Figure 5A , B Corticospinal axons of the axonal bundles of pyramidal tracts in medulla of P8 and P28 Xkr8 WT and cKO mice were visualized by Palmgren staining and quantified per each bundle , delineated by a dashed line ( Mann - Whitney test , n = 6 mice per genotype , the data are presented as median and quartiles ) . To label callosal projections in vivo , cholera toxin subunit B ( CTB ) was injected stereotactically into corpus callosum of Xkr8 WT and cKO brains at P30 - 32 . Back - labelled neurons were quantified in L4 of primary ( S1 ) and secondary ( S2 ) somatosensory cortex .

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

Figure 5A, B Corticospinal axons of the axonal bundles of pyramidal tracts in medulla of P8 and P28 Xkr8 WT and cKO mice were visualized by Palmgren staining and quantified per each bundle, delineated by a dashed line (Mann-Whitney test, n = 6 mice per genotype, the data are presented as median and quartiles).To label callosal projections in vivo, cholera toxin subunit B (CTB) was injected stereotactically into corpus callosum of Xkr8 WT and cKO brains at P30-32. Back-labelled neurons were quantified in L4 of primary (S1) and secondary (S2) somatosensory cortex.


Figure 6Xkr8 cKO neurons had increased mean sEPSC amplitude Data were analyzed by two - tailed Student ' s t - test ; n = 7 - 14 neurons per genotype . F - H Evoked excitatory postsynaptic currents ( eEPSC ) of Xkr8 cKO neurons showed a trend to decrease ( p = 0 . 08 ) ( F , G ) and the ratio of eEPSC and sEPSC amplitudes was lower in Xkr8 knockout neurons ( H ) . two - way repeated measures ANOVA ( H ) ; n = 7 - 14 neurons per genotype .

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Figure 6Xkr8 cKO neurons had increased mean sEPSC amplitude Data were analyzed by two-tailed Student's t-test ; n = 7-14 neurons per genotype.F-H Evoked excitatory postsynaptic currents (eEPSC) of Xkr8 cKO neurons showed a trend to decrease (p = 0.08) (F, G) and the ratio of eEPSC and sEPSC amplitudes was lower in Xkr8 knockout neurons (H). two-way repeated measures ANOVA (H); n = 7-14 neurons per genotype.


Figure 7A Brain regions with significantly increased functional connectivity in Xkr8 cKO mice compared to Xkr8 WT mice as identified by seed - based mapping of rsfMRI networks ( t - test , p < 0 . 05 FWE cluster - corrected , with cluster - defining threshold of t24 > 2 . 07 , p < 0 . 05 ; Colored regions represent the areas of the brain exhibiting increased functional connectivity in Xkr8 cKO compared to Xkr8 WT brains ; color scale represents the level of functional correlation ; abbreviations specify anatomical designations of identified regions . ACA : anterior cingulate cortex , AI : agranular insular area , AUD : auditory areas MOp : primary motor area , MOs : secondary motor area , PL : prelimbic area , SSp : primary somatosensory area , STR : striatum , STRd : striatum dorsal region , SUB : subiculum , TH : thalamus , VIS : visual areas , VISC : visceral area .

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Figure 7A Brain regions with significantly increased functional connectivity in Xkr8 cKO mice compared to Xkr8 WT mice as identified by seed-based mapping of rsfMRI networks (t-test, p < 0.05 FWE cluster-corrected, with cluster-defining threshold of t24 > 2.07, p < 0.05; Colored regions represent the areas of the brain exhibiting increased functional connectivity in Xkr8 cKO compared to Xkr8 WT brains; color scale represents the level of functional correlation; abbreviations specify anatomical designations of identified regions. ACA: anterior cingulate cortex, AI: agranular insular area, AUD: auditory areas MOp: primary motor area, MOs: secondary motor area, PL: prelimbic area, SSp: primary somatosensory area, STR: striatum, STRd: striatum dorsal region, SUB: subiculum, TH: thalamus, VIS: visual areas, VISC: visceral area.


Figure 1 ( B ) Quantification by micro - CT of mean tumor volumes in KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 18 mice , 21 mice , 21 mice , and 20 mice , respectively ) . ( C ) Quantification by micro - CT of total tumor volume per mouse in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection . ( D ) Depictions and quantitation of total tumor burden ( total tumor area / total lung area ) in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 21 weeks after infection . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . ( E ) Distribution of histological tumor grades in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) at 21 weeks after infection . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . ( F ) Assessment of the mitotic index of tumor cells by phosphorylated - histone H3 ( pHH3 ) - positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . ( G ) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . ( H ) Representative hematoxylin and eosin ( H & E ) and p - Fak1 immunohistochemical ( IHC ) staining of LUAD tumor sections 21 weeks after infection with control sgTom ( grade 1 depicted ) , sgNf1 . 1 ( grade 3 depicted ) , sgNf1 . 2 ( grade 3 depicted ) , or sgNf1 . 3 ( grade 3 depicted ) ( n = 50 tumors each ) . H & E scale bars ( low - magnification top row = 100 μm , high - magnification bottom row = 250 μm ) ; p - Fak1 IHC scale bars ( low - magnification top row = 250 μm , high - magnification bottom row = 500 μm ) . ( I ) Quantification of p - Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . ( J ) Quantification of p - Fak1 IHC signals in sgNf1 . 3 LUAD tumor sections analyzed by tumor grade . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . ( K ) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . ( L ) Quantification of Psat1 mRNA expression in sgNf1 . 3 LUAD tumor sections analyzed by tumor grade . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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

Figure 1(B) Quantification by micro-CT of mean tumor volumes in KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively).(C) Quantification by micro-CT of total tumor volume per mouse in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection.(D) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 21 weeks after infection. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(E) Distribution of histological tumor grades in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each) at 21 weeks after infection. For bar charts, data presented as means with error bars representing standard deviations (SDs).(F) Assessment of the mitotic index of tumor cells by phosphorylated-histone H3 (pHH3)-positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(G) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(H) Representative hematoxylin and eosin (H&E) and p-Fak1 immunohistochemical (IHC) staining of LUAD tumor sections 21 weeks after infection with control sgTom (grade 1 depicted), sgNf1.1 (grade 3 depicted), sgNf1.2 (grade 3 depicted), or sgNf1.3 (grade 3 depicted) (n = 50 tumors each). H&E scale bars (low-magnification top row = 100 μm, high-magnification bottom row = 250 μm); p-Fak1 IHC scale bars (low-magnification top row = 250 μm, high-magnification bottom row = 500 μm).(I) Quantification of p-Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(J) Quantification of p-Fak1 IHC signals in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(K) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(L) Quantification of Psat1 mRNA expression in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


Figure 2 ( A ) Western blotting analysis of p53 expression in various p53 / TP53 - WT cell lines incubated with the DNA - intercalating agent doxorubicin ( DOXO , 0 . 2 µg / ml for 6 hours ) to induce p53 stabilization ( n = 3 biological replicates ) . ( B , C ) Western blotting analysis confirming NF1 upregulation and p - FAK1 downregulation in ( B ) DOXO - treated PDKN1 cells and ( C ) DOXO - treated PDKN2 cells transduced with PGK - NF1 ( n = 3 biological replicates ) . ( D ) Western blotting analysis confirming NF1 downregulation and p - FAK1 upregulation in DOXO - treated SW1573 cells transduced with sgNf1 . 3 ( n = 3 biological replicates ) . ( E , F ) Western blotting analysis confirming Nf1 downregulation and p - Fak1 upregulation in ( E ) DOXO - treated LKR10 and ( F ) DOXO - treated LKR13 clones transduced with sgNf1 . 3 ( n = 3 biological replicates ) . ( G ) FAK1 target gene expression in DOXO - treated PDKN1 cells transduced with PGK - control or PGK - NF1 ( n = 3 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . ( H , I ) Subcutaneous tumor volumes of DOXO - treated PDKN1 and PDKN2 cells transduced with PGK - control or PGK - NF1 ( n = 18 each ) . ( J - L ) Subcutaneous tumor volumes of ( J ) SW1573 , ( K ) LKR10 , and ( L ) LKR13 cells transfected with sgTom or sgNf1 . 3 ( n = 18 each ) . ( M ) Depictions and quantitation of KrasG12D / + ; p53 + / + ( K - only ) autochthonous tumor burden ( total tumor area / total lung area ) in pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . ( N ) Analysis of tumor grades in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . ( O ) Quantification of Ki - 67 - positive nuclei per mm2 in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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

Figure 2(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates).(B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates).(D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates).(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates).(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).(J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each).(M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.(N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For bar charts, data presented as means with error bars representing standard deviations (SDs).(O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


Figure 3 ( A ) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . ( B ) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . ( C ) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell - titer glo ( relative luminescent units ) ( n = 4 biological replicates ) . ( D ) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2 . 0 or 0 . 5 mM glutamine ( n = 4 biological replicates ) . ( E ) Two patient - derived KRAS ; NF1 - mutant LUAD cell lines ( PDKN1 , PDKN2 ) , as well as the control patient - derived KRAS - mutant / NF1 - WT LUAD cell line ( PDK ) , were passaged for 14 population doublings prior to collection . The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines ( relative to the PDK control ) , with the degree of upregulation depicted by color as indicated in the legend . Quantification of the expression of the six key altered genes in cultured PDKN1 cells ( passaged for 14 population doublings ) vs . their respective original patient tumor samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . Quantification of the expression of the six key altered genes in cultured PDKN2 cells ( passaged for 14 population doublings ) vs . their respective original patient tumor samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . ( H ) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection . GAPDH was used as a loading control . ( I ) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) . ( J - L ) Cumulative population doublings of patient - derived KRAS - mutant LUAD cell lines that are either ( J , K ) NF1 - mutant ( PDKN1 and PDKN2 ) or ( L ) NF1 - WT ( PDK ) after selection with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) .

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

Figure 3(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.Quantification of the expression of the six key altered genes in cultured PDKN1 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).Quantification of the expression of the six key altered genes in cultured PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).(H) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control.(I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).(J-L) Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).


Figure 4Relative viability of KP and KPΔNF1 clones after CB - 839 treatment ( IC50 : 18 nM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . All data points are relative to vehicle - treated controls ( n = 4 biological replicates ) . Relative viability of KP and KPΔNF1 clones after AOA treatment ( IC50 : 6 µM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . All data points are relative to vehicle - treated controls ( n = 4 biological replicates ) . ( D ) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA after 6 d in culture ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) . ( E ) Relative viability of the indicated human KRAS - mutant lung cancer cell lines by trypan blue exclusion assay . Cells were incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA for 72 h ( n = 4 biological replicates ) . All data points are normalized to vehicle - treated cell lines . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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

Figure 4Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).Relative viability of KP and KPΔNF1 clones after AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).(D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).(E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines. For bar charts, data presented as means with error bars representing standard deviations (SDs).


Figure 5Quantification by micro - CT of mean tumor volumes in ( B ) CB - 839 - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice and 21 mice , respectively ) . Vehicle - treated KP mice are the same control group for ( B ) Quantification by micro - CT of mean tumor volumes in ( C ) AOA - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice and 21 mice , respectively ) . Vehicle - treated KP mice are the same control group for ( C ) . Quantification by micro - CT of total tumor volume per mouse in ( D ) CB - 839 - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection . Vehicle - treated KP mice are the same control group for ( D ) E ) Quantification by micro - CT of total tumor volume per mouse in , ( E ) AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection . Vehicle - treated KP mice are the same control group for ( E ) . Relative responses of KP mice tumors treated with ( F ) CB - 839 , or vehicle starting from week 8 post - infection and measured until week 20 . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Relative responses of KP mice tumors treated with ( G ) AOA , or vehicle starting from week 8 post - infection and measured until week 20 . Relative response = average tumor volume with treatment / average tumor volume with vehicle . ( H ) Depictions and quantitation of total tumor burden ( total tumor area / total lung area ) in CB - 839 - , AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 20 weeks post - infection . Vehicle - treated KP mice are the same control group in both boxplots . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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

Figure 5Quantification by micro-CT of mean tumor volumes in (B) CB-839-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (B)Quantification by micro-CT of mean tumor volumes in (C) AOA-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (C).Quantification by micro-CT of total tumor volume per mouse in (D) CB-839-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (D)E) Quantification by micro-CT of total tumor volume per mouse in , (E) AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (E).Relative responses of KP mice tumors treated with (F) CB-839, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.Relative responses of KP mice tumors treated with (G) AOA, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.(H) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in CB-839-, AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 20 weeks post-infection. Vehicle-treated KP mice are the same control group in both boxplots. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


Figure 6Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , ( B ) CB - 839 , starting from day 12 and measured until day 27 ( n = 6 tumors per cell line per treatment ) Relative response = average tumor volume with treatment / average tumor volume with vehicle . ) Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , C ) AOA starting from day 12 and measured until day 27 ( n = 6 tumors per cell line per treatment ) Relative response = average tumor volume with treatment / average tumor volume with vehicle . Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , CB - 839 , or AOA ( D ) the final masses of these tumors . or boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean . Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle , ( E ) CB - 839 starting from day 12 and measured until day 27 ( n = 24 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector . Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle , ( F ) AOA starting from day 12 and measured until day 27 ( n = 24 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector . Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle , ( G ) CB - 839 , with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle ( H ) AOA with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Relative responses of subcutaneous patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( I ) CB - 839 starting from day 12 and measured until day 30 ( n = 6 tumors per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Relative responses of subcutaneous patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle ( J ) AOA starting from day 12 and measured until day 30 ( n = 6 tumors per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Relative responses of orthotopic patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( K ) CB - 839 starting from day 12 and measured until day 27 ( n = 12 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector . Relative responses of orthotopic patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( L ) AOA starting from day 12 and measured until day 27 ( n = 12 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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

Figure 6Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, (B) CB-839, starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.) Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, C) AOA starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, CB-839, or AOA (D) the final masses of these tumors. or boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (E) CB-839 starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (F) AOA starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle, (G) CB-839, with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle (H) AOA with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (I) CB-839 starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle (J) AOA starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (K) CB-839 starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (L) AOA starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Figure 1A Indicated tail - anchored ( TA ) proteins were in vitro translated in rabbit reticulocyte lysate ( RRL ) using RNAs containing a stop codon . Translations were carried out in the presence of 2 μM HisTrx or HisTrx - SGTA ( HT - SGTA ) , followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration , samples resolved by SDS - PAGE and results visualised by phosphorimaging . Sec61β 3R carries three Arg residues within Sec61β transmembrane domain ( TMD ) which abolish its hydrophobic character and was used as a negative control for SGTA binding . A schematic representation of a TA - protein is also shown . Open circles indicate TA - proteins selectively bound by HisTrx - SGTA . RAMP4 - stress - associated endoplasmic reticulum protein 1 ; Syb2 - synaptobrevin 2 ; Synt5 - syntaxin 5 . B As for ( A ) but total translation products ( input ) were resolved by SDS - PAGE and results visualised by phosphorimaging . When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant . RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used . E As for ( D ) but total translation products were subjected to immunoprecipation with either anti - C99 antibody ( for the full - length variants ) or with anti - β - amyloid antibody ( for products lacking the TMD ) . A filled square indicates inefficiently translated APP - C99 - TMD immunoprecipitated with anti - β - amyloid antibody .

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

Figure 1A Indicated tail-anchored (TA) proteins were in vitro translated in rabbit reticulocyte lysate (RRL) using RNAs containing a stop codon. Translations were carried out in the presence of 2 μM HisTrx or HisTrx-SGTA (HT-SGTA), followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration, samples resolved by SDS-PAGE and results visualised by phosphorimaging. Sec61β 3R carries three Arg residues within Sec61β transmembrane domain (TMD) which abolish its hydrophobic character and was used as a negative control for SGTA binding. A schematic representation of a TA-protein is also shown. Open circles indicate TA-proteins selectively bound by HisTrx-SGTA. RAMP4 - stress-associated endoplasmic reticulum protein 1; Syb2 - synaptobrevin 2; Synt5 - syntaxin 5. B As for (A) but total translation products (input) were resolved by SDS-PAGE and results visualised by phosphorimaging. When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant. RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used. E As for (D) but total translation products were subjected to immunoprecipation with either anti-C99 antibody (for the full-length variants) or with anti-β-amyloid antibody (for products lacking the TMD). A filled square indicates inefficiently translated APP-C99-TMD immunoprecipitated with anti-β-amyloid antibody.


Figure 2A Full - length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx - SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide ( CHX ) or released with puromycin ( puro ) . Reactions were incubated with HisPur Cobalt resin , bound proteins eluted at high imidazole concentration , resolved by SDS - PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX - treated samples . total translation reactions were treated with RNaseA and immunoprecipitated with anti - C99 antibodyFollowing 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Full-length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx-SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide (CHX) or released with puromycin (puro). Reactions were incubated with HisPur Cobalt resin, bound proteins eluted at high imidazole concentration, resolved by SDS-PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX-treated samples.total translation reactions were treated with RNaseA and immunoprecipitated with anti-C99 antibodyFollowing 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.


Figure 3B 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B 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.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.


Figure 4Sec61β 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Sec61β 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).


Figure 5A , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, 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.


Figure 6A , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, 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.


Figure 7A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A 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.


Figure 2In 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2In 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.


Figure 3Boxplots 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Boxplots 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.


Figure 4Rose 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 .

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

Figure 4Rose 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.


Figure 5Boxplot 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 .

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

Figure 5Boxplot 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.


Figure 6Heatmap 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 .

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Figure 6Heatmap 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.


Figure 7COVID - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7COVID-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.


Figure 1A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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.


Figure 2A . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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).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).


Figure 3C - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C-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.