Figure 1 . Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( A ) Age - matched control Atg7 littermates ( Atg7flox / + ; nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) or Atg7 KO ( Atg7flox / flox ; nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) are shown . Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( B ) Control Atg5 littermates ( Atg5flox / + ; nestin - Cre ; denoted + ) at 16 ( open circles ; n = 4 ) and 26 wk ( open diamonds ; n = 3 ) or Atg5 KO ( Atg5flox / flox ; nestin - Cre ; denoted − ) at 16 ( closed circles ; n = 4 ) and 26 wk ( closed diamonds ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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 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

Figure 1.Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (A) Age-matched control Atg7 littermates (Atg7flox/+;nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4) or Atg7 KO (Atg7flox/flox;nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3) are shown.Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (B) Control Atg5 littermates (Atg5flox/+;nestin-Cre; denoted +) at 16 (open circles; n = 4) and 26 wk (open diamonds; n = 3) or Atg5 KO (Atg5flox/flox;nestin-Cre; denoted −) at 16 (closed circles; n = 4) and 26 wk (closed diamonds; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Figure 2 . Suppressed accumulation of Ub in Atg7 - / p62 - deficient brains . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured total ( A ) and isopeptide - linked ( B ) Ub from the soluble and insoluble fractions of autophagy - deficient brains . Age - matched control Atg7 littermates ( Atg7flox / + : nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) , p62 KO ( Atg7flox / flox : p62 − / − ; denoted − ) at 6 wk ( closed circles ; n = 3 ) , Atg7 KO ( Atg7flox / flox : nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) , or Atg7 - / p62 - DKO ( Atg7flox / flox : nestin - Cre : p62 − / − ) at 6 wk ( closed triangles ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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

Figure 2.Suppressed accumulation of Ub in Atg7-/p62-deficient brains. (A and B) Quantitative mass spectrometry analysis of P2UBA captured total (A) and isopeptide-linked (B) Ub from the soluble and insoluble fractions of autophagy-deficient brains. Age-matched control Atg7 littermates (Atg7flox/+:nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4), p62 KO (Atg7flox/flox:p62−/−; denoted −) at 6 wk (closed circles; n = 3), Atg7 KO (Atg7flox/flox:nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3), or Atg7-/p62-DKO (Atg7flox/flox:nestin-Cre:p62−/−) at 6 wk (closed triangles; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Figure 3 . Suppressed accumulation of Ub in Atg7 - / Nrf2 - deficient livers . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured p62 ( A ) , total ( A ) , and isopeptide - linked ( B ) Ub from the soluble and insoluble fraction of autophagy - deficient livers ( 28 d after pIpC injection ) . Control littermates ( F / F or F / F : Nrf2 + / − ; denoted + ; open circles ; n = 4 ) , Nrf2 KO ( F / F : Nrf2 − / − ; denoted − ; closed circles ; n = 4 ) , Atg7 KO ( F / F : Mx1 or F / F : Mx1 : Nrf2 + / − ; denoted − ; closed diamonds ; n = 6 ) , or Atg7 - / Nrf2 - DKO ( F / F : Mx1 : Nrf2 − / − ; closed triangles ; n = 6 ) are shown . Each symbol represents one animal . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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 3.Suppressed accumulation of Ub in Atg7-/Nrf2-deficient livers. (A and B) Quantitative mass spectrometry analysis of P2UBA captured p62 (A), total (A), and isopeptide-linked (B) Ub from the soluble and insoluble fraction of autophagy-deficient livers (28 d after pIpC injection). Control littermates (F/F or F/F:Nrf2+/−; denoted +; open circles; n = 4), Nrf2 KO (F/F:Nrf2−/−; denoted −; closed circles; n = 4), Atg7 KO (F/F:Mx1 or F/F:Mx1:Nrf2+/−; denoted −; closed diamonds; n = 6), or Atg7-/Nrf2-DKO (F/F:Mx1:Nrf2−/−; closed triangles; n = 6) are shown. Each symbol represents one animal. **, P ≤ 0.01; ***, P ≤ 0.005.


Figure 4 . ( B ) Elevated Ub mRNA levels from Atg5 - deficient brains . Total RNA was extracted from control Atg5 littermates ( Atg5flox / + ; nestin - Cre ) at 16 wk or Atg5 KO ( Atg5flox / flox ; nestin - Cre ) at 16 wk ( n = 4 for each genotype ; * * , P ≤ 0 . 01 ) and the amount of transcript from Ubc , Ubb , Uba52 , and Uba80 genes was quantified using real - time RT - PCR and normalized to actin . Error bars indicate SEM . au , arbitrary unit .

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

Figure 4.(B) Elevated Ub mRNA levels from Atg5-deficient brains. Total RNA was extracted from control Atg5 littermates (Atg5flox/+;nestin-Cre) at 16 wk or Atg5 KO (Atg5flox/flox;nestin-Cre) at 16 wk (n = 4 for each genotype; **, P ≤ 0.01) and the amount of transcript from Ubc, Ubb, Uba52, and Uba80 genes was quantified using real-time RT-PCR and normalized to actin. Error bars indicate SEM. au, arbitrary unit.


Figure 6 . ( B ) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line , m5 - 7 , as measured by the decreased levels of phosphatidylethanolamine - modified LC3 ( LC3 - II ) . ( C ) Western blot ( WB ) analysis of the accumulation of htt ( Q ) - GFP after autophagy shutoff . At steady - state ( t = 0 h ) , the levels of both htt ( Q25 ) - GFP and htt ( Q47 ) - GFP were virtually undetectable . After autophagy shutoff , there was a robust increase in the level of htt ( Q47 ) - GFP , as detected by GFP Western blot , whereas there was very little or no detectable increase in htt ( Q25 ) - GFP , GAPDH , LDH , or chFP . ( D ) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells . The ASI is the ratio of the relative increase in green and red fluorescence , as assessed by two - color flow cytometry , in response to dox . In control experiments , the ASI of two soluble , nonaggregating proteins , htt ( Q25 ) - GFP and chFP , was 1 . In contrast , the ASI of the aggregation - prone protein , htt ( Q47 ) - GFP , compared with chFP after autophagy shutoff ( + dox ; 100 h ) was 2 . Data represent three independent experiments performed on different days . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 . ( E ) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff ( + dox ) . The degree of cell death correlated directly with increased htt ( Q47 ) - GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt ( Q47 ) - GFP compared with htt ( Q25 ) - GFP ( * * * , P ≤ 0 . 005 ) . Data represent three independent experiments performed on different days . ( F ) AQUA using heavy isotope - labeled GFP standard in addition to the routine Ub standards after P2UBA pull - down of soluble and insoluble lysates from htt ( Q ) - GFP - IRES - chFP bicistronic expressing cells before ( − dox ) and after autophagy shutoff ( + dox ) . The experimental GFP peptide was only detected in two of the four experiments , and the data shown are representative of these . In the other two experiments , the experimental GFP peptide was below the threshold of detection .

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

Figure 6.(B) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line, m5-7, as measured by the decreased levels of phosphatidylethanolamine-modified LC3 (LC3-II).(C) Western blot (WB) analysis of the accumulation of htt(Q)-GFP after autophagy shutoff. At steady-state (t = 0 h), the levels of both htt(Q25)-GFP and htt(Q47)-GFP were virtually undetectable. After autophagy shutoff, there was a robust increase in the level of htt(Q47)-GFP, as detected by GFP Western blot, whereas there was very little or no detectable increase in htt(Q25)-GFP, GAPDH, LDH, or chFP.(D) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells. The ASI is the ratio of the relative increase in green and red fluorescence, as assessed by two-color flow cytometry, in response to dox. In control experiments, the ASI of two soluble, nonaggregating proteins, htt(Q25)-GFP and chFP, was 1. In contrast, the ASI of the aggregation-prone protein, htt(Q47)-GFP, compared with chFP after autophagy shutoff (+dox; 100 h) was 2. Data represent three independent experiments performed on different days. **, P ≤ 0.01; ***, P ≤ 0.005.(E) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff (+dox). The degree of cell death correlated directly with increased htt(Q47)-GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt(Q47)-GFP compared with htt(Q25)-GFP (***, P ≤ 0.005). Data represent three independent experiments performed on different days.(F) AQUA using heavy isotope-labeled GFP standard in addition to the routine Ub standards after P2UBA pull-down of soluble and insoluble lysates from htt(Q)-GFP-IRES-chFP bicistronic expressing cells before (−dox) and after autophagy shutoff (+dox). The experimental GFP peptide was only detected in two of the four experiments, and the data shown are representative of these. In the other two experiments, the experimental GFP peptide was below the threshold of detection.


Figure 2 ( A - C ) The relative levels of ( A ) canalicular membrane ( BA ) transporter Bsep , Mrp2 , Mdr1b , Mdr2 , Abcg5 and Abcg8 mRNA transcripts , ( B ) basolateral membrane BA transporter Mrp3 , Mrp4 , Ostβ , Ntcp and Oatp1a1 mRNA transcripts , and ( C ) BA synthetic enzyme Cyp7a1 , Cyp7b1 , Cyp8b1 , Cyp27a1 , and Cyp2c70 mRNA transcripts . Wild type , wild type mice , n = 6 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 7 . Data are shown as means ± SD . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test . ( D ) Western blot analysis of the relative levels of Bsep , Mrp2 , Mdr2 , Mrp3 , Mrp4 , Ostα / β , Ntcp , Cyp7a1 and Cyp8b1 protein expression in mouse livers . Contrary to the mRNA alterations , the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers , suggesting that the decreased these proteins were might be mediated by post - translational regulation in the livers of Sema7aR145W mutant mice . Wild type , wild type mice , n = 4 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 5 . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test . ( E , F ) Multiplex IF analyses of Bsep and Mrp2 expression in the livers ( E ) and primary hepatocytes in sandwich cultures ( F ) from WT and homozygous mice . Scale bar , 50 µm . ( G ) Multiplex IF analyses revealed that transfection with the SEMA7A _ WT or SEMA7A _ R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose - dependent manner ( 0 , 0 . 2 , 1 . 0 μg per well of 6 - well plate ; 1 . 5 ml culture medium per well ) . Scale bar , 50 µm . Together , the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis ( Appendix Table S16 ) . Subsequently , cholestasis triggered an adaptive response in the liver by down - regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up - regulating the expression of BA efflux transporters Mrp3 , Mrp4 and Ostα / β .

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 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 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

Figure 2(A-C) The relative levels of (A) canalicular membrane (BA) transporter Bsep, Mrp2, Mdr1b, Mdr2, Abcg5 and Abcg8 mRNA transcripts, (B) basolateral membrane BA transporter Mrp3, Mrp4, Ostβ, Ntcp and Oatp1a1 mRNA transcripts, and (C) BA synthetic enzyme Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and Cyp2c70 mRNA transcripts. Wild type, wild type mice, n=6; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=7. Data are shown as means ± SD. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.(D) Western blot analysis of the relative levels of Bsep, Mrp2, Mdr2, Mrp3, Mrp4, Ostα/β, Ntcp, Cyp7a1 and Cyp8b1 protein expression in mouse livers. Contrary to the mRNA alterations, the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers, suggesting that the decreased these proteins were might be mediated by post-translational regulation in the livers of Sema7aR145W mutant mice. Wild type, wild type mice, n=4; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=5. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.(E, F) Multiplex IF analyses of Bsep and Mrp2 expression in the livers (E) and primary hepatocytes in sandwich cultures (F) from WT and homozygous mice. Scale bar, 50 µm.(G) Multiplex IF analyses revealed that transfection with the SEMA7A_WT or SEMA7A_R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose-dependent manner (0, 0.2, 1.0 μg per well of 6-well plate; 1.5 ml culture medium per well). Scale bar, 50 µm. Together, the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis (Appendix Table S16). Subsequently, cholestasis triggered an adaptive response in the liver by down-regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up-regulating the expression of BA efflux transporters Mrp3, Mrp4 and Ostα/β.


Figure 3Treatment with UDCA ( 13 mg / kg / d ) and GSH ( 40 mg / kg / d ) for 2 weeks restored normal liver function in the child . However , when the treatment was ceased the levels of serum ALT , AST and TBA returned to abnormally high levels in the patient . Interestingly , treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST , and reduced serum TBA in the child patient .

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 3Treatment with UDCA (13 mg/kg/d) and GSH (40 mg/kg/d) for 2 weeks restored normal liver function in the child. However, when the treatment was ceased the levels of serum ALT, AST and TBA returned to abnormally high levels in the patient. Interestingly, treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST, and reduced serum TBA in the child patient.


Figure 1 C , Dose - response curves of BEAS - 2B and BEAS - 2B - KRAS cells treated for 72 hours with inhibitors of FGFR ( AZD4547 , BGJ398 ) and PLK1 ( BI2536 , BI6727 ) , alone or combination . Values of Fa and CI are shown underneath , with CI < 1 . 0 indicating synergistic effect . Data were from three independent experiments ( n = 3 ) , error bar : SD D , BEAS - 2B and BEAS - 2B - KRAS treated with the indicated drugs , alone or combination for 72 hours , were cultured in drug - free medium for additional 7 - 21 days . Surviving cells after the treatment were fixed and visualized by crystal violet staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 C, Dose-response curves of BEAS-2B and BEAS-2B-KRAS cells treated for 72 hours with inhibitors of FGFR (AZD4547, BGJ398) and PLK1 (BI2536, BI6727), alone or combination. Values of Fa and CI are shown underneath, with CI<1.0 indicating synergistic effect. Data were from three independent experiments (n=3), error bar: SD  D, BEAS-2B and BEAS-2B-KRAS treated with the indicated drugs, alone or combination for 72 hours, were cultured in drug-free medium for additional 7-21 days. Surviving cells after the treatment were fixed and visualized by crystal violet staining.


Figure 2 C , Clongenic assay of KRAS - mutant lung and pancreatic cells ( MIA PaCa - 2 , H2122 , H2009 , H23 , H358 and A549 ) treated with control ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination . D , Apoptotic assay of KRAS - mutant cancer cells ( H358 , H441 , A549 , and SW620 ) and KRAS - WT lung cancer cells ( EBC - 1 ) treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 48h . Data are presented as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons test . E , KRAS mutant lung ( A549 , H358 ， H441 ) , pancreatic ( MIAPaCa - 2 ) and colon ( SW620 ) cancer cells were transfected with control siRNAs , FGFR1 - and PLK1 - specific siRNAs , alone or combination . Cell viability was determined 72 h post transfection . Data are presented as mean ± SD ( n = 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , and ns P ＞ 0 . 05 by two - way ANOVA with Tukey ' s multiple comparisons test . ns , no significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 C, Clongenic assay of KRAS-mutant lung and pancreatic cells (MIA PaCa-2, H2122, H2009, H23, H358 and A549) treated with control (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination.  D, Apoptotic assay of KRAS-mutant cancer cells (H358, H441, A549, and SW620) and KRAS-WT lung cancer cells (EBC-1) treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 48h. Data are presented as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.  E, KRAS mutant lung (A549, H358，H441), pancreatic (MIAPaCa-2) and colon (SW620) cancer cells were transfected with control siRNAs, FGFR1- and PLK1-specific siRNAs, alone or combination. Cell viability was determined 72 h post transfection. Data are presented as mean ± SD (n=3). **p<0.01, ****p<0.0001, and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test. ns, no significance.


Figure 3 D , Clonogenic assay of H358 , A549 and MIA PaCa - 2 cells treated with AZD4547 , BI2536 , and NAC , alone or in combination as indicated . E , Immunoblots of H358 and A549 cells treated for 24 h with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of NAC ( 5 mM ) . clonogenic assay of H358 cells treated for 48 h with vehicle , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of Q - VD - OPh ( 20 µM ) . Data shown as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons 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 O O O O O O O O O O O O O

Figure 3 D, Clonogenic assay of H358, A549 and MIA PaCa-2 cells treated with AZD4547, BI2536, and NAC, alone or in combination as indicated.  E, Immunoblots of H358 and A549 cells treated for 24 h with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of NAC (5 mM).  clonogenic assay of H358 cells treated for 48 h with vehicle, AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Data shown as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.


Figure 4 B , C , Immunoblots ( B ) and viability assay ( C ) of H358 and A549 cells preincubated overnight with SP600125 ( 2 µM ) and SB203580 ( 1 µM ) and subsequently treated with AZD 4547 ( 5 µM ) and BI2536 ( 5 nM ) for 24 hours . Data were shown as mean ± SD ( n = 3 ) ; * * * P < 0 . 001 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . D , E , Immunoblots ( D ) and viability assay ( E ) of H358 and A549 cells transfected with E2F1 - specific or control siRNAs followed by treatment ( 48 h post transfection ) with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) for additional 24 hours . Data were shown as mean ± SD ( n = 3 ) . * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . F , H358 cells transfected with E2F1 - specific or control siRNAs for 24h were subsequently treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination for 48h before apoptotic assay . Data were shown as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 B, C, Immunoblots (B) and viability assay (C) of H358 and A549 cells preincubated overnight with SP600125 (2 µM) and SB203580 (1 µM) and subsequently treated with AZD 4547 (5 µM) and BI2536 (5 nM) for 24 hours. Data were shown as mean ± SD (n=3); ***P<0.001, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.  D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.  F, H358 cells transfected with E2F1-specific or control siRNAs for 24h were subsequently treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination for 48h before apoptotic assay. Data were shown as mean ± SD (n=3). *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


Figure 5 B , A549 cells stably expressing mCHerry - GFP - LC3B were exposed to vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 24h . Cell were then fixed and processed for immunofluorescence , scale bar = 10 µm . E , Flow cytometry - based ROS measure in H358 , A549 and MIA PaCa - 2 cells treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or the combination , in the presence or absence of HCQ ( 10 µM ) for 24h . Quantification of relative ROS levels was shown to the right . Data are presented as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . F , Immunoblots of H358 cells transfected with ATG5 - specific or control siRNAs for 48h and before treated for 24h with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) .

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

Figure 5 B, A549 cells stably expressing mCHerry-GFP-LC3B were exposed to vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. Cell were then fixed and processed for immunofluorescence, scale bar=10 µm.  E, Flow cytometry-based ROS measure in H358, A549 and MIA PaCa-2 cells treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or the combination, in the presence or absence of HCQ (10 µM) for 24h. Quantification of relative ROS levels was shown to the right. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.  F, Immunoblots of H358 cells transfected with ATG5-specific or control siRNAs for 48h and before treated for 24h with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536 (5 nM).


Figure 6 A , Growth curve of a KRAS - mutant lung cancer PDX ( BE564T ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 5 mice / group ) ; error bar : SD D , E , H & E and IHC analysis ( Ki67 and Caspase - 3 ) of PDX ( BE 564T ) tumors after the treatment ( D ) . Quantification of the IHC data for the positivity of Ki - 67 and Caspase - 3 are shown in ( E ) . * P < 0 . 05 and * * P < 0 . 01 by two - way ANOVA with Tukey ' s multiple comparisons test . Data are shown as mean ± SD ( error bar ) of three independent experiments ( n = 3 ) . F - K , Growth curves ( F , I ) of KRAS - mutant lung cancer PDXs ( PF139 , PF563 ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 4 mice / group ) ; error bar : SD Relative tumor volume ( G , J ) and weights ( H , K ) of the PDX tumors after the treatment . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by unpaired two - tailed t - test . Data are the mean ± SD ( error bar ) of tumor weights of each group ( 4 mice / group ) .

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

Figure 6 A, Growth curve of a KRAS-mutant lung cancer PDX (BE564T) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, ***P<0.001 and P＞0.05 (ns) by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD  D, E, H&E and IHC analysis (Ki67 and Caspase-3) of PDX (BE 564T) tumors after the treatment (D). Quantification of the IHC data for the positivity of Ki-67 and Caspase-3 are shown in (E). *P<0.05 and **P<0.01 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3).  F-K, Growth curves (F, I) of KRAS-mutant lung cancer PDXs (PF139, PF563) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (4 mice/group); error bar: SD Relative tumor volume (G, J) and weights (H, K) of the PDX tumors after the treatment. *P<0.05, **P<0.01, ***P<0.001 and P＞0.05 (ns) by unpaired two-tailed t-test. Data are the mean ± SD (error bar) of tumor weights of each group (4 mice/group).


Figure 7 F , Box plots showing the percentage of Ki67 - positive cells in different treatment groups . Data are based on IHC staining of lung tissue section of LSL - KrasG12D mice . * P < 0 . 05 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . The boxplots span from the first to third quartile , the whiskers extend to 1 . 5 × the interquartile range ( IQR ) . The horzontal line within the boxes represents the median of each group of 10 replicates ( n = 10 ) .

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

Figure 7 F, Box plots showing the percentage of Ki67-positive cells in different treatment groups. Data are based on IHC staining of lung tissue section of LSL-KrasG12D mice. *P<0.05 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. The boxplots span from the first to third quartile, the whiskers extend to 1.5× the interquartile range (IQR). The horzontal line within the boxes represents the median of each group of 10 replicates (n=10).


Figure 1A Representative IF image of NPCs stained for Nestin , FoxG1 and Pank2 . B NPCs differentiated into neurons by overexpressing Ngn2 ( one representative experiment is shown ) . Two weeks after the infection differentiated NPC were positive for neuronal markers βIII - tubulin ( Tuj1 ) , Map2 , NeuN and humannuclei ( hNu ) and synaptic markers , the voltage - gated Na + channels ( PanNav ) and the vesicular glutamate transporter 1 ( VGlut1 ) . C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β - actin antibodies ( arrows ) . Asterisk indicates nonspecific band . Data are representative of three independent experiments . D Plots showing the total dendritic length and branching points . Data presented as mean and + SEM from at least three independent experiments . A total of 38 neurons were counted for each sample . Statistics were determined by the t - test and resulted not significant . E Representative example of co - culture containing control ( green ) , PKAN ( red ) humanneurons , and E18 cortical miceneurons . Control and PKAN NPCs were infected with GFP - LV and tdT - LV expressing vectors , respectively , and differentiated for 8 weeks . Scale bars 20 μm . F Examples of electrophysiological properties of humanneurons : control individual ( top ) ; PKAN patient ( bottom ) . Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current - clamp mode . Middle traces show Na + and K + currents ( down - and upward - deflecting from baseline , respectively ) in response to a 60 mV step from a holding voltage of - 70 mV in voltage - clamp mode . Insets on the right display enlarged portions of the traces to magnify fast Na + currents . G Histogram with percentages of recorded cells showing repetitively firing ( z - test ) .

O B-PANEL_START 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 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 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

Figure 1A Representative IF image of NPCs stained for Nestin, FoxG1 and Pank2.B NPCs differentiated into neurons by overexpressing Ngn2 (one representative experiment is shown). Two weeks after the infection differentiated NPC were positive for neuronal markers βIII-tubulin (Tuj1), Map2, NeuN and humannuclei (hNu) and synaptic markers, the voltage-gated Na+ channels (PanNav) and the vesicular glutamate transporter 1 (VGlut1).C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β-actin antibodies (arrows). Asterisk indicates nonspecific band. Data are representative of three independent experiments.D Plots showing the total dendritic length and branching points. Data presented as mean and + SEM from at least three independent experiments. A total of 38 neurons were counted for each sample. Statistics were determined by the t-test and resulted not significant.E Representative example of co-culture containing control (green), PKAN (red) humanneurons, and E18 cortical miceneurons. Control and PKAN NPCs were infected with GFP-LV and tdT-LV expressing vectors, respectively, and differentiated for 8 weeks. Scale bars 20 μm.F Examples of electrophysiological properties of humanneurons: control individual (top); PKAN patient (bottom). Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current-clamp mode. Middle traces show Na+ and K+ currents (down- and upward-deflecting from baseline, respectively) in response to a 60 mV step from a holding voltage of -70 mV in voltage-clamp mode. Insets on the right display enlarged portions of the traces to magnify fast Na+ currents. G Histogram with percentages of recorded cells showing repetitively firing (z-test).


Figure 2A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM , the neuronal specific anti - NCAM antibody and the nuclear staining Hoechst . Left panel , basal conditions . Right panel , after addition of the mitochondrial uncoupler FCCP . Scale bar 20 μm . Plot showing the quantification of TMRM fluorescence signal from NCAM + neurons . Data presented as means + SEM of three independent experiments ( unpaired , two - tailed t - test ) . B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope . PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein . Scale bar 500nm . Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in > 30 field ( 200 mitochondria in total ) for each sample ( one - way ANOVA ) . C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls , PKAN patients and PKAN patients overexpressing PANK2 . The plots show OCR normalization to cell number . OCR was measured in basal conditions , and after oligomycin and FCCP addition . Bars indicate means + SEM of three independent experiments ( Two - way ANOVA ) .

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 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

Figure 2A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM, the neuronal specific anti-NCAM antibody and the nuclear staining Hoechst. Left panel, basal conditions. Right panel, after addition of the mitochondrial uncoupler FCCP. Scale bar 20 μm. Plot showing the quantification of TMRM fluorescence signal from NCAM+neurons. Data presented as means + SEM of three independent experiments (unpaired, two-tailed t-test).B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope. PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein. Scale bar 500nm. Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in >30 field (200 mitochondria in total) for each sample (one-way ANOVA).C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls, PKAN patients and PKAN patients overexpressing PANK2. The plots show OCR normalization to cell number. OCR was measured in basal conditions, and after oligomycin and FCCP addition. Bars indicate means + SEM of three independent experiments (Two-way ANOVA).


Figure 3A Representative images of neurons stained with the neuron specific anti - NCAM antibody , ROS sensible fluorescent probe DCF , and the nuclei dye Hoechst . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM positive controls - and PKAN humanneurons , infected or not with Ngn2 - PANK2 - LV . Data presented as means + SEM of at least three independent experiments ( One - way ANOVA ) . B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1 . Scale bar 20 μm . ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots . Data presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - 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 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 3A Representative images of neurons stained with the neuron specific anti-NCAM antibody, ROS sensible fluorescent probe DCF, and the nuclei dye Hoechst. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM positive controls- and PKAN humanneurons, infected or not with Ngn2-PANK2-LV. Data presented as means + SEM of at least three independent experiments (One-way ANOVA).B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1. Scale bar 20 μm. ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots. Data presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


Figure 4A Upper panel : representative images of in - gel enzymatic activity of mitochondrial and cytosolic aconitase ( mAco and cAco , respectively ) . The lower part of the gel was cut , stained with Coomassie blue , and a protein band was used as loading control ( Loading ) . Lower panel : quantification of mAco and cAco enzymatic activity by densitometry . B Upper panel : Western blot analysis of mitochondrial and cytosolic aconitases . Lower panel : quantification of mAco and cAco by densitometry . C Hemequantification by absorbance at 400 nm of the soluble cell lysates . D Upper panel : western blot analysis of transferrin receptor ( TfR1 ) and ferritin ( FtH ) . Lower panel : quantification of TfR1 or FtH normalized on actin by densitometry . All the data are presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - 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 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 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 O O O O

Figure 4A Upper panel: representative images of in-gel enzymatic activity of mitochondrial and cytosolic aconitase (mAco and cAco, respectively). The lower part of the gel was cut, stained with Coomassie blue, and a protein band was used as loading control (Loading). Lower panel: quantification of mAco and cAco enzymatic activity by densitometry.B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry.C Hemequantification by absorbance at 400 nm of the soluble cell lysates.D Upper panel: western blot analysis of transferrin receptor (TfR1) and ferritin (FtH). Lower panel: quantification of TfR1 or FtH normalized on actin by densitometry. All the data are presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


Figure 5A Representative images of co - cultures of control and PKAN NPCs infected with GFP - LV and tdT - LV expressing vectors , respectively , at the beginning ( Day 1 ) , after 120 ( Day 120 ) and 150 days of differentiation ( Day 150 ) . Scale bar 20 μm . Plots show the number of green and red humanneurons counted at different time points . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) . B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days . Repetitive firing activity ( left ) and relatively large Na + and K + currents ( right ) , were restored by CoA . The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs . CoA - treated neurons from control and PKAN patients ( z - test ) . C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst . Anti - NCAM antibody was used to detect neurons . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM + controls - and PKAN humanneurons differentiated or not in the presence of CoA ( 25 μM ) in the medium for 3 weeks . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) . D Oxygen consumption rate ( OCR ) with and without CoA on controls and PKAN patients . Basal and uncoupled ( FCCP ) respiration increased after CoA supplementation . Data presented as means + SEM of 24 independent replicates for each condition ( Two - way ANOVA ) . E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

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 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 O O O O 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

Figure 5A Representative images of co-cultures of control and PKAN NPCs infected with GFP-LV and tdT-LV expressing vectors, respectively, at the beginning (Day 1), after 120 (Day 120) and 150 days of differentiation (Day 150). Scale bar 20 μm. Plots show the number of green and red humanneurons counted at different time points. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days. Repetitive firing activity (left) and relatively large Na+ and K+ currents (right), were restored by CoA. The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs. CoA-treated neurons from control and PKAN patients (z-test).C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst. Anti-NCAM antibody was used to detect neurons. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM+ controls- and PKAN humanneurons differentiated or not in the presence of CoA (25 μM) in the medium for 3 weeks. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).D Oxygen consumption rate (OCR) with and without CoA on controls and PKAN patients. Basal and uncoupled (FCCP) respiration increased after CoA supplementation. Data presented as means + SEM of 24 independent replicates for each condition (Two-way ANOVA).E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


Figure 1Representative images of changes in intracellular calcium , as measured in fluorescence ratio images ( F340 / 380 ) ( A ) Examples show a TRPM8 - expressing DRG neuron ( left in A responding to both the TRPM8 agonist menthol ( 300 μM ) and to a cold stimulus from 32 ° C to 4 ° C a neuron that responds to cold ( right in A but does not express any of TRPM8 , TRPA1 or TRPC5 , as shown by lack of response to the TRPM8 agonist menthol ( 300 μM ) Calcium increase is due to influx from extracellular solutionexperiments showing an SCG neuron responding to the TRPA1 agonist AITC ( left in E and a novel cold - sensitive SCG neuron that responds to cold but does not express any of TRPM8 , TRPA1 or TRPC5 ( right in E Many glial cells are also visibly activated by AITC but not by cold .

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Figure 1Representative images of changes in intracellular calcium, as measured in fluorescence ratio images (F340/380) (A) Examples show a TRPM8-expressing DRG neuron (left in A responding to both the TRPM8 agonist menthol (300 μM) and to a cold stimulus from 32°C to 4°C a neuron that responds to cold (right in A but does not express any of TRPM8, TRPA1 or TRPC5, as shown by lack of response to the TRPM8 agonist menthol (300 μM) Calcium increase is due to influx from extracellular solutionexperiments showing an SCG neuron responding to the TRPA1 agonist AITC (left in E and a novel cold-sensitive SCG neuron that responds to cold but does not express any of TRPM8, TRPA1 or TRPC5 (right in E Many glial cells are also visibly activated by AITC but not by cold.


Figure 2 ( G ) Mean current voltage relation of 10 SCG neurons at 32 ° C ( dashed line ) , 4 ° C ( blue ) and 32 ° C ( red dots ) . ( H ) Difference trace from ( G ) .

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Figure 2(G) Mean current voltage relation of 10 SCG neurons at 32°C (dashed line), 4°C (blue) and 32°C (red dots). (H) Difference trace from (G).


Figure 3 ( C ) Mean cold - sensitive increase in calcium - dependent fluorescence ratio F340 / 380 in SCG neurons is significantly smaller in neurons recorded in voltage - clamp at - 60mV ( 0 . 56 ± 0 . 08 , mean ± SEM , n = 21 neurons ) than in neurons recorded in current - clamp mode ( 1 . 16 ± 0 . 29 , n = 11 neurons , p = 0 . 0165 , unpaired t - test ) . ( F ) Summary of results of experiments on SCG neurons ( mean ± SEM , n = 115 neurons , 4 separate cultures ) . YM58483 caused a significant decrease in cold - response amplitude ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s test ) , with only partial recovery . Temperature trace below . ( G ) In neurons with a small response to cold ( F340 / 380 increase < 0 . 2 ; n = 24 neurons ) , many responses are potentiated by verapamil ( 31 % of cold - insensitive neurons ) and are fully blocked by ORAI channel antagonist YM58483 ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s test ) .

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Figure 3(C) Mean cold-sensitive increase in calcium-dependent fluorescence ratio F340/380 in SCG neurons is significantly smaller in neurons recorded in voltage-clamp at -60mV (0.56 ±0.08, mean ± SEM, n=21 neurons) than in neurons recorded in current-clamp mode (1.16 ±0.29, n=11 neurons, p=0.0165, unpaired t-test).(F) Summary of results of experiments on SCG neurons (mean ±SEM, n=115 neurons, 4 separate cultures). YM58483 caused a significant decrease in cold-response amplitude (p<0.0001, RM one-way ANOVA + Dunnett's test), with only partial recovery. Temperature trace below. (G) In neurons with a small response to cold (F340/380 increase <0.2; n=24 neurons), many responses are potentiated by verapamil (31% of cold-insensitive neurons) and are fully blocked by ORAI channel antagonist YM58483 (p<0.0001, RM one-way ANOVA + Dunnett's test).


Figure 4 ( D ) Representative fluorescence images of SCG neurons from neonatal mice ( age P5 - 7 ) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1 . Neurons were cooled from 35 ° C to 7 ° C in the presence of 100μM verapamil . Electroporation was indicated by expression of mCherry ( # in left - hand panel ) ; in these neurons a cold response was absent ( # in right - hand panel ) while in neurons not expressing mCherry ( * ) cold responses were still observed . Scale bar 20μm . ( F ) Cold response amplitudes ( F340 / 380 , mean ± SEM ) . Successfully electroporated neurons ( mCherry positive , red bar , n = 111 neurons from four separate cultures ) had a significantly lower cold response amplitude than neurons not electroporated ( mCherry negative , black bar , n = 104 neurons from same cultures , p = 0 . 0082 , unpaired t - test ) . ( G ) Percentage of neurons that responded to cold ramp with F340 / 380 increase > 0 . 2 . Only 5 % of mCherry - positive neurons ( successfully electroporated , red bar ) responded to cold , compared to 19 % of mCherry - negative neurons in same cultures ( non - electroporated , black bar , p < 0 . 001 , chi - square test ) .

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Figure 4(D) Representative fluorescence images of SCG neurons from neonatal mice (age P5-7) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1. Neurons were cooled from 35°C to 7°C in the presence of 100μM verapamil. Electroporation was indicated by expression of mCherry (# in left-hand panel); in these neurons a cold response was absent (# in right-hand panel) while in neurons not expressing mCherry (*) cold responses were still observed. Scale bar 20μm.(F) Cold response amplitudes (F340/380, mean ±SEM). Successfully electroporated neurons (mCherry positive, red bar, n=111 neurons from four separate cultures) had a significantly lower cold response amplitude than neurons not electroporated (mCherry negative, black bar, n=104 neurons from same cultures, p=0.0082, unpaired t-test). (G) Percentage of neurons that responded to cold ramp with F340/380 increase >0.2. Only 5% of mCherry-positive neurons (successfully electroporated, red bar) responded to cold, compared to 19% of mCherry-negative neurons in same cultures (non-electroporated, black bar, p<0.001, chi-square test).


Figure 5 ( A ) Representative TIRF microscopy images ( imaging depth 100nm ) showing that cooling from 35 ° C to 13 ° C increases ORAI1 puncta formation and colocalization of ORAI1 with STIM1 in HEK293 cells transfected with ORAI1 - CFP and STIM1 - YFP . Cooling to 13 ° C causes the appearance of new ORAI1 puncta ( examples shown by * ) that are not present in the 35 ° C image and are colocalised with STIM1 . Cooling also causes STIM1 to move towards the cell surface in edge - on views ( arrows ) . To reduce cell motility , the experiment was conducted in nominal 0 [ Ca ] e , which potentially caused partial formation of STIM1 puncta before the images shown . Scale bar = 5μm . Temperature trace shown below . Mean fluorescence intensity adjusted to compensate for increase in intrinsic fluorescence of CFP and YFP caused by cooling ( see Fig . S12 ) .

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Figure 5(A) Representative TIRF microscopy images (imaging depth 100nm) showing that cooling from 35°C to 13°C increases ORAI1 puncta formation and colocalization of ORAI1 with STIM1 in HEK293 cells transfected with ORAI1-CFP and STIM1-YFP. Cooling to 13°C causes the appearance of new ORAI1 puncta (examples shown by *) that are not present in the 35°C image and are colocalised with STIM1. Cooling also causes STIM1 to move towards the cell surface in edge-on views (arrows). To reduce cell motility, the experiment was conducted in nominal 0[Ca]e, which potentially caused partial formation of STIM1 puncta before the images shown. Scale bar = 5μm. Temperature trace shown below. Mean fluorescence intensity adjusted to compensate for increase in intrinsic fluorescence of CFP and YFP caused by cooling (see Fig. S12).


Figure 6 ( A ) Representative immunofluorescence images showing adult mouse stellate ganglion ( SG ) sections stained for nNOS ( red ) , to label nitrergic neurons , βIII Tubulin ( green ) to label all neurons , and DAPI to label nuclei . nNOS is widely expressed in neurons of the stellate ganglion , but is absent from glial cells ( glial nuclei identifiable in merged image , similar results in n = 5 animals ) . Antibody against nNOS from Abcam ; similar results obtained with antibodies from ECM and Millipore . All three antibodies show nNOS widely expressed in SG cell bodies and neurites . ( B ) Adult mouse SCG ( n = 2 animals , upper ) and SG ( n = 5 animals , lower ) sections stained for nNOS ( red ) , tyrosine hydroxylase ( TH , green ) and DAPI ( blue ) to label nitrergic neurons , noradrenergic neurons and nuclei respectively . Many noradrenergic cells also express nNOS .

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Figure 6(A) Representative immunofluorescence images showing adult mouse stellate ganglion (SG) sections stained for nNOS (red), to label nitrergic neurons, βIII Tubulin (green) to label all neurons, and DAPI to label nuclei. nNOS is widely expressed in neurons of the stellate ganglion, but is absent from glial cells (glial nuclei identifiable in merged image, similar results in n=5 animals). Antibody against nNOS from Abcam; similar results obtained with antibodies from ECM and Millipore. All three antibodies show nNOS widely expressed in SG cell bodies and neurites.(B) Adult mouse SCG (n=2 animals, upper) and SG (n=5 animals, lower) sections stained for nNOS (red), tyrosine hydroxylase (TH, green) and DAPI (blue) to label nitrergic neurons, noradrenergic neurons and nuclei respectively. Many noradrenergic cells also express nNOS.


Figure 1 ( B ) The ratio of unique histone iCLIP - seq read population to unique histone RNA - seq read population was calculated and shown , with the unique histone RNA - seq read population set as " 1 " . Error bars represent standard deviations from three replicates of ALYREF iCLIP data . Statistical analysis was performed using Student ' s t - test . * * * P < 0 . 001 . ( C ) ALYREF RIP - qPCRs to examine ALYREF binding on histone mRNAs . Relative RIP efficiencies are shown . A tRNA was used as a negative control for ALYREF binding . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant . ( D ) The distribution of histone reads in 5 ' UTR , CDS and 3 ' UTR in rRNA - depleted nuclear RNA - seq and ALYREF - iCLIP - seq . ( E ) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA ' s contribution to the plot . ( F ) Screenshots of two histone mRNAs showing clustered ALYREF binding sites . The 3 ' UTR regions are highlighted . ( G ) Distribution profile of clustered ALYREF binding sites on histone mRNA 3 ' UTRs relative to the cleavage site ( CS ) . The CS is set as " 0 " in the x axis , and the relative position to the CS is marked . The y axis shows the total occurrence of binding sites in three repeats at each nucleotide .

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Figure 1(B) The ratio of unique histone iCLIP-seq read population to unique histone RNA-seq read population was calculated and shown, with the unique histone RNA-seq read population set as "1". Error bars represent standard deviations from three replicates of ALYREF iCLIP data. Statistical analysis was performed using Student's t-test. ***P< 0.001.(C) ALYREF RIP-qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P< 0.001, n.s., not significant.(D) The distribution of histone reads in 5' UTR, CDS and 3' UTR in rRNA-depleted nuclear RNA-seq and ALYREF-iCLIP-seq.(E) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.(F) Screenshots of two histone mRNAs showing clustered ALYREF binding sites. The 3' UTR regions are highlighted.(G) Distribution profile of clustered ALYREF binding sites on histone mRNA 3' UTRs relative to the cleavage site (CS). The CS is set as "0" in the x axis, and the relative position to the CS is marked. The y axis shows the total occurrence of binding sites in three repeats at each nucleotide.


Figure 2IPs from RNase A - treated S - phase HeLa cell lysate using the SLBP antibody or IgG ( A ) Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same blotsIPs from RNase A - treated S - phase HeLa cell lysate using the ALYREF antibody or IgG ( B ) . Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same blots ( C ) Pull - downs of in vitro translated 35S labelled SLBP and luciferase ( Cntl ) using MBP or MBP - ALYREF in the presence of RNase A . The proteins pulled down were visualized by coomassie staining ( left ) or phosphoimager ( right ) . 3 % of input was loaded . ( D ) GST - SLBP , GST - UAP56 and GST were used for pull - down of purified MBP - ALYREF or MBP in the presence of RNase A . Proteins pulled down were separated by SDS - PAGE , followed by coomassie staining . 37 . 5 % of input proteins were loaded . ( E ) ( Top ) Domain schematic representation of SLBP . ( Bottom ) Flag IPs from RNase A - treated HeLa cell lysate individually expressing indicated Flag tagged proteins , followed by western blotting using Flag and ALYREF antibodies . 3 % of input was loaded . The * indicates a band probably resulted from degradation of Flag - SLBP . The white line delineates the boundary where irrelevant lanes have been removed from the same blot ( E ) ( Top ) Domain schematic representation of SLBP . followed by western blotting using Flag and ALYREF antibodies . 3 % of input was loaded . The * indicates a band probably resulted from degradation of Flag - SLBP . The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA - SLBP stable expression cells transfected with plasmids expressing ALYREF fragments . ( G ) Western blotting to examine the KD efficiency of SLBP . GAPDH was used as a loading control . The white line delineates the boundary where irrelevant lanes have been removed from the same blotCntl - or SLBP - siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody . The immunoprecipitates were subjected to western analysis ( H ) Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant . Cntl - or SLBP - siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody . The immunoprecipitates were subjected to RT - qPCRs ( I ) . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant . ( J ) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA ' s contribution to the plot .

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Figure 2IPs from RNase A-treated S-phase HeLa cell lysate using the SLBP antibody or IgG (A) Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blotsIPs from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody or IgG (B). Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots(C) Pull-downs of in vitro translated 35S labelled SLBP and luciferase (Cntl) using MBP or MBP-ALYREF in the presence of RNase A. The proteins pulled down were visualized by coomassie staining (left) or phosphoimager (right). 3% of input was loaded.(D) GST-SLBP, GST-UAP56 and GST were used for pull-down of purified MBP-ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE, followed by coomassie staining. 37.5% of input proteins were loaded.(E) (Top) Domain schematic representation of SLBP. (Bottom) Flag IPs from RNase A-treated HeLa cell lysate individually expressing indicated Flag tagged proteins, followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot(E) (Top) Domain schematic representation of SLBP. followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA-SLBP stable expression cells transfected with plasmids expressing ALYREF fragments.(G) Western blotting to examine the KD efficiency of SLBP. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blotCntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to western analysis (H) Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to RT-qPCRs (I). Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.(J) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.


Figure 3 ( B ) List of histone genes with defective 3 ' - end processing detected by polyA + and polyA - RNA - seq . PolyA + ALYREF / Cntl shows polyA + RNA - seq RPM ratio of each histone gene in ALYREF KD versus Cntl . ( C ) Screenshots of two exemplified histone mRNAs with 3 ' - end processing defects are shown . Transcription direction is indicated . Black arrowheads and the corresponding lines mark the position of CS . ( D ) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3 ' READS + . ( E , F ) RT - qPCRs to detect histone mRNA levels in the polyA + ( E ) or the polyA - fraction ( F ) in cells treated with Cntl , ALYREF or ARS2 siRNA . The arrowhead and arrows indicate the cleavage site and primer location , respectively . The bars show the relative abundance of polyA + forms to the actin mRNA ( E ) and that of polyA - forms relative to the 18S rRNA ( F ) . Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Western blotting to examine ALYREF protein level ( G ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression cells treated with Cntl or ALYREF siRNA . The white line delineates the boundary where irrelevant lanes have been removed from the same blotRT - qPCRs to detect polyA + histone mRNA levels ( H ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression cells treated with Cntl or ALYREF siRNA . The bars show the relative abundance of histone mRNAs to the GAPDH mRNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 3(B) List of histone genes with defective 3'-end processing detected by polyA+ and polyA- RNA-seq. PolyA+ ALYREF/Cntl shows polyA+ RNA-seq RPM ratio of each histone gene in ALYREF KD versus Cntl.(C) Screenshots of two exemplified histone mRNAs with 3'-end processing defects are shown. Transcription direction is indicated. Black arrowheads and the corresponding lines mark the position of CS.(D) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3'READS+.(E, F) RT-qPCRs to detect histone mRNA levels in the polyA+ (E) or the polyA- fraction (F) in cells treated with Cntl, ALYREF or ARS2 siRNA. The arrowhead and arrows indicate the cleavage site and primer location, respectively. The bars show the relative abundance of polyA+ forms to the actin mRNA (E) and that of polyA- forms relative to the 18S rRNA (F). Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.Western blotting to examine ALYREF protein level (G) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The white line delineates the boundary where irrelevant lanes have been removed from the same blotRT-qPCRs to detect polyA+ histone mRNA levels (H) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The bars show the relative abundance of histone mRNAs to the GAPDH mRNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Figure 4IPs were carried out from RNase A - treated S - phase HeLa cell lysate using the Lsm11 antibody and IgG ( A ) followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same blotIPs were carried out from RNase A - treated S - phase HeLa cell lysate using the ALYREF antibody and IgG ( B ) , followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same blot ( C ) Pull - down of purified His - Cntl ( PGK1 ) or His - Lsm11 using equal amount of GST and GST - ALYREF proteins in the presence of RNase A . Proteins pulled down were separated by SDS - PAGE followed by coomassie staining . * indicates a nonspecific band . Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody . The immunoprecipitates were subjected western analysis ( D ) . * indicates the antibody heavy chain . Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody . The immunoprecipitates were subjected to RT - qPCRs ( E ) * indicates the antibody heavy chain . The bars show the relative IP efficiencies of histone pre - mRNAs to U7 snRNA . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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

Figure 4IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the Lsm11 antibody and IgG (A) followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blotIPs were carried out from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody and IgG (B), followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot(C) Pull-down of purified His-Cntl (PGK1) or His-Lsm11 using equal amount of GST and GST-ALYREF proteins in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE followed by coomassie staining. * indicates a nonspecific band.Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected western analysis (D). * indicates the antibody heavy chain.Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected to RT-qPCRs (E) * indicates the antibody heavy chain. The bars show the relative IP efficiencies of histone pre-mRNAs to U7 snRNA. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Figure 5 ( A ) ( Top ) Illustration of the HIST2H2AA3 reporter construct . ( Bottom ) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl , ALYREF , THOC2 ( THO ) and UAP56 . DAPI staining served as a nuclear marker . N and C indicate nuclear and cytoplasmic FISH signals , respectively . N / C ratios were determined for 20 cells in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . ( B ) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript - specific probe in HeLa cells depleted of Cntl , ALYREF , THOC2 ( THO ) and UAP56 . N and C indicate nuclear and cytoplasmic FISH signals , respectively . N / C ratios were calculated Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Western analysis to examine the purities of nuclear and cytoplasmic fractions ( C ) in Cntl and UAP56 KD cells . MTR4 and Tubulin were used as the nuclear and cytoplasmic marker , respectively . RT - qPCRs to examine N / C distribution of each histone mRNA ( D ) in Cntl and UAP56 KD cells . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . ( F ) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl , ALYREF and SLBP KD cells . Data information : Error bars represent standard deviations from biological repeats n = 2 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . ( G ) NXF1 iCLIP signals along the histone mRNA . The histone mRNA is divided into 10 fractions based on their mRNA size . Each fraction represents 10 % of each histone mRNA . The tenth fractions that contains the SL is boxed by green dashed lines .

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

Figure 5(A) (Top) Illustration of the HIST2H2AA3 reporter construct. (Bottom) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. DAPI staining served as a nuclear marker. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.(B) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript-specific probe in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were calculated Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.Western analysis to examine the purities of nuclear and cytoplasmic fractions (C) in Cntl and UAP56 KD cells. MTR4 and Tubulin were used as the nuclear and cytoplasmic marker, respectively.RT-qPCRs to examine N/C distribution of each histone mRNA (D) in Cntl and UAP56 KD cells. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.(F) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl, ALYREF and SLBP KD cells. Data information: Error bars represent standard deviations from biological repeats n=2 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.(G) NXF1 iCLIP signals along the histone mRNA. The histone mRNA is divided into 10 fractions based on their mRNA size. Each fraction represents 10% of each histone mRNA. The tenth fractions that contains the SL is boxed by green dashed lines.


Figure 6 ( A ) ( Top ) Illustration of the reporter constructs . The black and red lines indicate H2AA3 - SL and H2AA3 - SLD PCR products , respectively . The dashed line indicates the FISH probe . ( Bottom ) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3 - SL and H2AA3 - SLD PCR products . C and N indicate cytoplasmic and nuclear FISH signals , respectively . C / N ratios were determined for 20 cells in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 . ( B ) ( Top ) Illustration of the reporter constructs . The dashed line indicates the FISH probe . FISH to detect mRNA distribution at 24 hr after transfection of the H1C - SL - Rb ( cH1C ) and H1C - SLD - Rb ( pH1C ) constructs . C / N ratios were quantified Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 . ( Top ) Illustration of the reporter constructs . PCR products of cG - SL and cG - SLD were used instead . cG , β - globin cDNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 . H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . The immunoprecipitates were subjected to western analysis to detect ALYREF ( D ) H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . RT - qPCRs to detect RNAs ( E ) . The bars in the graph shows the relative ALYREF IP efficiency , with that for the H1C - SL - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 . ( F ) H1C - SLD - Rb construct with wild - type ( H1C - SLDWT - Rb ) or mutant ( H1C - SLDmut - Rb ) U7 - binding sequence , together with VSV M , was co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . The immunoprecipitates were subjected to western analysis ( left panel ) and RT - qPCRs ( right panel ) . The bars in the right graph show the relative ALYREF IP efficiency , with that for the H1C - SLDWT - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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

Figure 6(A) (Top) Illustration of the reporter constructs. The black and red lines indicate H2AA3-SL and H2AA3-SLD PCR products, respectively. The dashed line indicates the FISH probe. (Bottom) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3-SL and H2AA3-SLD PCR products. C and N indicate cytoplasmic and nuclear FISH signals, respectively. C/N ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.(B) (Top) Illustration of the reporter constructs. The dashed line indicates the FISH probe. FISH to detect mRNA distribution at 24 hr after transfection of the H1C-SL-Rb (cH1C) and H1C-SLD-Rb (pH1C) constructs. C/N ratios were quantified Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.(Top) Illustration of the reporter constructs. PCR products of cG-SL and cG-SLD were used instead. cG, β-globin cDNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis to detect ALYREF (D)H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. RT-qPCRs to detect RNAs (E). The bars in the graph shows the relative ALYREF IP efficiency, with that for the H1C-SL-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.(F) H1C-SLD-Rb construct with wild-type (H1C-SLDWT-Rb) or mutant (H1C-SLDmut-Rb) U7-binding sequence, together with VSV M, was co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis (left panel) and RT-qPCRs (right panel). The bars in the right graph show the relative ALYREF IP efficiency, with that for the H1C-SLDWT-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


Figure 7 ( A ) Western blotting to examine the expression levels of H2B and H4 in Cntl , ALYREF and ARS2 KD cells . GAPDH was used as a loading control . The white line delineates the boundary where irrelevant lanes have been removed from the same blot ( B ) Growth curve of Cntl and ALYREF KD cells . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * * P < 0 . 01 . ( C ) Cell cycle analysis of Cntl , ALYREF and ARS2 KD cells . Quantifications of cells in G0 / G1 ( 1N ) , G2 ( 2N ) and S - phase ( intermediate ) of the cell cycle are shown in the upper right corner of each histogram .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7(A) Western blotting to examine the expression levels of H2B and H4 in Cntl, ALYREF and ARS2 KD cells. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot(B) Growth curve of Cntl and ALYREF KD cells. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01.(C) Cell cycle analysis of Cntl, ALYREF and ARS2 KD cells. Quantifications of cells in G0/G1 (1N), G2 (2N) and S-phase (intermediate) of the cell cycle are shown in the upper right corner of each histogram.


Figure 4Results from multiplex ACE2 competition assay are shown for the three spike - derived antigens : RBD , S1 - domain ( S1 ) and homotrimeric spike ( Spike ) . Color - coded beads coated with the respective antigens were co - incubated with biotinylated ACE2 and dilution series of NM1267 ( 8 pM to 126 nM ) followed by measuring residual binding of ACE2 . MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2 - only control . IC50 values were calculated from a four - parametric sigmoidal model . Data are presented as mean ± s . d . of three technical replicates . E Neutralization potency of NM1267 was analyzed in Caco ‑ 2 cells using the SARS - CoV - 2 - mNG infectious clones . Infection rate normalized to virus - only infection control is illustrated as percent of infection ( % Infection ) . IC50 value was calculated from a four - parametric sigmoidal model and data are presented as mean ± s . e . m . of three biological replicates ( n = 3 ) .

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 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

Figure 4Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).


Figure 5B For the NeutrobodyPlex , antigen - coated beads comprising RBD , S1 or spike were co - incubated with serum samples from 5 patients and a dilution series of NM1267 ( 1 µM to 6 pM ) ( n = 1 ) . Mean fluorescent intensities ( MFI ) derived from antigen - bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum - only samples , illustrated as MFI ( % control ) , are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.


Figure 7A , B Total IgGs derived from spike - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . C , D Total IgGs derived from N - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . F

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 7A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F