Figure 3 ( A ) EGFR was not required for ligand - induced STING dimerization : MEFs were transfected with HA - tagged STING and treated with gefitinib or DMSO before cGAMP transfection , 4h later STING was immunoprecipitated with anti - HA antibodies and analyzed by Western blot . ( B ) EGFR was required for the activation of IRF3 , but not TBK1 : MEFs were pretreated with gefitinib for 1h , then treated with cGAMP for 2h ; cell lysate were subjected to Western blot analysis using the indicated antibodies . ( C ) Nuclear translocation of IRF3 , but not NFκB p65 , required EGFR : MEFs were pre - treated with gefitinib ( + ) or DMSO ( - ) for 1 hour before transfecting cGAMP . Cells were harvested and nuclei were isolated at the indicated time points ; total cell extracts ( T ) and nuclear extracts ( N ) were analyzed by Western blot . MEFs were pretreated with gefitinib for 1h , then treated with cGAMP for 2h ; ( D ) IRF3 , but not TBK1 , binding to STING required EGFR cell lysates were immunoprecipitated with anti - STING antibody and analyzed by Western blot . ( E ) STING - IRF3 interaction in human cells required EGFR : WT and EGFR - / - ( KO ) HeLa cells were transfected with cGAMP , 2h later cells were lysed , immunoprecipitated with anti - STING antibody and analyzed by Western blot . ( F ) STING and IRF3 co - localized in the endosomes of stimulated cells : STING - / - MEF cells were transfected with STING - HA for 24 hours then treated with cGAMP for 1h . The cells were then stained with the HA ( purple ) , CD63 ( red ) and IRF3 ( green ) antibodies . Imaging data were analyzed by the Fuji software to reveal co - localization as white dots . Scale bars represent 10 µ m . ( G ) EGFR was not required for ligand - induced phosphorylation of STING Ser366 : EGFR - / - HeLa cells or gefitinib - treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot . ( H ) Kinetics of STING and IRF3 co - localization in the endosomes of stimulated cells : STING - / - MEF cells were transfected with STING - HA for 24 hours and treated with cGAMP for the indicated time . The cells were then stained with the HA ( purple ) , CD63 ( red ) and IRF3 ( green ) antibodies and DAPI . Imaging data were analyzed by the Fuji software to reveal co - localization as white dots . Scale bars represent 10 µ m .

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

Figure 3(A) EGFR was not required for ligand-induced STING dimerization: MEFs were transfected with HA-tagged STING and treated with gefitinib or DMSO before cGAMP transfection, 4h later STING was immunoprecipitated with anti-HA antibodies and analyzed by Western blot.(B) EGFR was required for the activation of IRF3, but not TBK1: MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; cell lysate were subjected to Western blot analysis using the indicated antibodies.(C) Nuclear translocation of IRF3, but not NFκB p65, required EGFR: MEFs were pre-treated with gefitinib (+) or DMSO (-) for 1 hour before transfecting cGAMP. Cells were harvested and nuclei were isolated at the indicated time points; total cell extracts (T) and nuclear extracts (N) were analyzed by Western blot.MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; (D) IRF3, but not TBK1, binding to STING required EGFR cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.(E) STING-IRF3 interaction in human cells required EGFR: WT and EGFR-/- (KO) HeLa cells were transfected with cGAMP, 2h later cells were lysed, immunoprecipitated with anti-STING antibody and analyzed by Western blot.(F) STING and IRF3 co-localized in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours then treated with cGAMP for 1h. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.(G) EGFR was not required for ligand-induced phosphorylation of STING Ser366: EGFR-/- HeLa cells or gefitinib-treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot.(H) Kinetics of STING and IRF3 co-localization in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours and treated with cGAMP for the indicated time. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies and DAPI. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.


Figure 4 ( A ) STING was Tyr - phosphorylated in cGAMP - treated cells . Raw 264 . 7 cells were tranfected with cGAMP ; after 60 min , cell lysates were immuno - precipitated with anti - pTyr antibodies and then subjected to anti - STING Western blot . ( B ) STING Tyr - phosphorylation was inhibited by gefitinib . MEF cells were pre - treated with gefitinib before cGAMP - stimulation for 60 min . Cell lysates were immuno - precipitated with anti - pTyr antibody and then analyzed by anti - STING Western blot . NTC : no treatment control . ( C ) STING Tyr - phosphorylation required EGFR . Raw 264 . 7 cells expressing EGFR shRNA ( KD ) or non - target shRNA ( CO ) were transfected with cGAMP for 2 hours ; cell lysates were analyzed by Western blot before or after immunoprecipitation with anti - pTyr antibodies . ( D ) cGAMP stimulation triggered EGFR - binding to STING . Raw 264 . 7 cells were transfected with cGAMP for 1h ; cell lysates were immunoprecipitated with anti - STING antibody and analyzed by Western blot . ( E ) STING co - localized with EGFR after cGAMP stimulation . STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24hours and stimulated with cGAMP for 1h , cells were fixed and labeled with EGFR ( Red ) and Calnexin ( Purple ) antibodies . Co - localization was analyzed by Fuji and co - localized dots are shown in white . Scale bars represent 10 µ m . ( F ) STING - EGFR interaction was transient . Raw 264 . 7 cell lysates were prepared at indicated time points after cGAMP transfection , immunoprecipitated with anti - EGFR antibodies and subjected to Western blot . ( G ) Gefitinib did not block , but prolonged STING - EGFR interaction . Raw 264 . 7 cells were pre - treated with gefitinib or DMSO for 1 hour before cGAMP stimulation . Cell lysates were prepared at indicated time points , immuno - precipitated with anti - STING antibody and subjected to Western blot . ( H ) Poly I : C did not activate EGFR . STING - / - MEF cells were transfected with cGAMP or Poly ( I : C ) ; after 3h , p - EGFR were detected by Western blot . ( I ) Kinetics of EGFR activation . Raw 264 . 7 cells were treated with cGAMP for the indicated time . Cell lysates were analyzed for p - EGFR via Western blot . ( J ) STING - bound EGFR was phosphorylated . Raw 264 . 7 cells were stimulated for 1h , cell lysates were immunoprecipitated with anti - STING antibody and subjected to Western blot . ( K ) Kinetics of phosphorylation . Raw 264 . 7 cells were treated with cGAMP for the indicated time . Cell lysates were subjected to Western blot using the indicated antibodies . ( L ) Poly I : C did not trigger Tyr or Ser phosphorylation of STING . HeLa cells were transfected with cGAMP or Poly ( I : C ) ; after 3h , cell lysates were analyzed by Western blot using the indicated antibodies .

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

Figure 4(A) STING was Tyr-phosphorylated in cGAMP-treated cells. Raw 264.7 cells were tranfected with cGAMP; after 60 min, cell lysates were immuno-precipitated with anti-pTyr antibodies and then subjected to anti-STING Western blot.(B) STING Tyr-phosphorylation was inhibited by gefitinib. MEF cells were pre-treated with gefitinib before cGAMP-stimulation for 60 min. Cell lysates were immuno-precipitated with anti-pTyr antibody and then analyzed by anti-STING Western blot. NTC: no treatment control.(C) STING Tyr-phosphorylation required EGFR. Raw 264.7 cells expressing EGFR shRNA (KD) or non-target shRNA (CO) were transfected with cGAMP for 2 hours; cell lysates were analyzed by Western blot before or after immunoprecipitation with anti-pTyr antibodies.(D) cGAMP stimulation triggered EGFR-binding to STING. Raw 264.7 cells were transfected with cGAMP for 1h; cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.(E) STING co-localized with EGFR after cGAMP stimulation. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours and stimulated with cGAMP for 1h, cells were fixed and labeled with EGFR (Red) and Calnexin (Purple) antibodies. Co-localization was analyzed by Fuji and co-localized dots are shown in white. Scale bars represent 10 µ m.(F) STING-EGFR interaction was transient. Raw 264.7 cell lysates were prepared at indicated time points after cGAMP transfection, immunoprecipitated with anti-EGFR antibodies and subjected to Western blot.(G) Gefitinib did not block, but prolonged STING-EGFR interaction. Raw 264.7 cells were pre-treated with gefitinib or DMSO for 1 hour before cGAMP stimulation. Cell lysates were prepared at indicated time points, immuno-precipitated with anti-STING antibody and subjected to Western blot.(H) Poly I:C did not activate EGFR. STING-/- MEF cells were transfected with cGAMP or Poly (I:C); after 3h, p-EGFR were detected by Western blot.(I) Kinetics of EGFR activation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were analyzed for p-EGFR via Western blot.(J) STING-bound EGFR was phosphorylated. Raw 264.7 cells were stimulated for 1h, cell lysates were immunoprecipitated with anti-STING antibody and subjected to Western blot.(K) Kinetics of phosphorylation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were subjected to Western blot using the indicated antibodies.(L) Poly I:C did not trigger Tyr or Ser phosphorylation of STING. HeLa cells were transfected with cGAMP or Poly (I:C); after 3h, cell lysates were analyzed by Western blot using the indicated antibodies.


Figure 5 ( A ) In the absence of EGFR , after cGAMP stimulation , STING translocated out of the ER . STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h and transfected with cGAMP for 2 . 5h ; cells were fixed and labeled with Calreticulin ( ER marker , Red ) antibody . Co - localization ( Col ) is indicated by white dots . STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h and transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by white dots . ( B ) STING was present in the ERGIC of stimulated cells in the presence or the absence of gefitinib . , the cells were labeled with P58 ( ERGIC marker , Red ) antibody . MEF cells were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h and transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by white dots . ( C ) STING translocated to the autophagosomes in the absence of EGFR . the cells were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( E ) In gefitinib - treated cells , STING was detected in the autophagosomes as early as 0 . 5h . The procedures were as in C . Co - localization ( Col ) is indicated by white dots . STING translocated to the autophagosomes in the absence of EGFR . , the cells were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( D ) No STING was detected in the autophagosomes even after 3h after stimulation if EGFR was not inhibited . ( F ) STING translocation to late endosomes required EGFR activity . STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24hours , pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time periods ; cells were fixed and labeled with CD63 ( late endosome marker , Red ) antibody .

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

Figure 5(A) In the absence of EGFR, after cGAMP stimulation, STING translocated out of the ER. STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h; cells were fixed and labeled with Calreticulin (ER marker, Red) antibody. Co-localization (Col) is indicated by white dots.STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (B) STING was present in the ERGIC of stimulated cells in the presence or the absence of gefitinib. , the cells were labeled with P58 (ERGIC marker, Red) antibody.MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (C) STING translocated to the autophagosomes in the absence of EGFR. the cells were labeled with LC3 (Autophagosome marker, Red) antibody. (E) In gefitinib-treated cells, STING was detected in the autophagosomes as early as 0.5h. The procedures were as in C.Co-localization (Col) is indicated by white dots. STING translocated to the autophagosomes in the absence of EGFR. , the cells were labeled with LC3 (Autophagosome marker, Red) antibody. (D) No STING was detected in the autophagosomes even after 3h after stimulation if EGFR was not inhibited.(F) STING translocation to late endosomes required EGFR activity. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time periods; cells were fixed and labeled with CD63 (late endosome marker, Red) antibody.


Figure 6 ( A ) EGFR phosphorylates STING in vitro . Purified STING - GFP was incubated with increasing amounts of EGFR for in vitro kinase assays ; the products were analyzed on phos - tag gel by Western blot using GFP antibody ( upper panel ) . The substrate was analyzed , before the assay incubations , by the same method ( lower panel ) . products were analyzed on phos - tag gel by Western blot using GFP antibody ( upper panel ) . The substrate was analyzed , before the assay incubations , by the same method ( lower panel ) . ( B ) Y245F STING mutant is not a substrate of EGFR . WT and the indicated mutants of STING - GFP were purified and used as substrates for EGFR ( C ) LC - MS / MS analysis of STING identifies phosphorylated Y245 . STING - GFP was purified from cGAMP - stimulated cells , subjected to protease digestion and LC - MS / MS analysis . Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified . The MS / MS spectra for the phosphorylated peptide VYSNSIpYELLENGQR is shown . This doubly charged peptide has an observed m / z of 932 . 9266 Da and is within - 0 . 96 ppm of the expected mass . The mass difference between the y8 and y9 ions is consistent with phosphorylation at Y245 . ( E ) Gefitinib pretreatment inhibits phosphorylation of Y245 . STING - GFP - 293XL cells were pretreated with gefitinib and then treated with cGAMP . LC - MS / MS analysis was done , as above , for detecting pY245 ( n = 3 ) . ( F ) EGFR is required for STING Tyr phosphorylation . STING - GFP was expressed in 293XL cells in which EGFR expression had been knocked down . STING phosphorylation was analyzed by LC - MS / MS ( G ) Y245F STING mutant cannot trigger IFNβ mRNA induction . RNA was isolated from cells , expressing WT or Y245F STING , 3h after stimulation and subjected to qRT - PCR analysis ( n = 3 ) . ( H ) Y245F STING is phosphorylated on S366 after stimulation of cells . Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the indicated time . ( I ) TBK1 , but not IRF3 , can bind to Y245F STING and be phosphorylated in cGAMP - stimulated cells . Lysates from cells expressing the WT or the mutant STING were analyzed by Western blot after ( upper panel ) or before ( lower panel ) GFP pull down . ( J ) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL cells expressing WT or mutant STING . ( K ) Inhibition of EGFR by gefitinib induced more LC3II after 1 . 5h cGAMP treatment of L929 cells .

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

Figure 6(A) EGFR phosphorylates STING in vitro. Purified STING-GFP was incubated with increasing amounts of EGFR for in vitro kinase assays; the products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel).products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel). (B) Y245F STING mutant is not a substrate of EGFR. WT and the indicated mutants of STING-GFP were purified and used as substrates for EGFR(C) LC-MS/MS analysis of STING identifies phosphorylated Y245. STING-GFP was purified from cGAMP-stimulated cells, subjected to protease digestion and LC-MS/MS analysis. Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified. The MS/MS spectra for the phosphorylated peptide VYSNSIpYELLENGQR is shown. This doubly charged peptide has an observed m/z of 932.9266 Da and is within -0.96 ppm of the expected mass. The mass difference between the y8 and y9 ions is consistent with phosphorylation at Y245.(E) Gefitinib pretreatment inhibits phosphorylation of Y245. STING-GFP-293XL cells were pretreated with gefitinib and then treated with cGAMP. LC-MS/MS analysis was done, as above, for detecting pY245 (n=3).(F) EGFR is required for STING Tyr phosphorylation. STING-GFP was expressed in 293XL cells in which EGFR expression had been knocked down. STING phosphorylation was analyzed by LC-MS/MS(G) Y245F STING mutant cannot trigger IFNβ mRNA induction. RNA was isolated from cells, expressing WT or Y245F STING, 3h after stimulation and subjected to qRT-PCR analysis (n=3).(H) Y245F STING is phosphorylated on S366 after stimulation of cells. Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the indicated time.(I) TBK1, but not IRF3, can bind to Y245F STING and be phosphorylated in cGAMP-stimulated cells. Lysates from cells expressing the WT or the mutant STING were analyzed by Western blot after (upper panel) or before (lower panel) GFP pull down.(J) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL cells expressing WT or mutant STING.(K) Inhibition of EGFR by gefitinib induced more LC3II after 1.5h cGAMP treatment of L929 cells.n


Figure 7 ( A ) STING - / - MEF cells were transfected with mSTING WT - GFP and Y244F - GFP ( Green ) for 24h , pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time ; cells were fixed and labeled with LC3 ( autophagosome marker , Red ) antibody . Co - localization ( Col ) is indicated by white dots . ( A ) STING - / - MEF cells were transfected with mSTING WT - GFP and Y244F - GFP ( Green ) for 24h , pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time ; cells were fixed and labeled with CD63 ( late endosome marker ) .

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

Figure 7(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with LC3 (autophagosome marker, Red) antibody. Co-localization (Col) is indicated by white dots.(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with CD63 (late endosome marker).


Figure 1A - C . Immunodetection of BES1 - CFP from heat - treated plants . Plants pre - grown at 21 ° C were ( A ) exposed for 60 minutes to the indicated temperatures , ( B ) to 45 ° C for the indicated period of time , or ( C ) to 45 ° C and , following sampling after 30 minutes ( lane 2 ) and 60 minutes ( lane 3 ) , returned to 21 ° C for 3 hours ( lane 4 ) . CBB , Coomassie Brilliant Blue stained gel . D . Subcellular localization of BES1 - CFP in roots of 7 - day - old plants after heat shock . 35S : BES1 - CFP seedlings were grown at 21 ° C and either left untreated or exposed for 60 minutes to 45 ° C . Left : representative photos . Right : quantification of the nuclear / cytoplasmic signal . Data show the mean ± SD . n = 25 . The asterisks indicate significant differences to the untreated plants by Student ' s t - test ( * * * P ≤ 0 . 001 ) . F . Heat stress resistance of bes1 - 1 , bes1 - 2 and bes1 - D as compared to wild - type . For basal resistance assays ( top charts ) plants were directly exposed to 45 ° C for the indicated time . For acquired resistance ( bottom charts ) , plants were primed with the respective temperatures for 120 minutes before transfer to 45 ° C for 150 minutes . Data show the mean ± SE . n = 7 . The asterisks indicate significant differences compared to wild - type by 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 O O O O O O O O O O O O O O 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 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

Figure 1A-C. Immunodetection of BES1-CFP from heat-treated plants. Plants pre-grown at 21°C were (A) exposed for 60 minutes to the indicated temperatures, (B) to 45°C for the indicated period of time, or (C) to 45°C and, following sampling after 30 minutes (lane 2) and 60 minutes (lane 3), returned to 21°C for 3 hours (lane 4). CBB, Coomassie Brilliant Blue stained gel.D. Subcellular localization of BES1-CFP in roots of 7-day-old plants after heat shock. 35S:BES1-CFP seedlings were grown at 21°C and either left untreated or exposed for 60 minutes to 45°C. Left: representative photos. Right: quantification of the nuclear/cytoplasmic signal. Data show the mean ±SD. n=25. The asterisks indicate significant differences to the untreated plants by Student's t-test (***P≤0.001).F. Heat stress resistance of bes1-1, bes1-2 and bes1-D as compared to wild-type. For basal resistance assays (top charts) plants were directly exposed to 45°C for the indicated time. For acquired resistance (bottom charts), plants were primed with the respective temperatures for 120 minutes before transfer to 45°C for 150 minutes. Data show the mean ±SE. n=7. The asterisks indicate significant differences compared to wild-type by Student's t-test (*P≤0.05; **P≤0.01; ***P≤0.001).


Figure 2B . Immunodetection of BES1 - CFP from heat - stressed plants , treated with epiBL or BRZ . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 1 μM 24 - epiBL or 5 μM BRZ and then heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes ; lane 4 : 45 ° C for 60 minutes and recovery at 21 ° C for 180 minutes . C , D . Immunodetection of BES1 - CFP from heat - treated bri1 - 1 or det2 - 1 plants . Left : phenotypes of 10 - day - old plants of all lines as compared to wild - type Col - 0 ( wt ) . Right : Immunoblots from the lines grown and treated as in B . F . Immunodetection of BES1 - CFP from heat - treated bin2 - 1 plants .

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

Figure 2B. Immunodetection of BES1-CFP from heat-stressed plants, treated with epiBL or BRZ. 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 1 μM 24-epiBL or 5 μM BRZ and then heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes; lane 4: 45°C for 60 minutes and recovery at 21°C for 180 minutes.C, D. Immunodetection of BES1-CFP from heat-treated bri1-1 or det2-1 plants. Left: phenotypes of 10-day-old plants of all lines as compared to wild-type Col-0 (wt). Right: Immunoblots from the lines grown and treated as in B.F. Immunodetection of BES1-CFP from heat-treated bin2-1 plants.


Figure 3A . Immunodetection of BES1 - CFP from heat - stressed plants , treated with ABA or Fluridone ( Flu ) . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 5 μM ABA or 10 μM Fluridone and heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue . C . Immunodetection of native BES1 from heat - stressed and epiBL - treated abitM plants . abitM and wild - type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue .

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

Figure 3A. Immunodetection of BES1-CFP from heat-stressed plants, treated with ABA or Fluridone (Flu). 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 5 μM ABA or 10 μM Fluridone and heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.C. Immunodetection of native BES1 from heat-stressed and epiBL-treated abitM plants. abitM and wild-type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.


Figure 4C . ChIP to determine BES1 - CFP enrichment on the promoters of HSP70 . 3 , HSP70 . 4 and HSP90 . 1 following heat stress . 35S : BES1 - CFP and wild - type plants grown at 21 ° C were either left untreated or treated with 45 ° C for 60 minutes . BES1 - CFP was immuno - precipitated with α - GFP beads . Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated . TA3 ( 70 . 3p and 90 . 1p ) or 5s rRNA ( 70 . 4p ) were used for normalization . UBQ5 served as an internal control . Data show the mean ± SE . n = 3 biological replicates measured in 3 technical repeats . Significant differences as compared to wild - type at 21 ° C , calculated by Student ' s t - test , are shown ( * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4C. ChIP to determine BES1-CFP enrichment on the promoters of HSP70.3, HSP70.4 and HSP90.1 following heat stress. 35S:BES1-CFP and wild-type plants grown at 21°C were either left untreated or treated with 45°C for 60 minutes. BES1-CFP was immuno-precipitated with α-GFP beads. Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated. TA3 (70.3p and 90.1p) or 5s rRNA (70.4p) were used for normalization. UBQ5 served as an internal control. Data show the mean ±SE. n=3 biological replicates measured in 3 technical repeats. Significant differences as compared to wild-type at 21°C, calculated by Student's t-test, are shown (*P≤0.05; **P≤0.01; ***P≤0.001).


Figure 5A . EMSAs showing in vitro DNA interaction of recombinant BES1 and / or HSFA1 with HSE - containing fragments of the HSP70 . 3 and HSP90 . 1 promoters . Unlabeled competitor fragments with functional HSEs ( C1 + HSE ) or mutated versions ( C2 - HSE ) were used for competition studies in 100x molar access ( lanes 6 and 7 ) . B . BiFC assays showing cells co - transformed with nYFP - BES1 and cYFP - HSFA1a . The fusion proteins were co - expressed in N . benthamiana leaves and fluorescence was visualized . Two nuclei are highlighted and shown in a 6 - fold magnification . Scale bar = 20 μm . C . LUC assays in protoplasts of wild - type , bes1 - D or the hsfA1qM plants . LUC reporter constructs , driven by 2 kb of the HSP70 . 3 , HSP70 . 4 and HSP90 . 1 promoters , were tested either alone ( 0 , with empty effector plasmid ) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed . Data show the mean ± SD . n = 5 . Statistically significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined by Student ' s 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 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

Figure 5A. EMSAs showing in vitro DNA interaction of recombinant BES1 and/or HSFA1 with HSE-containing fragments of the HSP70.3 and HSP90.1 promoters. Unlabeled competitor fragments with functional HSEs (C1 +HSE) or mutated versions (C2 -HSE) were used for competition studies in 100x molar access (lanes 6 and 7).B. BiFC assays showing cells co-transformed with nYFP-BES1 and cYFP-HSFA1a. The fusion proteins were co-expressed in N. benthamiana leaves and fluorescence was visualized. Two nuclei are highlighted and shown in a 6-fold magnification. Scale bar=20 μm.C. LUC assays in protoplasts of wild-type, bes1-D or the hsfA1qM plants. LUC reporter constructs, driven by 2 kb of the HSP70.3, HSP70.4 and HSP90.1 promoters, were tested either alone (0, with empty effector plasmid) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed. Data show the mean ±SD. n=5. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.


Figure 6A , B . Constitutive and BR - responsive growth of bes1 - DxhsfA1qM seedlings . Hypocotyl elongation of 7 - day - old dark - grown ( A ) and light - grown ( B ) seedlings on medium containing either 1 μM epi - BL , 1 μM BRZ or DMSO as a control ( c ) . Left : Mean and SD of 30 plants . Significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined with a Student ' s t - test . Right : photos of representative plants . C . Expression of BR - regulated genes in bes1 - DxhsfA1qM , as compared to its parents and respective wild - types analyzed by qPCRs . Data show the mean ± SD . n = 3 biological repeats , each measured in 3 technical replicates , normalized to UBC . Statistically significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined by Student ' s 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 O O O 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 6A, B. Constitutive and BR-responsive growth of bes1-DxhsfA1qM seedlings. Hypocotyl elongation of 7-day-old dark-grown (A) and light-grown (B) seedlings on medium containing either 1 μM epi-BL, 1 μM BRZ or DMSO as a control (c). Left: Mean and SD of 30 plants. Significant difference at P≤0.05 of results is indicated with different letters and was determined with a Student's t-test. Right: photos of representative plants.C. Expression of BR-regulated genes in bes1-DxhsfA1qM, as compared to its parents and respective wild-types analyzed by qPCRs. Data show the mean ±SD. n=3 biological repeats, each measured in 3 technical replicates, normalized to UBC. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.


Figure 1 ( B ) The degree of polymerisation of Z α1 - antitrypsin after 72 hours at 37 ° C , as determined by an end - point immunoassay using the 2C1 monoclonal antibody , in varying concentrations of compound ( shown in panel A ) . Modification of the phenyl and indole heterocycle of GSK425 ( pIC50 6 . 5 ) resulted in an ~ 100 fold increase in potency and the discovery of the 2 - oxindole GSK716 ( pIC50 8 . 3 ) . Data presented as mean ± SD , n = 2 ( GSK425 ) and n = 25 ( GSK716 ) . ( C ) GSK716 binds to Z α1 - antitrypsin with a high affinity mean pKD of 8 . 5 ± 0 . 12 ( n = 18 ) as determined by a competition binding assay with a fluorescently labelled derivative . There was a 50 - fold lower affinity for plasma - purified wild - type M α1 - antitrypsin , with a mean pKD of 6 . 8 ± 0 . 18 ( n = 10 ) . Data presented as mean ± SD . ( D ) The compound bound to monomeric but not polymeric Z α1 - antitrypsin ( Z α1 - AT ) as reported by fluorescence polarisation of an Alexa - 488 - labelled variant of GSK716 . ( E ) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1 - antitrypsin based on changes in intrinsic tryptophan fluorescence . Based on the concentration dependence ( inset ) , the second - order rate constant of association was found to be 4 . 1 x 104 M - 1 s - 1 . ( F ) The association of GSK716 with M α1 - antitrypsin , giving a second - order rate constant of 2 . 1 x 102 M - 1 s - 1 .

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

Figure 1(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).(C) GSK716 binds to Z α1-antitrypsin with a high affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716.(E) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1. (F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1.


Figure 2 ( G ) CHO - K1 Tet - On cells expressing Z α1 - antitrypsin were induced with doxycycline ( 0 . 5μg / ml ) for 48 h . Cells were incubated with 10μM GSK716 ( or 0 . 1 % v / v DMSO for the control ) during the induction . Culture media containing either the experimental compound or DMSO were changed every 24 h . After the induction , cells were labelled for 10 minutes with 35S Met / Cys and chased at the indicated times . Culture media were collected and cells lysed in 1 % v / v NP - 40 buffer . Intracellular fractions and culture media from cells expressing Z α1 - antitrypsin were immunoprecipitated either with a mAb against total α1 - antitrypsin ( 3C11 ) or with a polymer - specific mAb ( 2C1 ) . Samples were resolved by 4 - 12 % w / v acrylamide SDS - PAGE and detected by autoradiography . ( H ) The graphs show the effect of GSK716 on intracellular and extracellular Z α1 - antitrypsin ( mean ± SEM , n = 2 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (G) CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE and detected by autoradiography. (H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2)


Figure 4 ( B ) M ( above ) and Z ( below ) α1 - antitrypsin , at a concentration of 0 . 2mg / ml in PBS + 5 % v / v glycerol , were heated at a range of temperatures between 48 ° C - 65 ° C for 4 hours in the presence and absence of GSK716 , as indicated . The oligomerisation state was determined by non - denaturing PAGE electrophoresis ; the lane denoted ' M ' contains the unheated monomeric control . ( C ) M and Z α1 - antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride ( gdnhcl ) in the presence and absence of GSK716 , with bis - ANS dye added to report the presence of the unfolded intermediate . The normalised fluorescence intensity data were fitted with an equation describing a three - state unfolding curve . Values shown are the mean of three independent experiments and the error bars represent ± SEM . ( D ) Top panel , the association of GSK716 induced a marked quenching and blue - shift of the α1 - antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein ( inset graph ) . The association of 10µM GSK716 with four α1 - antitrypsin variants that vary in their propensity to polymerise , ranging from inert ( M ) , to mild ( S ) , moderate ( Baghdad denoted ' B ' ) and severe ( Z ) . Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0 . 2mg / ml protein are shown . Bottom panel , the half - time of association calculated from three such independent experiments ( error bars are ± SEM ) show a correspondence with the polymerisation propensity of the four variants . The change in Z α1 - antitrypsin fluorescence was faster than the dead - time of the apparatus ( ~ 10s ) and scaling was with reference to unbound intensity .

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

Figure 4(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.


Figure 5 ( B ) M α1 - antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS - PAGE . The position of covalent α1 - antitrypsin - chymotrypsin complex ( cpx ) , native ( nat ) and cleaved ( clv ) is shown . ( C ) The inhibitory activity of α1 - antichymotrypsin against chymotrypsin ( ach + chym ) , neuroserpin against trypsin ( neus + tryp ) , and antithrombin against thrombin ( atiii + thrombin ) was determined in the presence and absence of GSK716 . Error bars reflect ± SD of two independent experiments .

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

Figure 5(B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown.(C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.


Figure 6Z α1 - antitrypsin transgenic animals were dosed with 10 , 30 or 100 mg / kg GSK716 three times a day . Total concentrations of GSK716 were determined by LC - MS / MS ( Table 2 ) . ( A ) 100mg / kg GSK716 resulted in a mean 7 - fold increase in circulating monomeric Z α1 - antitrypsin levels demonstrating robust target engagement in the liver . The observed steady state total and free compound levels of GSK716 in the transgenic Z α1 - antitrypsin mouse were well - predicted by the in silico PK model built on : ( 1 ) in vitro metabolic clearance data , ( 2 ) plasma protein binding data , ( 3 ) in vivo PK data from wild type mice and ( 4 ) a term comprising a 5μM circulating sink for drug with an affinity of 1 . 5 nM , representing the Z α1 - antitrypsin within blood . The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay . Interestingly , 30mg / kg and 10mg / kg groups also gave significant , dose - dependent increases in circulating Z α1 - antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period . Significance at p < 0 . 05 by a student ' s t test is denoted by * , shown as pairwise comparisons ( each animal is compared with itself , treated vs pre - treatment ) and between groups ( compound treated vs vehicle ) , with n = 7 , 8 and 8 per group for day 6 dose level 10 , 30 and 100mg / Kg respectively ; day 15 , n = 10 ; day 21 , n = 5 . Data presented as mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.


Figure 1A . Confocal images of live control ( top ) and U18 - treated ( bottom ) tsA201 cells expressing P4M - YFP . Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo / lysosomal compartments , respectively . B . Quantification of perinuclear P4M - YFP mean fluorescence intensity normalized to the cytoplasm . Control : n = 19 ; U18 : n = 16 . K . Heat map of PtdIns , PtdInsP and PtdInsP2 levels from 4 different murine brain regions . The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild - type brains ; n = 3 for both WT and NPC1I1061T animals .

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

Figure 1A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively. B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16.K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.


Figure 2C . Confocal images of control and U18 - treated tsA201 cells fixed and stained for OSBP ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of OSBP and TGN46 regions . D . Quantification of OSBP signal in TGN46 regions ( OSBPTGN46 ) . Control : n = 40 ; U18 : n = 39 . J . Confocal images of control and U18 - treated GFP11 - SAC1 HEK293 cells ( top ) and control and NPC1I1061T patient fibroblasts immunolabeled with anti - SAC1 antibody ( bottom ) . Insets show an enlarged view of SAC1 centered on a perinuclear region . K . Quantification analyses of GFP11 - SAC1 ( top ; Control : n = 10 ; U18 : n = 10 ) and anti - SAC1 ( bottom ; Control : n = 7 ; NPC1I1061T : n = 8 ) mean fluorescence intensities in Golgi normalized to the cytoplasm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions. D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39. J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region. K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm.


Figure 3C . Left : Confocal images of control ( left ) and U18 - treated ( right ) tsA201 cells expressing PI4KIIα - GFP . Bottom panels show an enlarged view of the probe centered on a perinuclear region . Right : Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm . Control : n = 12 ; U18 : n = 14 . D . Same as ( C ) only PI4KIIIβ . Control : n = 12 ; U18 : n = 15 . E . Confocal images of control and U18 - treated tsA201 cells fixed and stained for PI4KIIα ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of PI4KIIα and TGN46 regions . F . Quantification of PI4KIIα signal in TGN46 regions ( PI4KIIα TGN46 ) . Control : n = 36 ; U18 : n = 36 .

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 O O O

Figure 3C. Left: Confocal images of control (left) and U18-treated (right) tsA201 cells expressing PI4KIIα -GFP. Bottom panels show an enlarged view of the probe centered on a perinuclear region. Right: Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm. Control: n=12; U18: n=14. D. Same as (C) only PI4KIIIβ. Control: n=12; U18: n=15. E. Confocal images of control and U18-treated tsA201 cells fixed and stained for PI4KIIα (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of PI4KIIα and TGN46 regions. F. Quantification of PI4KIIα signal in TGN46 regions (PI4KIIα TGN46). Control: n=36; U18: n=36.


Figure 4B , Confocal images of wild - type and PI4KIIα - depleted ( sgPI4K2A ) HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in a Golgi region . C , Quantitative analyses of P4M - YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions . Control : n = 19 ; U18 : n = 13 , sgPI4K2A : n = 19 ; sgPI4K2A + U18 : n = 17 ; sgDHHC3 : n = 11 ; sgDHHC3 + U18 : n = 11 . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups . H . Control or GFP - DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ . Bottom panels show enlarged views of DHHC3 , PI4KIIα , or PI4KIIIβ at Golgi regions . I . Quantification of PI4KIIα ( left ; control : n = 42 , DHHC3 : n = 24 ) , or PI4KIIIβ ( right ; control : n = 39 , DHHC3 : n = 26 ) intensity relative to cytoplasm . M . Confocal images of control ( top row ) , NPC1I1061T ( middle row ) , and NPC1I1061T treated with sgDHHC3 ( bottom row ) patient cells expressing P4M , LAMP1 and CAS9 . Insets are zoomed regions from LAMP1 positive areas . N . Quantification of P4M changes at endomembranes . Control : n = 149 , U18 : n = 251 , sgPI4KIIa + U18 : n = 131 , sgDHHC3 + U18 : n = 271 . Each n represents a single enodolysosome .

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

Figure 4B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm. M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.


Figure 5C . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3 . Black and orange squares show expanded views of anti - ACBD3 in Golgi and endomembrane compartments , respectively . D . Analyses of ACBD3 intensity distributed in Golgi ( left graph ; Control : n = 9 ; NPC1I1061T : n = 9 ) or in endomembranes ( right graph ; Control : n = 1360 ; NPC1I1061T : n = 1645 from 15 cells ) , normalized to the cytoplasm . I . Confocal images of control and sgACBD3 HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments . J . Quantitative analyses of P4M - YFP mean fluorescence intensity at Golgi ( left graph ) or endomembranes ( right graph ) normalized to the cytoplasm . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups . For left graph ; Control : n = 19 ; U18 : n = 13 ; sgPI4K3B : n = 12 ; sgPI4K3B + U18 : n = 11 ; sgPI4K2A + sgPI4K3B : n = 12 ; sgPI4K2A + sgPI4K3B + U18 : n = 12 ; sgACBD3 : n = 15 ; sgACBD3 + U18 : n = 16 . For right graph ; control : n = 149 ; U18 : n = 251 ; sgACBD3 : n = 138 ; sgACBD3 + U18 : n = 190 . Please note that ' control ' and ' U18 ' datasets in Fig 5J are identical to those in Fig . 4C . Data for all CRISPR experiments were collected on the same days .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5C. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively. D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm.I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments. J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18 : n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.


Figure 6A . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts expressing P4M - YFP . Black and orange squares show expanded views of PtdIns4P in Golgi and endo / lysosomal compartments respectively . C . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody . Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images . White dashed lines represent the outline of each cell . D . Quantification of mTORC1 mean integrated density normalized bycell area in control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts . Control : n = 15 ; NPC1I1061T : n = 14 ; NPC1I1061T + sgACBD3 : n = 11 .

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

Figure 6A. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts expressing P4M-YFP. Black and orange squares show expanded views of PtdIns4P in Golgi and endo/lysosomal compartments respectively.C. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody. Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images. White dashed lines represent the outline of each cell. D. Quantification of mTORC1 mean integrated density normalized bycell area in control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts. Control: n=15; NPC1I1061T: n=14; NPC1I1061T+sgACBD3: n=11.


Figure 7B . Confocal images of tsA201 cells expressing PI4KIIα - GFP . Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . C . Quantification of PI4KIIα - GFP mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . D , E . Same as ( B , C ) but in tsA - 201 cells transfected with PI4KIIIβ - GFP . Control : n = 18 , U18 : n = 22 , U18 + PF : n = 23 . Confocal images of tsA201 cells Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . F , G . Same as ( B , C ) but in tsA - 201 cells transfected with P4M - YFP . Control : n = 39 , U18 : n = 42 , U18 + PF : n = 32 . H , I . Same as ( F , G ) but in wild - type and SCAP - / - CHO cells . Control : n = 12 , U18 : n = 11 , SCAP - / - + U18 : n = 10 .

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 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

Figure 7B. Confocal images of tsA201 cells expressing PI4KIIα-GFP. Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. C. Quantification of PI4KIIα-GFP mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. D, E. Same as (B, C) but in tsA-201 cells transfected with PI4KIIIβ-GFP. Control: n=18, U18: n=22, U18+PF: n=23.Confocal images of tsA201 cells Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. F, G. Same as (B, C) but in tsA-201 cells transfected with P4M-YFP. Control: n=39, U18: n=42, U18+PF: n=32. H, I. Same as (F, G) but in wild-type and SCAP-/- CHO cells. Control: n=12, U18: n=11, SCAP-/- +U18: n=10.


Figure 8A . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI - M6PR . Insets show expanded views of each immunolabeling in the Golgi region ( upper insets ) or centered on endomembranes ( bottom inset , merged signals ) . Dashed grey and white lines represent the outline of each cell . B . Analysis of the mean area of M6P and Giantin colocalizing puncta ( top graph ; Control : n = 23 ; NPC1I1061T : n = 18 ) and of their mean fluorescence intensity ( bottom graph ; Control : n = 18 ; NPC1I1061T : n = 18 ) in control and NPC1I1061T patient fibroblasts . C . Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1 - GFP . Insets show enlarged views of ARF1 in the Golgi area . Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows . D . Quantification of ΑRF - 1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts . Control : n = 10 ; NPC1I1061T : n = 11 .

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

Figure 8A. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI-M6PR. Insets show expanded views of each immunolabeling in the Golgi region (upper insets) or centered on endomembranes (bottom inset, merged signals). Dashed grey and white lines represent the outline of each cell. B. Analysis of the mean area of M6P and Giantin colocalizing puncta (top graph; Control: n=23; NPC1I1061T: n=18) and of their mean fluorescence intensity (bottom graph; Control: n=18; NPC1I1061T: n=18) in control and NPC1I1061T patient fibroblasts. C. Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1-GFP. Insets show enlarged views of ARF1 in the Golgi area. Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows. D. Quantification of ΑRF-1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts. Control: n=10; NPC1I1061T: n=11.


Figure 1Human tissue sections were probed with Siglec ‐ 5 Fc and Siglec ‐ 5 R119A Fc to determine the presence of potential sialic acid ‐ independent ligands . Positive signals ( in blue ) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts , as well as in pancreatic tissues . Pancreatic tissue lysate or lung BAL were incubated with Siglec ‐ 5 R119A Fc ( Sig5 ‐ RA Fc ) or Siglec ‐ 7 Fc ( Sig7Fc ) immobilized onto Protein G beads . The proteins captured by these complexes were identified by mass spectrometric analysis . Hsp70 was the only protein that was captured by the Sig5 ‐ RA Fc bead complex from pancreas lysate and bronchoalveolar lavage , but not captured by Sig7Fc bead complex .

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

Figure 1Human tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands. Positive signals (in blue) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts, as well as in pancreatic tissues.Pancreatic tissue lysate or lung BAL were incubated with Siglec‐5 R119A Fc (Sig5‐RA Fc) or Siglec‐7 Fc (Sig7Fc) immobilized onto Protein G beads. The proteins captured by these complexes were identified by mass spectrometric analysis. Hsp70 was the only protein that was captured by the Sig5‐RA Fc bead complex from pancreas lysate and bronchoalveolar lavage, but not captured by Sig7Fc bead complex.


Figure 2Co ‐ localization of Hsp70 with Siglec ‐ 5 and Siglec ‐ 14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec ‐ 5 R119A Fc or Siglec ‐ 14 R119A Fc ( red ) with an anti ‐ Hsp70 antibody ( green ) . The co ‐ localized signals for the Siglec ‐ Fc probes with an anti ‐ Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid ‐ independent ligands of Siglec ‐ 5 and Siglec ‐ 14 in the lung bronchiole . White dashes mark the lung bronchiole epithelium . All images are representative of at least 2 - 3 replicates .

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

Figure 2Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green). The co‐localized signals for the Siglec‐Fc probes with an anti‐Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid‐independent ligands of Siglec‐5 and Siglec‐14 in the lung bronchiole. White dashes mark the lung bronchiole epithelium. All images are representative of at least 2-3 replicates.


Figure 3Immunoblot probed with Siglec ‐ 5 Fc and Siglec ‐ 14 Fc to detect direct binding to Hsp70 . B6N domain of GBS β ‐ protein was used as a positive control , and clusterin was used as the negative control . ELISA demonstrates direct binding of Siglec ‐ 5 Fc and Siglec ‐ 14 Fc , but not Siglec ‐ 7 Fc , to Hsp70 . Bovine serum albumin ( BSA ) was used as the negative control . Siglec ‐ 5 or Siglec ‐ 14 Fc immobilized on magnetic beads was incubated with heat ‐ shocked HEK293A cell lysates . A Western blot of the captured proteins using an anti ‐ Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec ‐ 5 and Siglec ‐ 14 . HEK293A cells were transfected with expression plasmids for Siglec ‐ 7 , Siglec ‐ 5 or Siglec ‐ 14 , and then subsequently incubated with ( red ) or without ( gray ) recombinant Hsp70 ( bottom panels ) . Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec ‐ 5 and Siglec ‐ 14 , but not Siglec ‐ 7 . Histogram of Siglec expression after transfection is shown as blue lines ( top panels ) . Using flow cytometry , undifferentiated or PMA ‐ differentiated THP1 cells with empty vector ( EV ) , Siglec ‐ 5 ( Sig5 ) , or Siglec ‐ 14 ( Sig14 ) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface . The mean fluorescence intensity ( MFI ) of signals from an anti ‐ Hsp70 antibody on unpermeabilized THP1 cells is 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Immunoblot probed with Siglec‐5 Fc and Siglec‐14 Fc to detect direct binding to Hsp70. B6N domain of GBS β‐protein was used as a positive control, and clusterin was used as the negative control.ELISA demonstrates direct binding of Siglec‐5 Fc and Siglec‐14 Fc, but not Siglec‐7 Fc, to Hsp70. Bovine serum albumin (BSA) was used as the negative control.Siglec‐5 or Siglec‐14 Fc immobilized on magnetic beads was incubated with heat‐shocked HEK293A cell lysates. A Western blot of the captured proteins using an anti‐Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec‐5 and Siglec‐14.HEK293A cells were transfected with expression plasmids for Siglec‐7, Siglec‐5 or Siglec‐14, and then subsequently incubated with (red) or without (gray) recombinant Hsp70 (bottom panels). Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec‐5 and Siglec‐14, but not Siglec‐7. Histogram of Siglec expression after transfection is shown as blue lines (top panels).Using flow cytometry, undifferentiated or PMA‐differentiated THP1 cells with empty vector (EV), Siglec‐5 (Sig5), or Siglec‐14 (Sig14) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface. The mean fluorescence intensity (MFI) of signals from an anti‐Hsp70 antibody on unpermeabilized THP1 cells is shown.


Figure 4A comparison of the relative binding affinities between Siglec ‐ 5 WT versus R119A Fc , and Siglec ‐ 14 WT versus R119A Fc to Hsp70 by ELISA . The arginine ‐ mutated Siglecs display lower affinity for Hsp70 in comparison with their wild ‐ type sequence . A lysine ‐ to ‐ alanine mutation in residue 134 in Siglec ‐ 14 Fc ( K134A ) also abrogates its affinity for Hsp70 , as determined by ELISA . Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec ‐ 5 and Siglec ‐ 14 with Siglec ‐ 7 . The Siglec ‐ 14 R119A and K134A double mutant ( DM ) Fc reduces the interaction with Hsp70 to background levels . Antibodies against Siglec ‐ 5 and Siglec ‐ 14 neutralized the interaction between Siglec ‐ 14 Fc to Hsp70 , as determined by ELISA . Co ‐ incubation of B6N peptide with Siglec ‐ 5 and Siglec ‐ 14 Fc decreased binding of each Siglec to Hsp70 in an ELISA .

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

Figure 4A comparison of the relative binding affinities between Siglec‐5 WT versus R119A Fc, and Siglec‐14 WT versus R119A Fc to Hsp70 by ELISA. The arginine‐mutated Siglecs display lower affinity for Hsp70 in comparison with their wild‐type sequence.A lysine‐to‐alanine mutation in residue 134 in Siglec‐14 Fc (K134A) also abrogates its affinity for Hsp70, as determined by ELISA. Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec‐5 and Siglec‐14 with Siglec‐7. The Siglec‐14 R119A and K134A double mutant (DM) Fc reduces the interaction with Hsp70 to background levels.Antibodies against Siglec‐5 and Siglec‐14 neutralized the interaction between Siglec‐14 Fc to Hsp70, as determined by ELISA.Co‐incubation of B6N peptide with Siglec‐5 and Siglec‐14 Fc decreased binding of each Siglec to Hsp70 in an ELISA.


Figure 5Secretion of pro ‐ inflammatory cytokine TNFα by THP1 cells transfected with an empty vector ( EV ) or Siglec ‐ 5 ( Sig5 ) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg / ml Hsp70 or LPS alone . Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone . Similarly , IL ‐ 8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg / ml Hsp70 . Secretion of IL ‐ 8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone . THP1 Sig5 cells were incubated with Hsp70 , and the levels of SHP ‐ 1 recruitment to Siglec ‐ 5 were evaluated by immunoprecipitation of Siglec ‐ 5 and Western blot for SHP ‐ 1 . Higher levels of SHP ‐ 1 were co ‐ immunoprecipitated with Siglec ‐ 5 when the cells were incubated with Hsp70 . Western blots demonstrate phosphorylated p38 ( p ‐ p38 ) was reduced in THP1Siglec ‐ 5cells when exposed to both LPS and Hsp70 in comparison with LPS alone . This reduction was also greater in THP1Siglec ‐ 5cells in comparison with the control THP1cells . Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control ( total p38 ) . Secretion of pro ‐ inflammatory cytokine IL ‐ 8 by PMA ‐ differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng / ml LPS with an anti ‐ Hsp70 antibody or isotype ‐ matched antibody . Secretion of IL ‐ 8 was increased in only THP1 Sig5 cells exposed to LPS and an anti ‐ Hsp70 antibody in comparison with the isotype control antibody , but this increase was not replicated in THP1 EV cells .

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

Figure 5Secretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone. Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone.Similarly, IL‐8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70. Secretion of IL‐8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone.THP1 Sig5 cells were incubated with Hsp70, and the levels of SHP‐1 recruitment to Siglec‐5 were evaluated by immunoprecipitation of Siglec‐5 and Western blot for SHP‐1. Higher levels of SHP‐1 were co‐immunoprecipitated with Siglec‐5 when the cells were incubated with Hsp70.Western blots demonstrate phosphorylated p38 (p‐p38) was reduced in THP1Siglec‐5cells when exposed to both LPS and Hsp70 in comparison with LPS alone. This reduction was also greater in THP1Siglec‐5cells in comparison with the control THP1cells. Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control (total p38).Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng/ml LPS with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was increased in only THP1 Sig5 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this increase was not replicated in THP1 EV cells.


Figure 6Secretion of TNFα by THP1 cells transfected with a Siglec ‐ 5 or Siglec ‐ 14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin ‐ neutralizing agent polymyxin B . Only THP1 Siglec ‐ 14 cells exhibited significant secretion of TNFα in an Hsp70 dose ‐ dependent manner . IL ‐ 8 secretion was also evaluated after THP1Siglec ‐ 5 and Siglec ‐ 14cells were exposed to Hsp70 . Similarly , only THP1Siglec ‐ 14cells exhibited significant secretion of IL ‐ 8 in an Hsp70 dose ‐ dependent manner . Western blot analysis demonstrates a greater increase in phosphorylated p38 ( p ‐ p38 ) from THP1 Siglec ‐ 14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time ‐ dependent manner . Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control . Exposure to Hsp70 also yielded greater degradation of the NF ‐ κB inhibitor , IκBα , in THP1 Siglec ‐ 14 cells in comparison with control THP1 cells , augmentation of the pro ‐ inflammatory response . Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control . Secretion of pro ‐ inflammatory cytokine IL ‐ 8 by PMA ‐ differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti ‐ Hsp70 antibody or isotype ‐ matched antibody . Secretion of IL ‐ 8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti ‐ Hsp70 antibody in comparison with the isotype control antibody , but this change was not observed in THP1 EV cells .

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

Figure 6Secretion of TNFα by THP1 cells transfected with a Siglec‐5 or Siglec‐14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin‐neutralizing agent polymyxin B. Only THP1 Siglec‐14 cells exhibited significant secretion of TNFα in an Hsp70 dose‐dependent manner.IL‐8 secretion was also evaluated after THP1Siglec‐5 and Siglec‐14cells were exposed to Hsp70. Similarly, only THP1Siglec‐14cells exhibited significant secretion of IL‐8 in an Hsp70 dose‐dependent manner.Western blot analysis demonstrates a greater increase in phosphorylated p38 (p‐p38) from THP1 Siglec‐14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time‐dependent manner. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.Exposure to Hsp70 also yielded greater degradation of the NF‐κB inhibitor, IκBα, in THP1 Siglec‐14 cells in comparison with control THP1 cells, augmentation of the pro‐inflammatory response. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this change was not observed in THP1 EV cells.


Figure 7TNFα secretion from SIGLEC14 + / + monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin ‐ neutralizing compound polymyxin B . Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B , suggesting a mechanism independent of endotoxin contaminants . DnaK , the E . coli homolog of human Hsp70 , did not induce significant secretion of TNFα above background levels . TNFα secretion from SIGLEC14 + / + monocytes by Hsp70 was reduced when the cells were exposed to an anti ‐ Siglec ‐ 5 / Siglec ‐ 14 blocking antibody . Human monocytes isolated from SIGLEC14 + / + , SIGLEC14 + / − , and SIGLEC14 − / − individuals were exposed to varying concentrations of Hsp70 , and TNFα secretion was evaluated . The amount of TNFα secreted was normalized to the respective cells ' response to 1 ng / ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals . TNFα secretion from SIGLEC14 − / − monocytes stimulated with 15 ng / ml LPS and various concentrations of Hsp70 . TNFα secretion was reduced with increasing concentrations of Hsp70 .

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 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

Figure 7TNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B. Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B, suggesting a mechanism independent of endotoxin contaminants. DnaK, the E. coli homolog of human Hsp70, did not induce significant secretion of TNFα above background levels.TNFα secretion from SIGLEC14+/+ monocytes by Hsp70 was reduced when the cells were exposed to an anti‐Siglec‐5/Siglec‐14 blocking antibody.Human monocytes isolated from SIGLEC14+/+, SIGLEC14+/−, and SIGLEC14−/− individuals were exposed to varying concentrations of Hsp70, and TNFα secretion was evaluated. The amount of TNFα secreted was normalized to the respective cells' response to 1 ng/ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals.TNFα secretion from SIGLEC14−/− monocytes stimulated with 15 ng/ml LPS and various concentrations of Hsp70. TNFα secretion was reduced with increasing concentrations of Hsp70.


Figure 1A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone ( HF ) for 5 h . B Quantification of ATF4 and P - eIF2α from experiments as in ( A ) . Data are mean ± SD ( n = 3 biological replicates ) . * p ≤ 0 . 0414 , * * p ≤ 0 . 0019 , * * * * p < 0 . 0001 , as determined by one - way ANOVA with Dunnett ' s multiple comparison test . Data information : Representative results of at least three independent experiments are shown in each panel . C Similar to ( A ) , but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel . D Similar to ( A ) , but using lysates from HeLa cells treated with 62 . 5 nM and 312 . 5 nM HF for indicated times . Data information : Representative results of at least three independent experiments are shown in each panel .

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

Figure 1A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone (HF) for 5 h. B Quantification of ATF4 and P-eIF2α from experiments as in (A). Data are mean ± SD (n = 3 biological replicates). *p ≤ 0.0414, **p ≤ 0.0019, ****p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparison test. Data information: Representative results of at least three independent experiments are shown in each panel.C Similar to (A), but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.D Similar to (A), but using lysates from HeLa cells treated with 62.5 nM and 312.5 nM HF for indicated times. Data information: Representative results of at least three independent experiments are shown in each panel.


Figure 2A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts ( MEFs ) of indicated genotype after treatment with 12 . 5 nM or 200 nM HF for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel . B Similar to ( A ) , but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h . C Similar to ( A ) , but with high HF concentrations . Data information : Representative results of at least three independent experiments are shown in each panel .

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

Figure 2A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts (MEFs) of indicated genotype after treatment with 12.5 nM or 200 nM HF for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.B Similar to (A), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h. C Similar to (A), but with high HF concentrations. Data information: Representative results of at least three independent experiments are shown in each panel.


Figure 4B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF . C Similar to ( B ) , but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF . Data information : Representative results of at least three independent experiments are shown in each panel . D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8 - activating enzyme inhibitor MLN4924 ( 1 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel . E . Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG - 132 ( 10 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel .

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

Figure 4B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF. C Similar to (B), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF. Data information: Representative results of at least three independent experiments are shown in each panel.D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8-activating enzyme inhibitor MLN4924 (1 μM). Data information: Representative results of at least three independent experiments are shown in each panel.E. Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG-132 (10 μM). Data information: Representative results of at least three independent experiments are shown in each panel.