = 3 biological replicates ) ) . MGDG , monogalactosyldiacylglycerol ; DGDG , digalactosyldiacylglycerol ; PC , phosphatidylcholine ; PE , phosphatidylethanolamine ; PG , phosphatidylglycerol ; PS , phosphatidylserine ; PA , phosphatidic acid .

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

Total lipid levels in WT, pldα6 and COM without and with 10 μM GA3. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and lipids were extracted and profiled using ESI-tandem mass spectrometry. Values are means ± SD (n = 3 biological replicates)). MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PA, phosphatidic acid.


Phospholipid species in WT , pldα6 and COM . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected and the different phospholipid species including carbon number and double bond number were determined . Values are means ± SD ( n = 3 biological replicates ) . GA3 was dissolved in ethanol and seedlings treated with the same ethanol concentration were used as control .

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

Phospholipid species in WT, pldα6 and COM. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and the different phospholipid species including carbon number and double bond number were determined. Values are means ± SD (n = 3 biological replicates). GA3 was dissolved in ethanol and seedlings treated with the same ethanol concentration were used as control.