LC3immunoblot ( E ) immunofluorescent labeling ( F - K ) of hippocampaldendrites ( brackets ) in 8 - 9 - wk - old PS1 / APP NTg mice show LC3 - II elevation ( P < 0 . 05 ) in 8 - wk - old PS1 / APP compared with NTg mice ( E ) . ( D ) * , P < 0 . 001 . ( E ) * , P < 0 . 05 . Error bars represent . LC3 immunoreactivity in pyramidal celldendrites is increased in 9 - mo - old ( F - H ) 9 - wk - old ( I - K ) PS1 / APPmice frequently exhibits a punctate labeling pattern , which is more evident at 9 mo than at 9 wk ( H K , arrows ) is uncommon in NTg mice ( F I ) . Bars ( F , G , I , J ) , 20 μm ; ( H K ) , 10 μm .

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LC3immunoblot and analysis (E) and immunofluorescent labeling (F-K) of the hippocampaldendrites (brackets) in 8-9-wk-old PS1/APP and NTg mice show LC3-II elevation (P < 0.05) in 8-wk-old PS1/APP compared with NTg mice (E). (D) *, P < 0.001. (E) *, P < 0.05. Error bars represent SEM. LC3 immunoreactivity in pyramidal celldendrites is increased in 9-mo-old (F-H) and 9-wk-old (I-K) PS1/APPmice and frequently exhibits a punctate labeling pattern, which is more evident at 9 mo than at 9 wk (H and K, arrows) and is uncommon in NTg mice (F and I). Bars (F, G, I, and J), 20 μm; (H and K), 10 μm.


Immunolocalization of PS1 in plaques AVs within dystrophic neurites in AD PS1 / APP mice . Cingulate cortex from 9 - mo - old PS1 / APP mice immunolabeled with PS1 antibody NT1 showed that PS1 localized to plaques ( A ) . At higher magnification , anti - PS1 antibodies strongly labeled neuritic profiles that were distributed within plaque corona ( B ) .

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Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques (A). At higher magnification, anti-PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona (B).


PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1 / APP ( C D ) Arrowheads identify tubulovesicular labeling . PS1 ( C - F , arrows ) was localized on outer limiting of AV but not in mitochondria ( Mito ) or on plasma membranes ( PM ) .

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PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1/APP animals (C and D)Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM).


PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain ( E F ) by IEM . Arrowheads identify tubulovesicular labeling . PS1 ( C - F , arrows ) was localized on outer limiting of AV but not in mitochondria ( Mito ) or on plasma membranes ( PM ) . IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD ( F ) .

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PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain (E and F) by IEM. Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM). IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD (F).


Induction of macroautophagy in L / APP , SH - SY5Y , N2a . ( A B ) EM showing changes in number of AVs ( arrows ) in L / APP - overexpressing APP695 ( L / APP ) grown in complete medium ( A , top left ) or in medium lacking Leu His ( A , top right ) for 6 h in SH - SY5Y grown in presence ( A , bottom left ) or absence ( A , bottom right ) of serum . At higher magnification , early late AVs with typical morphologies are seen in a Leu / His - deprived L / APP ( B ) .

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Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B).


( C ) immunofluorescent labeling of large vesicles by AV marker monodansylcadaverine ( 0 . 5 μg / ml for 30 min ; left ) LC3 antibody ( middle , L / APP ; right , SH - SY5Y ) in macroautophagy - induced ( bottom ) , which are much less abundant in grown in complete medium ( top ) .

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(C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top).


( D ) Western blots confirm cytochemical evidence for increased LC3 - II as well as phospho - mTOR ( P - 2481 ) but not total mTOR after macroautophagy induction by Leu His deprivation or 10 nM rapamycin ( Rap ) macroautophagy inhibition by 5 mM 3MA in L / APP , N2a , SH - SY5Y . Immunoblots for LC3 in SH - SY5Y P - 2481 mTOR in L / APP have been spliced but are derived from same blot .

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(D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.


Aβ generation in after autophagic modulation . of Aβ40 ( A ) , Aβ42 ( B ) , βCTF ( C ) , APP ( D ) measured by sandwich ELISA after incubation of L / APP ( 6 h ) in conditions that block autophagy ( + Leu , + His , + Leu / + His , 5 mM 3MA ) , activate macroautophagy ( − Leu , − His , − Leu / − His , rapamycin ) , or do not affect autophagy ( complete media enrichment of deprivation of Gly or Val ) . Values reported as difference of control ± ; * , P < 0 . 05 ( at least ) .

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Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least).


In similar experiments , Aβ40 Aβ42 from SH - SY5Y ( E F ) N2a ( G H ) after various treatments . Error bars represent .

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In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.


Immunolocalization of Aβ in AVs from L / APP PS1 / APPbrains γ - secretase components ( PS1 nicastrin ) in L / APP . Immunogold localization of Aβ40 ( A ) , Aβ42 ( B ) , PS1 ( C ) , nicastrin ( D ) , in absence of antibody ( E ) in L / APP grown for 6 h in absence of Leu His . ( F ) of gold particle frequency in AV or tubulovesicular compartments ( TBV ) , which comprise 27 . 0 ± 11 . 0 19 . 2 ± 7 . 2 % , respectively , of total . Error bars represent .

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Immunolocalization of Aβ in AVs from L/APP cells and PS1/APPbrains and γ-secretase components (PS1 and nicastrin) in L/APP cells. Immunogold localization of Aβ40 (A), Aβ42 (B), PS1 (C), nicastrin (D), and in the absence of primary antibody (E) in L/APP cells grown for 6 h in the absence of Leu and His. (F) Quantification of gold particle frequency in AV or tubulovesicular compartments (TBV), which comprise 27.0 ± 11.0 and 19.2 ± 7.2%, respectively, of the total cell area. Error bars represent SEM.


( G H ) IEM followed by silver stain enhancement for Aβ40 was performed in 9 - mo - old PS1 / APP mice .

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(G and H) IEM followed by silver stain enhancement for Aβ40 was performed in 9-mo-old PS1/APP mice.


( A B ) Ultrastructure of AVs ( A1 A2 ) lysosomes in subcellular fractions from serum - deprived L / APP ( A ) Western blot ( B ) for LC3 - II , rab24 , APP , βCTF , PS1 ( PS1 - terminal fragment ) , nicastrin ( NCT ) in L / APP subcellular fractions . AVs , A1 A2 ; L , lysosomes ; E , tubulovesicular compartments ( Golgi / ER / endosomes ) ; P , postnuclear pellet ; C , cytosol ; M , mitochondria . Empty lanes in original blot have been removed from figure noted with a .

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(A and B) Ultrastructure of AVs (A1 and A2) and lysosomes in subcellular fractions from serum-deprived L/APP cells (A) and Western blot analysis (B) for LC3-II, rab24, APP, βCTF, PS1 (PS1 amino-terminal fragment), and nicastrin (NCT) in L/APP subcellular fractions. AVs, A1 and A2; L, lysosomes; E, tubulovesicular compartments (Golgi/ER/endosomes); P, postnuclear pellet; C, cytosol; M, mitochondria. Empty lanes in the original blot have been removed from the figure and noted with a white line.


( C ) Rates of cleavage of fluorogenic substrate in subcellular fractions from L / APP grown in − serum media ( left ) or − Leu / − His media ( right ) . PNP , postnuclear pellet .

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(C) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L/APP cells grown in −serum media (left) or −Leu/−His media (right). PNP, postnuclear pellet.


( D E ) Proportions of total recovered γ - secretase activity in different subcellular fractions after serum deprivation ( D ) or in uninduced ( + serum ) or induced ( − Leu / − His ) ( E ) . Comparison of + serum versus − Leu − His conditions shows redistribution of γ - secretase activity from tubulovesicular to AV fractions after macroautophagic induction . G , Golgi ; A , AV .

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(D and E) Proportions of the total recovered cell γ-secretase activity in different subcellular fractions after serum deprivation (D) or in uninduced (+serum) or induced (−Leu/−His) cells (E). Comparison of +serum versus −Leu and −His conditions shows the redistribution of γ-secretase activity from the tubulovesicular to AV fractions after macroautophagic induction. G, Golgi; A, AV.


( F ) Liquid chromatography mass spectrometry of fluorogenic substrate after incubation with purified AVs . Selected ion chromatogram corresponding to different cleavage products is displayed . Numbers in graph refer to cleavage / acid site from Aβ peptide .

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(F) Liquid chromatography mass spectrometry analysis of the fluorogenic substrate after incubation with purified AVs. Selected ion chromatogram corresponding to different cleavage products is displayed. Numbers in graph refer to the cleavage/amino acid site from the Aβ peptide.


( G ) γ - Secretase activity in AV lysosome fractions from mice blastocysts in which PS1 PS2 genes were deleted ( PS KO ; BD8 ) or in which human PS1 was stably transfected into PS KO blastocysts ( hPS1 ; BD8 / hPS1 ) . Numbers on x axis are in minutes .

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(G) γ-Secretase activity in AV and lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted (PS KO; BD8) or in which human PS1 was stably transfected into the PS KO blastocysts (hPS1; BD8/hPS1). Numbers on x axis are in minutes.


( H ) Aβ40 Aβ42 in medium from these as detected by sandwich ELISA . Values are given as means ± ( error bars ) .

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(H) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA. Values are given as means ± SEM (error bars).


EdU - labeled viral forms large foci in ThP1 infected by HIV - 1 . EdU are shown in red , DAPI in blue . ( A ) Left : nucleus of an infected at 48 h p . i . displays three large bright EdU foci ( colored arrows ) , as well as small dim ( grey arrows ) . Center : Magnified views have colored borders matching colors of arrows . 3D show that EdU foci are located within nucleoplasm ( Movie EV1 , Appendix Fig . S2 ) . Right : nucleus of an uninfected displays no EdU signal . ( B ) Graph shows EdU profiles along lines shown in A , with colors matching corresponding arrows .

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EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1. EdU images are shown in red, DAPI images in blue. (A) Left: nucleus of an infected cell at 48 h p.i. displays three large and bright EdU foci (colored arrows), as well as small and dim punctae (grey arrows). Center: Magnified views have colored borders matching the colors of the arrows. 3D images show that EdU foci are located within the nucleoplasm (Movie EV1, Appendix Fig. S2). Right: nucleus of an uninfected cell displays no EdU signal. (B) Graph shows EdU intensity profiles along lines shown in A, with colors matching the corresponding arrows.


( C ) Dual - color image of an infected with EdU ( red ) immunolabeled lamin ( grey ) . Curves to right plot profile of EdU lamin along dashed blue . EdU peaks do not coincide with lamin enrichment . See also Fig . EV1A , B .

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(C) Dual-color image of an infected cell with EdU (red) and immunolabeled lamin (grey). Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line. The EdU intensity peaks do not coincide with lamin enrichment. See also Fig. EV1A,B.


( D ) Multi - color of an infected showing EdU ( red ) with CA ( yellow ) integrase ( green ) . colocalization of EdU foci with CA IN ( arrows ) confirms that EdU specifically labels viral shows presence of these in vDNA foci . See also Appendix Fig . S3 . ( E , F ) vDNA foci are much larger than predicted of single viral genomes .

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(D) Multi-color images of an infected cell showing EdU (red) with CA (yellow) and integrase (green). The colocalization of nuclear EdU foci with CA and IN (arrows) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci. See also Appendix Fig. S3. (E,F) vDNA foci are much larger than the predicted size of single viral genomes.


( E ) Left image shows an observed , EdU - labeled vDNA focus . Right : simulations of a single linear or circular 10 Kb long chromatinized polymer chain corresponding predicted in diffraction - limited ( ~ 300 nm resolution ) microscopy . ( F ) Boxplots show distribution of sizes ( FWHM , full width at half maximum ) of n = 40 measured vDNA foci ( for MOI 100 ) compared to sizes predicted for linear ( left ) circular ( right ) chains on n = 100 simulated configurations each . Blue lines in boxes define medians , top bottom limits define upper lower quartiles , respectively . Whiskers show full data range , except for outliers . Grey dots are data points . differences are highly significant ( Wilcoxon data vs . model : * * * p ≈ 3 ⋅ 10 − 20 , circular vs . linear model : * * * p ≈ 10 − 17 ) .

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(E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17).


( G ) of vDNA foci ( arrows ) in ThP1 infected with multiplicities of infection ( MOI ) 10 , 50 100 . for MOI 10 are shown at two different contrast to reveal dimmer spots . Scale bars , 3 µm .

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(G) Images of vDNA foci (arrows) in ThP1 cells infected with multiplicities of infection (MOI) 10, 50 and 100. Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots. Scale bars, 3 µm.


( H ) Boxplots compare peak intensities inside vDNA foci for MOIs 10 , 50 100 . Central lines in boxes define medians , top bottom limits define upper lower quartiles , respectively . Whiskers show full data range , except for outliers . Grey dots are data points . Intensities increase significantly with MOI ( Wilcoxon : MOI 10 vs . MOI 50 : * * * p ≈ 5 ⋅ 10 − 16 ; MOI 50 vs . MOI 100 : * * * p ≈ 5 ⋅ 10 − 5 ; MOI 10 vs . MOI 100 : * * * p ≈ 2 ⋅ 10 − 23 ) . Data from one experiment . A replicate showed similar results .

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(H) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10, 50 and 100. Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Intensities increase significantly with MOI (Wilcoxon tests: MOI 10 vs. MOI 50: *** p ≈ 5 ⋅ 10 − 16; MOI 50 vs. MOI 100: ***p ≈ 5 ⋅ 10 − 5; MOI 10 vs. MOI 100: ***p ≈ 2 ⋅ 10 − 23). Data from one experiment. A replicate showed similar results.


( I ) Histograms show number of foci per nucleus for MOIs of 10 ( blue ) , 50 ( green ) 100 ( red ) .

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(I) Histograms show the number of foci per nucleus for MOIs of 10 (blue), 50 (green) and 100 (red).


vDNA foci are also foci of vRNA . ( A ) Dual - color image of an infected ThP1 showing viral ( red ) visualized by - FISH ( green ) . Arrows show foci containing both vDNA vRNA . bottom row shows boxed region magnified , with vDNA vRNA displayed separately in combination . See also Appendix Fig . S6 .

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vDNA foci are also foci of vRNA. (A) Dual-color image of an infected ThP1 cell showing the viral DNA (red) and the RNA visualized by RNA-FISH (green). Arrows show foci containing both vDNA and vRNA. The bottom row shows the boxed region magnified, with vDNA and vRNA displayed separately and in combination. See also Appendix Fig. S6.


( C ) Scatter plot shows intensities of vDNA vRNA in colocalizing foci with Spearman correlation r associated p - value .

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(C) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p-value.


In ThP1 co - infected with a HIV1 - GFP a HIV1 - LUC , foci contain mixtures of both . ( D ) Dual - color image shows - FISH against GFP ( red ) - FISH against LUC ( green ) . Arrows show foci containing both GFP vRNA LUC vRNA . bottom row shows boxed region magnified , with GFP vRNA LUC vRNA displayed separately in combination . See Appendix Fig . S7 .

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In ThP1 cells co-infected with a HIV1-GFP virus and a HIV1-LUC virus, nuclear foci contain mixtures of both viruses. (D) Dual-color image shows RNA-FISH against GFP (red) and RNA-FISH against LUC (green). Arrows show foci containing both GFP vRNA and LUC vRNA. The bottom row shows the boxed region magnified, with GFP vRNA and LUC vRNA displayed separately and in combination. See Appendix Fig. S7.


( F ) Scatter plot shows intensities of GFP LUC in colocalizing foci with Spearman correlation r associated p - value .

(F) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p-value.


of infected ( right ) or uninfected ( left ) ThP1 showing vDNA ( EdU ) in red , nucleus ( DAPI ) in blue , selected factors in green . ( A ) Green image shows immunolabeling of CPSF6 .

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Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6.


( B ) Green image shows - FISH against NEAT1 .

(B) Green image shows RNA-FISH against NEAT1.


( C ) Green image shows immunolabeling of SC35 . See also Appendix Figs . S9 - 14 .

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(C) Green image shows immunolabeling of SC35. See also Appendix Figs. S9-14.


( D ) Boxplots show Pearson correlations r between vDNA ( EdU ) CPSF6 , NEAT1 or SC35 in 17 - 19 regions of interest ( ROIs ) . Central lines in boxes define medians , top bottom limits define upper lower quartiles , respectively . Whiskers show full data range , except for outliers . Grey dots are data points . Significance of positive or negative correlations was assessed using Costes method of random ROI shifts . Highly significant ( p < 0 . 01 ) positive correlations between vDNA CPSF6 intensities are found in 16 out of 19 ROIs ; highly significant negative correlations between vDNA NEAT1 are found in 19 / 19 ROIs , highly significant positive correlations between vDNA SC35 are found for 16 / 17 ROIs . Data are from one experiment . For CPSF6 , experiment was repeated with similar results .

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

(D) Boxplots show Pearson correlations r between vDNA (EdU) and CPSF6, NEAT1 or SC35 in 17-19 regions of interest (ROIs). Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts. Highly significant (p<0.01) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs; highly significant negative correlations between vDNA and NEAT1 are found in 19/19 ROIs, and highly significant positive correlations between vDNA and SC35 are found for 16/17 ROIs. Data are from one experiment. For CPSF6, the experiment was repeated with similar results.


( E ) profiles of EdU DAPI along dotted lines in vDNA above .

(E) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above.


( A ) Image of a ThP1 infected with an integration - deficient HIV - 1 carrying mutation D116A in catalytic site of IN . EdU - labeled vDNA is shown in red vRNA detected by - FISH in green . nucleus ( DAPI ) is shown in blue . See Appendix Fig . S12 for a larger image region .

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(A) Image of a ThP1 cell infected with an integration-deficient HIV-1 carrying the mutation D116A in the catalytic site of IN. The EdU-labeled vDNA is shown in red and the vRNA detected by RNA-FISH in green. The nucleus (DAPI) is shown in blue. See Appendix Fig. S12 for a larger image region.


( C ) Scatter plot shows vDNA vRNA intensities in colocalizing vDNA vRNA clusters with Spearman correlation r associated p - value .

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(C) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p-value.


( D ) ThP1 were infected with HIV - 1 at an MOI of 20 , in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI . Green : vRNA ( - FISH ) . Red : vDNA ( EdU ) . ( E ) Scatter plots show intensities of vDNA vRNA in detected vRNA clusters .

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(D) ThP1 cells were infected with HIV-1 at an MOI of 20, in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Red: vDNA (EdU). (E) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters.


( F ) Image of a ThP1 infected by HIV - 1 with EU labeled in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI . Green : vRNA ( - FISH ) . Grey : EU - labeled vRNA . See Appendix Fig . S19 for a larger image region . ( G ) profiles of EU - FISH along dotted lines in panel F ( top ) .

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

(F) Image of a ThP1 cell infected by HIV-1 with EU labeled RNA in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Grey: EU-labeled vRNA. See Appendix Fig. S19 for a larger image region. (G) Intensity profiles of EU and RNA-FISH along the dotted lines in panel F (top).


left panel shows timeline of drug exposure experiments . ( A - D ) show vRNA vDNA in an infected ThP1 ( MOI 20 ) for each of four experimental conditions . See Appendix Fig . S15 - S17 for of larger regions . ( A , B ) ThP1 were exposed to NVP for 48 h ( A ) or 72 h after infection ( B ) , then NVP was washed out , were fixed for click chemistry 24 h later . ( C ) were exposed to PF74 for 72 h after infection , then fixed for click chemistry . ( D ) were exposed to NVP for 24 h after infection , then NVP was washed out , were exposed to PF74 , before being fixed for click chemistry 24 h later . ( E , F , H ) Scatter plots show vDNA vRNA intensities in colocalizing clusters with Spearman correlation r associated p - values . Replicates of four experiments yielded similar results . ( G ) qPCR measures synthesis ( left ) import ( right ) at 72 h p . i . in absence of drug treatment , or after exposure to NVP or PF74 . Bars error bars define mean standard deviation , respectively . Samples were analyzed in triplicate two biological replicates were performed .

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The left panel shows the timeline of drug exposure experiments. (A-D) Images show vRNA and vDNA in an infected ThP1 cell (MOI 20) for each of four experimental conditions. See Appendix Fig. S15-S17 for images of larger regions. (A,B) ThP1 cells were exposed to NVP for 48 h (A) or 72 h after infection (B), then NVP was washed out, and cells were fixed for click chemistry 24 h later. (C) Cells were exposed to PF74 for 72 h after infection, then fixed for click chemistry. (D) Cells were exposed to NVP for 24 h after infection, then NVP was washed out, and cells were exposed to PF74, before being fixed for click chemistry 24 h later. (E,F,H) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p-values. Replicates of the four experiments yielded similar results. (G) qPCR measures DNA synthesis (left) and nuclear import (right) at 72 h p.i. in absence of drug treatment, or after exposure to NVP or PF74. Bars and error bars define mean and standard deviation, respectively. Samples were analyzed in triplicate and two biological replicates were performed.


left panel shows timeline of infection / or drug exposure experiments . ( A - D ) of GFP positive at 3 7 days post - infection analyzed by FACS in absence of EdU . ( A ) Uninfected control . ( B ) Untreated , infected ThP1 fixed at 3 d ( 72 h ) p . i . ( C ) Infected were exposed to NVP for 72 h , then NVP was washed out were cultured for another 96 h fixed at 7 d p . i . ( D ) Infected were cultured for 72 h p . i . , then NVP was washed out were exposed to import inhibitor PF74 for another 96 h fixed at 7 d p . i . ( E - H ) Multicolor of ThP1 infected with a GFP - reporter . vRNA ( - FISH ) is labeled in red , GFP in green , nuclei ( DAPI ) in blue . ( E ) Uninfected control . ( F ) Untreated , infected ThP1 fixed at 3 d ( 72 h ) p . i . ( G ) Infected were exposed to NVP for 72 h , then NVP was washed out were cultured for another 96 h fixed at 7 d p . i . ( H ) Infected were cultured for 72 h p . i . , then NVP was washed out were exposed to import inhibitor PF74 for another 96 h fixed at 7 d p . i . experiment was repeated three times .

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The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Percentage of GFP positive cells at 3 and 7 days post-infection analyzed by FACS in absence of EdU. (A) Uninfected control cells. (B) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (C) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (D) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. (E-H) Multicolor images of ThP1 cells infected with a GFP-reporter virus. vRNA (RNA-FISH) is labeled in red, GFP in green, and nuclei (DAPI) in blue. (E) Uninfected control cells. (F) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (G) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (H) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. The experiment was repeated three times.


left panel shows timeline of infection / or drug exposure experiments . ( A - D ) Multicolor of vDNA , vRNA , DAPI / or GFP in monocyte derived macrophages ( MDMs ) from two different donors , infected with HIV1 . from donor 1 were infected with VSV - G - pseudotyped HIV - 1ΔEnv Vpx carrying a GFP reporter ( A ) from donor 2 were infected with a VSV - G - pseudotyped HIV - 1ΔEnv without Vpx ( B - D ) . ( A , B ) Infected were left untreated fixed at 6 d p . i . ( C ) Infected were treated with 10 µM Nevirapine ( NVP ) throughout experiment fixed at 6 d p . i . ( D ) Infected were exposed to NVP for 3 d p . i . , then NVP was washed out were cultured for another 4 d fixed at 7 d p . i . Arrows in ( A - D ) show position of selected vRNA foci . ( E ) Boxplot shows of foci in untreated infected from donor 2 imaged at 7 d p . i . ( B ) , measured by FWHM of n = 24 foci . red defines median , top bottom limits define upper lower quartiles , respectively . Whiskers show full data range , except for outliers . Grey dots are data points . ( F ) Boxplot shows Pearson ' s r , measuring colocalization of with foci in same ( n = 20 ) . Center defines median , top bottom limits define upper lower quartiles , respectively . Whiskers show full data range , except for outliers . Grey dots are data points .

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

The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Multicolor images of vDNA, vRNA, DAPI and/or GFP in monocyte derived macrophages (MDMs) from two different donors, infected with HIV1. Cells from donor 1 were infected with VSV-G-pseudotyped HIV-1ΔEnv Vpx carrying a GFP reporter (A) and cells from donor 2 were infected with a VSV-G-pseudotyped HIV-1ΔEnv without Vpx (B-D). (A,B) Infected cells were left untreated and fixed at 6 d p.i. (C) Infected cells were treated with 10 µM Nevirapine (NVP) throughout the experiment and fixed at 6 d p.i. (D) Infected cells were exposed to NVP for 3 d p.i., then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p.i. Arrows in (A-D) show the position of selected vRNA foci. (E) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p.i. (B), measured by the FWHM of n=24 foci. The red line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (F) Boxplot shows Pearson's r, measuring colocalization of DNA with RNA foci in the same cells (n=20). Center line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points.


( G ) FACS of Gag positive from donor 2 in different conditions . Red : uninfected . Blue : untreated infected at 7 d p . i . as in B . Orange : were treated with NVP for 7 d as in C . Green : infected were exposed to NVP for 3 d , then NVP was washed out were cultured for another 4 d as in D . Median of intensities ( MFI ) for each sample are shown in table .

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(G) FACS analysis of Gag positive cells from donor 2 in different conditions. Red: uninfected cells. Blue: untreated infected cells at 7 d p.i. as in B. Orange: cells were treated with NVP for 7 d as in C. Green: infected cells were exposed to NVP for 3 d, then NVP was washed out and cells were cultured for another 4 d as in D. Median of fluorescence intensities (MFI) for each sample are shown in the table.


Macrophages were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( A ) Immunoblot of co - immunoprecipitation of lysates with BECN1 specific antibody using antibody to PIK3C3 BECN1 with antibody to β - actin as loading control .

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Macrophages were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Immunoblot of co-immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β-actin as loading control.


( B ) were incubated with 10 µg / mL pepstatin A for 4 h prior to lysis . Immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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(B) Cells were incubated with 10 µg/mL pepstatin A for 4 h prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( C ) were fixed permeabilized then stained with antibody to LC3B ( green ) Hoechst 33342 ( blue ) . Scale bars indicate 10 µm . Left , representative microscopy . When are not present , LC3B gives rise to a diffuse pattern . Right , of with LC3B . Bar charts are presented as means ± s . e . m . , n = 6 .

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(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 6.


( D ) Immunoblots of SQSTM1 using antibody to SQSTM1 or β - actin . * p < 0 . 05 .

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(D) Immunoblots of SQSTM1 using antibody to SQSTM1 or β-actin. * p<0.05.


Macrophages transduced with non - specific scrambled shRNA ( shNS ) or TLR8 shRNA ( shTLR8 ) were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( A ) were lysed immunoblot performed with antibodies to TLR8 β - actin .

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Macrophages transduced with non-specific scrambled shRNA (shNS) or TLR8 shRNA (shTLR8) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Cells were lysed and immunoblot performed with antibodies to TLR8 and β-actin.


( B ) Immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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(B) Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( C ) were fixed permeabilized then stained with antibody to LC3B ( green ) Hoechst 33342 ( blue ) . Scale bars indicate 10 µm . Left , representative microscopy from three donors are shown . When are not present , LC3B gives rise to a diffuse pattern . Right , of with LC3B . Bar charts are presented as means ± s . e . m . , n = 4 . * p < 0 . 05 .

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(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images from three donors are shown. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 4. * p<0.05.


A - B ) Macrophages were treated for 24 h with CL097 , ssRNA40 or ssRNA41 . ( A ) Top , qRT - PCR for CYP27B1 after 6 h . Bottom , immunoblots of CYP27B1 using antibody to CYP27B1 or β - actin .

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A-B) Macrophages were treated for 24 h with CL097, ssRNA40 or ssRNA41. (A) Top, qRT-PCR for CYP27B1 after 6 h. Bottom, immunoblots of CYP27B1 using antibody to CYP27B1 or β-actin.


( B ) Top , qRT - PCR for VDR after 6 h . Bottom , immunoblots of VDR using antibody to VDR or β - actin .

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(B) Top, qRT-PCR for VDR after 6 h. Bottom, immunoblots of VDR using antibody to VDR or β-actin.


( C - D ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , CYP27B1 shRNA ( shCYP27B1 ) or VDR shRNA ( shVDR ) were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( C ) Left , immunoblots performed with antibodies to CYP27B1 , VDR β - actin . Right , immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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(C-D) Macrophages transduced with non-specific scrambled shRNA (shNS), CYP27B1 shRNA (shCYP27B1) or VDR shRNA (shVDR) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (C) Left, immunoblots performed with antibodies to CYP27B1, VDR and β-actin. Right, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( D ) were fixed permeabilized then stained with antibody to LC3B . Bar charts represent of with LC3B are presented as means ± s . e . m . , n = 4 .

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(D) Cells were fixed and permeabilized then stained with antibody to LC3B. Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s.e.m., n = 4.


( E ) Macrophages were treated for 24 h with CL097 , ssRNA40 , rapamycin in presence of 45 nmol / L or 100 nmol / L 25D3 lysed . Immunoblots of LC3B isoforms SQSTM1 using antibody to LC3B , SQSTM1 or β - actin . Bar charts are reported as mean ± s . e . m . , n = 4 . * p < 0 . 05 .

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(E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or β-actin. Bar charts are reported as mean ± s.e.m., n = 4. * p<0.05.


Macrophages transduced with non - specific scrambled shRNA ( shNS ) or CEBPB shRNA ( shCEBPB ) were treated for 24 h with CL097 , ssRNA40 , or ssRNA41 . ( A ) were lysed immunoblot performed with antibodies to CEBPB β - actin .

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Macrophages transduced with non-specific scrambled shRNA (shNS) or CEBPB shRNA (shCEBPB) were treated for 24 h with CL097, ssRNA40, or ssRNA41. (A) Cells were lysed and immunoblot performed with antibodies to CEBPB and β-actin.


B ) Immunoblots of CYP27B1 expression using antibody to CYP27B1 or β - actin .

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B) Immunoblots of CYP27B1 expression using antibody to CYP27B1 or β-actin.


TLR8 agonists inhibit HIV replication . Macrophages were incubated with ( A ) ssRNA40 or ( B ) CL097 for 24 h before infection with HIV for 3 h . were then washed incubated with ssRNA40 or CL097 for 10 d . Extracellular release of HIV p24 antigen into supernatant at days 0 , 3 , 5 , 7 , 10 was detected by ELISA . Results are reported as mean ± s . e . m . , n = 5 . * p < 0 . 05 .

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TLR8 agonists inhibit HIV replication.Macrophages were incubated with (A) ssRNA40 or (B) CL097 for 24 h before infection with HIV for 3 h. Cells were then washed and incubated with ssRNA40 or CL097 for 10 d. Extracellular release of HIV p24 antigen into the cell supernatant at days 0, 3, 5, 7, 10 was detected by ELISA. Results are reported as mean ± s.e.m., n = 5. * p<0.05.


( A ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , TLR8 shRNA ( shTLR8 ) , CYP27B1 shRNA ( shCYP27B1 ) , or VDR shRNA ( shVDR ) were treated with CL097 for 24 h before infection with HIV with continuous CL097 treatment . Top , immunoblot performed using antibodies raised to TLR8 , CYP27B1 , VDR β - actin . Bottom , ELISA performed for extracellular release of HIV p24 antigen over 10 d . Results are reported as mean ± s . e . m . , n = 4 .

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(A) Macrophages transduced with non-specific scrambled shRNA (shNS), TLR8 shRNA (shTLR8), CYP27B1 shRNA (shCYP27B1), or VDR shRNA (shVDR) were treated with CL097 for 24 h before infection with HIV with continuous CL097 treatment. Top, immunoblot analysis performed using antibodies raised to TLR8, CYP27B1, VDR and β-actin. Bottom, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4.


( B ) Macrophages were incubated with ssRNA40 or CL097 in presence of 45 or 100 nmol / L 25D3 for 24 h before infection with HIV . were then washed incubated with 25D3 ssRNA40 or CL097 for 10 d Extracellular release of HIV p24 antigen was detected by ELISA . Results are reported as mean ± s . e . m . , n = 6 . * p < 0 . 05 .

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(B) Macrophages were incubated with ssRNA40 or CL097 in the presence of 45 or 100 nmol/L 25D3 for 24 h before infection with HIV. Cells were then washed and incubated with 25D3 and ssRNA40 or CL097 for 10 d Extracellular release of HIV p24 antigen was detected by ELISA. Results are reported as mean ± s.e.m., n = 6. * p<0.05.


TLR8 - mediated inhibition of HIV is autophagy dependent . ( A - B ) Macrophages transduced with BECN1 shRNA ( shBECN1 ) ( A ) or ATG5 shRNA ( shATG5 )

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TLR8-mediated inhibition of HIV is autophagy dependent.(A-B) Macrophages transduced with BECN1 shRNA (shBECN1) (A) or ATG5 shRNA (shATG5)


( B ) were incubated with CL097 for 24 h before infection with HIV with continuous CL097 treatment for 10 d . Right , immunoblot performed using antibodies raised to BECN1 , ATG5 β - actin . Left , ELISA performed for extracellular release of HIV p24 antigen over 10 d . Results are reported as mean ± s . e . m . , n = 4 .

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(B) were incubated with CL097 for 24 h before infection with HIV with continuous CL097 treatment for 10 d. Right, immunoblot analysis performed using antibodies raised to BECN1, ATG5 and β-actin. Left, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4.


( C ) Macrophages were pretreated with bafilomycin A1 ( Baf A1 ) , SID 26681509 ( SID ) or vehicle control before incubation with CL097 or ssRNA40 for 24 h before infection with HIV . were then washed incubated with TLR8 agonists inhibitors for 10 d ELISA performed for extracellular HIV p24 antigen release over 10 d . Results are reported as mean ± s . e . m . , n = 5 . * p < 0 . 05 ; # p < 0 . 001 .

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(C) Macrophages were pretreated with bafilomycin A1 (Baf A1), SID 26681509 (SID) or vehicle control before incubation with CL097 or ssRNA40 for 24 h before infection with HIV. Cells were then washed and incubated with TLR8 agonists and inhibitors for 10 d and ELISA performed for extracellular HIV p24 antigen release over 10 d. Results are reported as mean ± s.e.m., n = 5. * p<0.05; # p<0.001.


( A ) Macrophages were treated with CL097 , ssRNA40 , ssRNA41 for 6 h after which qRT - PCR for CAMP was performed . Results are reported as mean ± s . e . m . , n = 4 .

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(A) Macrophages were treated with CL097, ssRNA40, ssRNA41 for 6 h after which qRT-PCR for CAMP was performed. Results are reported as mean ± s.e.m., n = 4.


( B ) Macrophages were treated for 24 h with CL097 or ssRNA40 in presence of 45 nmol / L or 100 nmol / L 25D3 . Left , qRT - PCR for CAMP performed after 6 h . Results are reported as mean ± s . e . m . , n = 4 . Right , after 24 h were stained with anti - CAMP antibodies analyzed by flow cytometry . Representative histograms from three donors are shown .

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(B) Macrophages were treated for 24 h with CL097 or ssRNA40 in the presence of 45 nmol/L or 100 nmol/L 25D3. Left, qRT-PCR for CAMP performed after 6 h. Results are reported as mean ± s.e.m., n = 4. Right, after 24 h cells were stained with anti-CAMP antibodies and analyzed by flow cytometry. Representative histograms from three donors are shown.


( C ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) or CAMP shRNA ( shCAMP ) were incubated with CL097 , rapamycin ( Rapa ) or vehicle control for 24 h . Top , immunoblot performed using antibodies raised to CAMP β - actin . Bottom , immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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(C) Macrophages transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) were incubated with CL097, rapamycin (Rapa) or vehicle control for 24 h. Top, immunoblot analysis performed using antibodies raised to CAMP and β-actin. Bottom, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( D ) After 24 h treatment with CL097 or ssRNA40 , macrophages from part C were also washed infected with HIV incubated with CL097 , ssRNA40 or vehicle control for 10 d . ELISA was performed for extracellular release of HIV p24 antigen over 10 d . Results are reported as mean ± s . e . m . , n = 6 . * p < 0 . 05 .

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(D) After 24 h treatment with CL097 or ssRNA40, macrophages from part C were also washed and infected with HIV and incubated with CL097, ssRNA40 or vehicle control for 10 d. ELISA was performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 6. * p<0.05.


Possible correlation between AR - UPR - associated gene expression was assessed in global gene expression data available in TCGA Prostate Adenocarcinoma cohort ( n = 190 ) ( http : / / www . cbioportal . org / public - portal / index . do ) . Tumors were stratified according to AR status into three groups , that is ARlow ( n = 60 ) , ARmedium ( n = 70 ) , ARhigh ( n = 60 ) . of UPR gene expression in three groups were compared using Pearson ' s correlation by R software presented as a . There were significant differences between three groups ( Supplementary Table S2 ) .

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Possible correlation between AR- and UPR-associated gene expression was assessed in the global gene expression data available in the TCGA Prostate Adenocarcinoma cohort (n = 190) (http://www.cbioportal.org/public-portal/index.do). Tumors were stratified according to AR status into three groups, that is ARlow (n = 60), ARmedium (n = 70), and ARhigh (n = 60). The levels of UPR gene expression in the three groups were compared using Pearson's correlation analysis by the R software and presented as a heatmap. There were significant differences between the three groups (Supplementary Table S2).


expression profiles of some prominent UPR genes from data in ( A ) , including ERN1 ( IRE1 ) , EDEM1 , ATF6 , DNAJC3 ( P58IPK ) , are presented . P - values of different genes are given .

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The expression profiles of some prominent UPR genes from the data in (A), including ERN1 (IRE1), EDEM1, ATF6, and DNAJC3 (P58IPK), are presented. P-values of the different genes are given.


A , B mRNA expression in LNCaP for indicated genes were investigated using quantitative PCR ( qPCR ) . Controls were treated with vehicle for 84 h set to 100 . Data represent mean of three independent experiments in triplicate , bars represent SE . P - values ranged between 1 . 66 × 10 − 5 0 . 025 , IRE1α expression in R1881 48 h was * P = 0 . 013 with respect to vehicle - treated using unpaired Student ' s t - .

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A, B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR (qPCR). Controls were treated with vehicle for 84 h and set to 100. Data represent the mean of three independent experiments in triplicate, and bars represent SE. P-values ranged between 1.66 × 10−5 and 0.025, and IRE1α expression in R1881 48 h was *P = 0.013 with respect to vehicle-treated cells using unpaired Student's t-test.


C - E IRE1α , XBP - 1S , XBP - 1U mRNA in CWR22xenografts grown in nude mice collected at indicated times after castration . value at t = 0 was set to 1 . Columns represent mean of at least three independent tumors for each time point , bars represent SE . P - values ranged between 5 . 7 × 10 − 10 0 . 0002 . IRE1α expression at 48 h post - castration was 3 . 17 × 10 − 6 with respect to t = 0 using unpaired Student ' s t - .

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C-E IRE1α, XBP-1S, and XBP-1U mRNA in CWR22xenografts grown in nude mice and collected at the indicated times after castration. The value at t = 0 was set to 1. Columns represent the mean of at least three independent tumors for each time point, and bars represent SE. P-values ranged between 5.7  ×  10−10 and 0.0002. IRE1α expression at 48 h post-castration was 3.17 × 10−6 with respect to t = 0 using unpaired Student's t-test.


F , G expression in LNCaP upon treatment with R1881 for indicated times by Western blot . Data presented are representative of three independent experiments .

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F, G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis. Data presented are representative of three independent experiments.


After starvation , LNCaP were transfected with control ( CTRL ) siRNA or AR siRNA . were then treated with R1881 for indicated times . Controls were treated with vehicle for 48 h . A Expression level of AR mRNA upon siRNA treatment for 48 h assessed by qPCR in LNCaP . Expression in transfected with CTRL siRNA was set to 1 . Bars represent SE with * P = 0 . 001 indicating significant difference between AR siRNA - control siRNA - transfected using paired Student ' s t - .

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After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.A Expression level of AR mRNA upon siRNA treatment for 48 h assessed by qPCR in LNCaP cells. Expression in cells transfected with CTRL siRNA was set to 1. Bars represent SE with *P = 0.001 indicating significant difference between AR siRNA- and control siRNA-transfected cells using paired Student's t-test.


After starvation , LNCaP were transfected with control ( CTRL ) siRNA or AR siRNA . were then treated with R1881 for indicated times . Controls were treated with vehicle for 48 h . B - E Same as in ( A ) , but mRNA expression of indicated UPR genes were determined by qPCR at indicated time points after R1881 stimulation . Expression in transfected with CTRL siRNA was set to 100 . Bars represent SE . P - values are shown indicating significant difference between AR siRNA - control siRNA - transfected using unpaired Student ' s t - .

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After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.B-E Same as in (A), but mRNA expression levels of the indicated UPR genes were determined by qPCR at indicated time points after R1881 stimulation. Expression in cells transfected with CTRL siRNA was set to 100. Bars represent SE. P-values are shown indicating significant difference between AR siRNA- and control siRNA-transfected cells using unpaired Student's t-test.


After starvation , LNCaP were transfected with control ( CTRL ) siRNA or AR siRNA . were then treated with R1881 for indicated times . Controls were treated with vehicle for 48 h . F Expression of indicated under conditions indicated on top label was determined by Western blot . Representative blots for three independent experiments are shown .

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After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.F Expression of the indicated proteins under conditions indicated on the top label was determined by Western blot analysis. Representative blots for three independent experiments are shown.


A , B LNCaP ( A ) or VCaP ( B ) were cultured treated with vehicle ( C ) for 48 h or R1881 for 24 48 h . were then fixed , ChIP was performed as described in Materials Methods using AR antibody . data shown are representative of one experiment in duplicate . Error bars represent SE . * P < 0 . 01 for LNCaP * P < 0 . 04 for VCaP indicate significant difference between C ( control ) R1881 using unpaired Student ' s t - .

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A, B LNCaP (A) or VCaP cells (B) were cultured and treated with vehicle (C) for 48 h or R1881 for 24 and 48 h. The cells were then fixed, and ChIP assay was performed as described in Materials and Methods using AR antibody. The data shown are representative of one experiment in duplicate. Error bars represent SE. *P < 0.01 for LNCaP and *P < 0.04 for VCaP indicate significant difference between C (control) and R1881 using unpaired Student's t-test.


A Knockdown of IRE1α or XBP - 1 leads to a decrease in survival . LNCaP were transfected with siRNA targeting either IRE1α or XBP - 1 ( 5 nM ) starved in 2 % CT - FCS medium for 3 days before viability was measured using CCK - 8 . graph is representative of one experiment in triplicate was repeated three times with similar results . Error bars represent SD with * P = 6 . 6 × 10 − 5 4 . 5 × 10 − 5 for comparison between Ctrl siRNA against IRE1α XBP - 1 , respectively , using paired Student ' s t - .

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A Knockdown of IRE1α or XBP-1 leads to a decrease in cell survival. LNCaP cells were transfected with siRNA targeting either IRE1α or XBP-1 (5 nM) and starved in 2% CT-FCS medium for 3 days before cell viability was measured using the CCK-8 assay. The graph is representative of one experiment in triplicate and was repeated three times with similar results. Error bars represent SD with *P = 6.6 × 10−5 and 4.5 × 10−5 for comparison between Ctrl and siRNA against IRE1α and XBP-1, respectively, using paired Student's t-test.


B XBP - 1 rescues growth defect of siIRE1α - transfected LNCaP . LNCaP were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent . One day after siRNA transfection , were transfected with either vector control ( Empty ) or Flag - XBP - 1S ( XBP - 1S ) . Three days after transfection , were harvested for western blot or cultured for three more days before being applied to proliferation using CCK - 8 reagent . data are representative of two experiments in triplicate . Error bars represent SE . * P = 0 . 02 , * * P = 8 . 54 × 10 − 7 using paired Student ' s t - .

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B XBP-1 rescues the growth defect of siIRE1α-transfected LNCaP cells. LNCaP cells were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent. One day after siRNA transfection, the cells were transfected with either vector control (Empty) or Flag-XBP-1S (XBP-1S). Three days after transfection, cells were harvested for western blot analysis or cultured for three more days before being applied to cell proliferation assay using the CCK-8 reagent. The data are representative of two experiments in triplicate. Error bars represent SE. *P = 0.02, **P = 8.54 × 10−7 using paired Student's t-test.


C , D IRE1α XBP - 1 knockdown inhibits clonogenic capacity of LNCaP . Control LN - Scr ( Scr ) , LN - shIRE1 ( shIRE1 ) , or LN - shXBP1 ( shXBP - 1 ) were cultured for 3 . colonies formed were stained with crystal violet photographed . extent of IRE1α XBP - 1 knockdown was determined by Western blot . covered by colonies was quantified using Gene Tools software ( SynGene ) . data are representative of three experiments in triplicate . Error bars represent . * P = 4 . 38 × 10 − 7 * * P = 3 . 39 × 10 − 20 using paired Student ' s t - .

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C, D IRE1α and XBP-1 knockdown inhibits clonogenic capacity of LNCaP cells. Control LN-Scr (Scr), LN-shIRE1 (shIRE1), or LN-shXBP1 (shXBP-1) cells were cultured for 3 weeks. The colonies formed were stained with crystal violet and photographed. The extent of IRE1α and XBP-1 knockdown was determined by Western blot analysis. The area covered by colonies was quantified using the Gene Tools software (SynGene). The data are representative of three experiments in triplicate. Error bars represent SEM. *P = 4.38 × 10−7 and **P = 3.39 × 10−20 using paired Student's t-test.


E Growth of xenograftedLNCaPtumors in nude mice . LNCaP expressing shRNA against IRE1α ( LN - shIRE1 ) , XBP1 ( LN - shXBP - 1 ) , or control shRNA ( LN - Scr ) were subcutaneously implanted into both flanks of nude mice ( 6 mice per group ) . was measured at indicated time points . Representative pictures of tumors at harvest are shown . Error bars indicate . * P = 0 . 03 for shIRE1 at week 7 , P = 0 . 02 for shXBP - 1 at week 7 , * * P = 0 . 01 for both shIRE1 shXBP - 1 at week 8 using unpaired Student ' s t - .

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E Growth analysis of xenograftedLNCaPtumors in nude mice. LNCaP cells expressing shRNA against IRE1α (LN-shIRE1), XBP1 (LN-shXBP-1), or control shRNA (LN-Scr) were subcutaneously implanted into both flanks of male nude mice (6 mice per group). Tumor size was measured at the indicated time points. Representative pictures of the tumors at harvest are shown. Error bars indicate SEM. *P = 0.03 for shIRE1 at week 7, P = 0.02 for shXBP-1 at week 7, **P = 0.01 for both shIRE1 and shXBP-1 at week 8 using unpaired Student's t-test.


F PCNA immunostaining in tumors from bearing LN - shIRE1 , LN - shXBP - 1 , or LN - Scr tumors . Scale bars : 100 μm .

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F PCNA immunostaining in tumors from animals bearing LN-shIRE1, LN-shXBP-1, or LN-Scr tumors. Scale bars: 100 μm.


LNCaP xenografts were grown in nude mice until palpable . Mice were then intraperitoneally injected with 0 . 5 mg / kg toyocamycin or saline ( Vehicle ) ( numbers : n = 15 , orn = 10 , respectively ) twice weekly . sizes were measured weekly with calipers . Error bars indicate . * P = 0 . 04 ; * * P = 0 . 0004 ; * * * P = 0 . 002 using unpaired Student ' s t - . Same procedure was repeated on VCaP xenografts , with similar findings as in LNCaP xenografts ( numbers : n = 9 , n = 11 ) . Error bars indicate . * P = 0 . 02 * * P = 0 . 0006 using unpaired Student ' s t - .

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LNCaP xenografts were grown in nude mice until palpable. Mice were then intraperitoneally injected with 0.5 mg/kg toyocamycin or saline (Vehicle) (tumor numbers:n = 15, orn = 10, respectively) twice weekly.Tumor sizes were measured weekly with calipers. Error bars indicate SEM. *P = 0.04; **P = 0.0004; ***P = 0.002 using unpaired Student's t-test.Same procedure was repeated on VCaP xenografts, with similar findings as in LNCaP xenografts (tumor numbers: n = 9, n = 11). Error bars indicate SEM. *P = 0.02 and **P = 0.0006 using unpaired Student's t-test.


XBP - 1S mRNA in LNCaP xenografts from treated with toyocamycin or vehicle . Tumors were harvested , - extracted , qPCR performed . Error bars indicate . * P = 0 . 04 for LNCaP * * P = 0 . 02 for VCaP using unpaired Student ' s t - .

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XBP-1S mRNA levels in LNCaP xenografts from animals treated with toyocamycin or vehicle. Tumors were harvested, RNA-extracted, and qPCR performed. Error bars indicate SEM. *P = 0.04 for LNCaP and **P = 0.02 for VCaP using unpaired Student's t-test.


PCNA in tumors from treated with either toyocamycin or saline . Scale bar : 100 μm .

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PCNA staining in tumors from animals treated with either toyocamycin or saline. Scale bar: 100 μm.


XBP - 1S expression was determined by in two different cohorts of human prostatectomy samples . Representative pictures of benign samples .

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XBP-1S expression was determined by IHC in two different cohorts of human prostatectomy samples.Representative pictures of benign and tumor samples.


Tissue microarrays ( Wang et al , 2010 ) containing 25 benign 283 samples were stained with a XBP - 1S specific antiserum scored by a pathologist . P - value indicates difference between XBP - 1S ( strong moderate ) in normal vs cancer using Mann - Whitney .

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Tissue microarrays (Wang et al, 2010) containing 25 benign and 283 tumor samples were stained with a XBP-1S specific antiserum and scored by a pathologist. The P-value indicates the difference between XBP-1S staining (strong and moderate) in normal vs cancer cells using Mann-Whitney test.


XBP - 1S expression was determined by of a neoadjuvant hormone therapy ( NHT ) tissue microarray containing samples from hormone naïve ( untreated ) ( n = 25 ) , that received NHT for 1 - 6 months ( n = 33 ) , that received NHT for 7 - 12 months ( n = 50 ) , as indicated . Representative are shown . Scale bars : 100 μm . Quantitative presentation of data from ( C ) . * P = 0 . 006 in unpaired Student ' s t - .

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XBP-1S expression was determined by IHC of a neoadjuvant hormone therapy (NHT) tissue microarray containing samples from hormone naïve (untreated) (n = 25), patients that received NHT for 1-6 months (n = 33), and patients that received NHT for 7-12 months (n = 50), as indicated. Representative images are shown. Scale bars: 100 μm.Quantitative presentation of the data from (C). *P = 0.006 in unpaired Student's t-test.


( A ) Raw 264 . 7 were pre - treated with DMSO or EGFR inhibitor gefitinib ( 10 µM ) before cGAMP transfection ; after 5h , IFNβ mRNA was measured by qRT - PCR ( n = 3 ) .

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(A) Raw 264.7 cells were pre-treated with DMSO or the EGFR inhibitor gefitinib (10 µM) before cGAMP transfection; after 5h, IFNβ mRNA was measured by qRT-PCR (n=3).


( B ) WT EGFR knockdown Raw 264 . 7 were transfected with cGAMP ; 4 . 5h post treatment , IFNβ mRNA was measured ( n = 3 ) .

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(B) WT and EGFR knockdown Raw 264.7 cells were transfected with cGAMP; 4.5h post treatment, IFNβ mRNA was measured (n=3).


( C ) WT EGFR - / - BMDMs were transfected with cGAMP ; 5h post transfection , IFNα mRNA induction was measured ( n = 4 ) .

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(C) WT and EGFR-/- BMDMs were transfected with cGAMP; 5h post transfection, IFNα mRNA induction was measured (n=4).


( D ) WT EGFR - / - HeLa were transfected with cGAMP ; after 5h IFNβ mRNA was measured ( n = 3 ) .

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(D) WT and EGFR-/- HeLa cells were transfected with cGAMP; after 5h IFNβ mRNA was measured (n=3).


MEF were pre - treated with gefitinib for 1h then transfected with cGAMP ; 4 . 5h post treatment , total was harvested for . ( E ) - seq ( n = 2 ) . For - seq , poly T enriched cDNA library was prepared 30 M single - end 50 bp reads were analyzed . heat map for some of genes is shown .

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MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (E) RNA-seq analysis (n=2). For RNA-seq analysis, poly T enriched cDNA library was prepared and 30 M single-end 50 bp reads were analyzed. The heat map for some of the genes is shown.


MEF were pre - treated with gefitinib for 1h then transfected with cGAMP ; 4 . 5h post treatment , total was harvested for . ( G ) - seq results were confirmed by qRT - PCR of a gefitinib - sensitive an insensitive mRNA ( n = 3 ) .

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MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (G) The RNA-seq analysis results were confirmed by qRT-PCR analysis of a gefitinib-sensitive and an insensitive mRNA (n=3).


( H ) MEF were pretreated with gefitinib for 1h followed with cGAMP or Poly ( I : C ) transfection ; 4 . 5h post treatment , total was harvested IFNα mRNA induction was measured ( n = 3 ) .

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(H) MEF cells were pretreated with gefitinib for 1h and followed with cGAMP or Poly (I:C) transfection; 4.5h post treatment, total RNA was harvested and IFNα mRNA induction was measured (n=3).


( A ) MEF were pre - treated with gefitinib or DMSO for 1 hour , then infected with HSV1 at m . o . i . of 1 . 4 hours post infection ( h . p . i . ) , IFNβ mRNA induction was monitored by qRT - PCR ( n = 3 ) . Uninfected were used as negative control ( U ) .

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(A) MEF cells were pre-treated with gefitinib or DMSO for 1 hour, and then infected with HSV1 at m.o.i. of 1. 4 hours post infection (h.p.i.), IFNβ mRNA induction was monitored by qRT-PCR (n=3). Uninfected cells were used as negative control (U).


( B ) was extracted from gefitinib - treated or untreated infected MEFs 0 16 h . p . i . viral was measured by qPCR ( n = 3 ) .

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(B) DNA was extracted from gefitinib-treated or untreated infected MEFs 0 and 16 h.p.i. and viral DNA was measured by qPCR (n=3).


( C ) WT EGFR KO HeLa were infected with HSV1 , was extracted at 4 h . p . i . IFNα mRNA induction was measured ( n = 3 ) .

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(C) WT and EGFR KO HeLa cells were infected with HSV1, RNA was extracted at 4 h.p.i. and IFNα mRNA induction was measured (n=3).


( D ) Survival of uninfected mice treated with gefitinib HSV1 - infected mice treated or untreated with gefitinib .

(D) Survival of uninfected mice treated with gefitinib and HSV1-infected mice treated or untreated with gefitinib.


( E ) Survival of WT EGFRfl + / - LysMCre + / + mice infected with HSV1 .

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(E) Survival of WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1.


WT EGFRfl + / - LysMCre + / + mice infected with HSV1 . HSV1 titers in brains of live mice at day 9 were determined .

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WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1. HSV1 titers in the brains of the live mice at day 9 were determined.


( A ) EGFR was not required for ligand - induced STING dimerization : MEFs were transfected with HA - tagged STING treated with gefitinib or DMSO before cGAMP transfection , 4h later STING was immunoprecipitated with anti - HA antibodies analyzed by Western blot .

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(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 activation of IRF3 , but not TBK1 : MEFs were pretreated with gefitinib for 1h , then treated with cGAMP for 2h ; lysate were subjected to Western blot using indicated antibodies .

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(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 ) translocation of IRF3 , but not NFκB p65 , required EGFR : MEFs were pre - treated with gefitinib ( + ) or DMSO ( - ) for 1 hour before transfecting cGAMP . were harvested nuclei were isolated at indicated time points ; total extracts ( T ) extracts ( N ) were analyzed by Western blot .

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(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 lysates were immunoprecipitated with anti - STING antibody analyzed by Western blot .

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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 required EGFR : WT EGFR - / - ( KO ) HeLa were transfected with cGAMP , 2h later were lysed , immunoprecipitated with anti - STING antibody analyzed by Western blot .

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(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 IRF3 co - localized in endosomes of stimulated : STING - / - MEF were transfected with STING - HA for 24 hours then treated with cGAMP for 1h . were then stained with HA ( purple ) , CD63 ( red ) IRF3 ( green ) antibodies . Imaging data were analyzed by Fuji software to reveal co - localization as dots . Scale bars represent 10 µ m .

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(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 or gefitinib - treated untreated WT HeLa were treated with cGAMP for indicated time lysates were analyzed by Western blot .

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(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 IRF3 co - localization in endosomes of stimulated : STING - / - MEF were transfected with STING - HA for 24 hours treated with cGAMP for indicated time . were then stained with HA ( purple ) , CD63 ( red ) IRF3 ( green ) antibodies DAPI . Imaging data were analyzed by Fuji software to reveal co - localization as dots . Scale bars represent 10 µ m .

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(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.


( A ) STING was Tyr - phosphorylated in cGAMP - treated . Raw 264 . 7 were tranfected with cGAMP ; after 60 min , lysates were immuno - precipitated with anti - pTyr antibodies then subjected to anti - STING Western blot .

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(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 were pre - treated with gefitinib before cGAMP - stimulation for 60 min . lysates were immuno - precipitated with anti - pTyr antibody then analyzed by anti - STING Western blot . NTC : no treatment control .

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(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 expressing EGFR shRNA ( KD ) or non - target shRNA ( CO ) were transfected with cGAMP for 2 hours ; lysates were analyzed by Western blot before or after immunoprecipitation with anti - pTyr antibodies .

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(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 were transfected with cGAMP for 1h ; lysates were immunoprecipitated with anti - STING antibody analyzed by Western blot .

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(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 were transfected with STING - GFP ( Green ) for 24hours stimulated with cGAMP for 1h , were fixed labeled with EGFR ( Red ) Calnexin ( Purple ) antibodies . Co - localization was analyzed by Fuji co - localized dots are shown in . Scale bars represent 10 µ m .

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(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 lysates were prepared at indicated time points after cGAMP transfection , immunoprecipitated with anti - EGFR antibodies subjected to Western blot .

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(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 were pre - treated with gefitinib or DMSO for 1 hour before cGAMP stimulation . lysates were prepared at indicated time points , immuno - precipitated with anti - STING antibody subjected to Western blot .

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(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 were transfected with cGAMP or Poly ( I : C ) ; after 3h , p - EGFR were detected by Western blot .

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(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 were treated with cGAMP for indicated time . lysates were analyzed for p - EGFR via Western blot .

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(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 were stimulated for 1h , lysates were immunoprecipitated with anti - STING antibody subjected to Western blot .

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(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 were treated with cGAMP for indicated time . lysates were subjected to Western blot using indicated antibodies .

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(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 were transfected with cGAMP or Poly ( I : C ) ; after 3h , lysates were analyzed by Western blot using indicated antibodies .

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(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.


( A ) In absence of EGFR , after cGAMP stimulation , STING translocated out of ER . STING - / - MEF were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h transfected with cGAMP for 2 . 5h ; were fixed labeled with Calreticulin ( ER marker , Red ) antibody . Co - localization ( Col ) is indicated by dots .

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(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 were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by dots . ( B ) STING was present in ERGIC of stimulated in presence or absence of gefitinib . , were labeled with P58 ( ERGIC marker , Red ) antibody .

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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 were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by dots . ( C ) STING translocated to autophagosomes in absence of EGFR . were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( E ) In gefitinib - treated , STING was detected in autophagosomes as early as 0 . 5h . procedures were as in C .

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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 dots . STING translocated to autophagosomes in absence of EGFR . , were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( D ) No STING was detected in autophagosomes even after 3h after stimulation if EGFR was not inhibited .

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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 were transfected with STING - GFP ( Green ) for 24hours , pretreated with gefitinib for 1h transfected with cGAMP for indicated time periods ; were fixed labeled with CD63 ( late endosome marker , Red ) antibody .

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(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.


( A ) EGFR phosphorylates STING in vitro . Purified STING - GFP was incubated with increasing amounts of EGFR for in vitro kinase assays ; products were analyzed on phos - tag gel by Western blot using GFP antibody ( upper panel ) . substrate was analyzed , before incubations , by same method ( lower panel ) .

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(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 ) . substrate was analyzed , before incubations , by same method ( lower panel ) . ( B ) Y245F STING mutant is not a substrate of EGFR . WT indicated mutants of STING - GFP were purified used as substrates for

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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 of STING identifies phosphorylated Y245 . STING - GFP was purified from cGAMP - stimulated , subjected to protease digestion LC - MS / MS . Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified . MS / MS spectra for phosphorylated peptide VYSNSIpYELLENGQR is shown . This doubly charged peptide has an observed m / z of 932 . 9266 Da is within - 0 . 96 ppm of expected mass . mass difference between y8 y9 ions is consistent with phosphorylation at Y245 .

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(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.