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 .

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(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 .

(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.


( E ) Gefitinib pretreatment inhibits phosphorylation of Y245 . STING - GFP - 293XL were pretreated with gefitinib then treated with cGAMP . LC - MS / MS was done , as above , for detecting pY245 ( n = 3 ) .

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(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 in which EGFR expression had been knocked down . STING phosphorylation was analyzed by LC - MS /

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(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 . was isolated from , expressing WT or Y245F STING , 3h after stimulation subjected to qRT - PCR ( n = 3 ) .

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(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 . Phosphorylation of S366 was detected in expressing WT or mutant STING after stimulation for indicated time .

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(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 be phosphorylated in cGAMP - stimulated . Lysates from expressing WT or mutant STING were analyzed by Western blot after ( upper panel ) or before ( lower panel ) GFP pull down .

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(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 expressing WT or mutant STING .

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(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 .

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(K) Inhibition of EGFR by gefitinib induced more LC3II after 1.5h cGAMP treatment of L929 cells.n


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

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(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 were transfected with mSTING WT - GFP Y244F - GFP ( Green ) for 24h , pretreated with gefitinib for 1h transfected with cGAMP for indicated time ; were fixed labeled with CD63 ( late endosome marker ) .

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(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).


A Whole mount immunofluorescence of a P8 Syne4 - / - organ of Corti showing intact hair , as labelled with myosin VIIa . B Whole mount immunofluorescence of WT Syne4 - / - organ of Corti from 8 , 16 , 32 kHz regions at P8 P14 .

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A Whole mount immunofluorescence of a P8 Syne4-/- organ of Corti showing intact hair cells, as labelled with myosin VIIa. B Whole mount immunofluorescence of WT and Syne4-/- organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14.


C Inner outer hair of Syne4 - / - organ of Corti at P8 , P10 , P12 , P14 .

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C Inner and outer hair cell counts of Syne4-/- organ of Corti at P8, P10, P12, and P14.


D FM1 - 43 uptake performed on P8 + 1 DIV ( days - in - vitro ) WT Syne4 - / - organ of Corti . Top shows OHC plane bottom shows plane .

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D FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4-/- organ of Corti. Top shows OHC plane and bottom shows IHC plane.


B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV . GFP at P1 showing complete transduction of both inner outer HC . Myosin VIIa was used to label hair . C Examples of 8 , 16 , 32 kHz regions of an organ of Corti of a mouse injected with AAV . GFP . Top shows OHC plane , bottom shows plane , right shows YZ orthogonal projection . Black asterisks show bright Deiters . D of GFP of inner outer HC from 3 injected mice , normalized to average of HC in a control , un - injected mouse .

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B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. C Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. D Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse.


E Transduction rates of AAV9 - PHP . B at 8 , 16 , 32kHz regions on GFP . A total of 162 841 OHC were analyzed from 3 injected mice 1 control littermate .

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E Transduction rates of AAV9-PHP.B at 8, 16, and 32kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate.


F for FLAG at at P14 of organ of Corti of a mouse injected at P1 with AAV . Syne4 .

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F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4.


G of FLAG DAPI along a centered at envelope . Eight OHC were measured .

G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured.


H 3D surface projection of two adjacent OHC from a mouse injected with AAV . Syne4 . In left myosin VIIa DAPI signals were removed to only show FLAG .

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H 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.


A Whole - mount immunofluorescence of 8 kHz region from P14 organ of Corti from WT , Syne4 - / - , Syne4 - / - mice injected with AAV . Syne4 . B 3D surface projection of OHC at P14 from 8kHz region of WT , Syne4 - / - mice Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , arrows denote cuticular plate .

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A Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4-/-, and Syne4-/- mice injected with AAV.Syne4. B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4-/- mice and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate.


C Image of nucleus position of OHC . show an arc fitted through apical surface , nucleus , basal end of in 3D . X , Y , Z axes show position in μm .

C Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm.


D of from cuticular plate . A total of 45 135 OHC from 3 WT mice , 45 81 OHC from 3 Syne4 - / - mice , 30 90 OHC from 2 Syne4 - / - mice injected with AAV . Syne4 were measured .

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D Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4-/- mice, 30 IHC and 90 OHC from 2 Syne4-/- mice injected with AAV.Syne4 were measured.


A Representative example of ABR traces obtained at 4w from a WT , Syne4 - / - mouse , Syne4 - / - mouse injected with AAV . Syne4 in response to an 18 kHz stimulus .

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A Representative example of ABR traces obtained at 4w from a WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4 in response to an 18 kHz stimulus.


B ABR thresholds at 4w of WT , Syne4 - / - , Syne4 - / - mice injected with AAV . Syne4 , Syne4 - / - mice injected with AAV . GFP , n = 7 for WT , n = 8 for Syne4 - / - , n = 20 for Syne4 - / - + AAV . Syne4 , n = 6 for Syne4 - / - + AAV . GFP . C of P1 - N1 amplitude delta from ( B ) , n = 4 for WT , n = 4 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 . D of P1 latency from ( B ) , n = 4 for WT , n = 2 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 .

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B ABR thresholds at 4w of WT, Syne4-/-, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=7 for WT, n=8 for Syne4-/-, n=20 for Syne4-/- + AAV.Syne4, n=6 for Syne4-/- + AAV.GFP. C Quantification of P1-N1 amplitude delta from (B), n=4 for WT, n=4 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4. D Quantification of P1 latency from (B), n=4 for WT, n=2 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4.


E DPOAE thresholds at 4w , n = 6 for WT , n = 5 for Syne4 - / - , n = 6 for Syne4 - / - + AAV . Syne4 . F ABR threshold at 12w , n = 6 for WT , n = 8 for Syne4 - / - , n = 16 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP .

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E DPOAE thresholds at 4w, n=6 for WT, n=5 for Syne4-/-, n=6 for Syne4-/- + AAV.Syne4. F ABR threshold at 12w, n=6 for WT, n=8 for Syne4-/-, n=16 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP.


A Whole mount immunofluorescence at 12w of organ of Corti of a Syne4 - / - mouse injected with AAV . Syne4 mouse injected with AAV . Syne4 . B Whole mount immunofluorescence at 12w of 12 kHz region of WT , Syne4 - / - mouse , Syne4 - / - mouse injected with AAV . Syne4 .

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A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4-/- mouse injected with AAV.Syne4 mouse injected with AAV.Syne4. B Whole mount immunofluorescence at 12w of the 12 kHz region of WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4.


C OHC , n = 3 for WT , n = 4 for Mut , n = 6 for Mut + AAV . Syne4 . D Correlation between HC ABR threshold at 12kHz .

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C IHC and OHC counts, n=3 for WT, n=4 for Mut, n=6 for Mut + AAV.Syne4. D Correlation between HC count and ABR threshold at 12kHz.


E 3D surface projection of an OHC at 12w from 12kHz region of WT Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , arrows denote cuticular plate . F Image of nucleus position of OHC . G of from cuticular plate . A total of 30 OHC from 3 WT mice 38 OHC from 4 Syne4 - / - mice injected with AAV . Syne4 were measured .

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E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate. F Image analysis of nucleus position of OHC. G Quantification of nuclear distance from the cuticular plate. A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4-/- mice injected with AAV.Syne4 were measured.


B , C of activity level cumulative freezing duration at 12w during seconds 90 - 210 of day 2 in WT , Syne4 - / - mice , Syne4 - / - mice injected with AAV . Syne4 , Syne4 - / - mice injected with AAV . GFP , n = 14 for WT , n = 16 for Syne4 - / - , n = 9 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP . ( C ) Activity level . ( B ) Cumulative freezing duration .

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B, C Quantification of activity level and cumulative freezing duration at 12w during seconds 90-210 of day 2 in WT, Syne4-/- mice, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=14 for WT, n=16 for Syne4-/-, n=9 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP. (C) Activity level. (B) Cumulative freezing duration.


F - I ChIP assays with antibodies against H3K9me3 , H4K20me3 , H3K4me3 H3K9Acetyl were performed as in ( D ) . Only ' C ' position signals were showed normalized to Input .

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F-I ChIP assays with antibodies against H3K9me3, H4K20me3, H3K4me3 and H3K9Acetyl were performed as in (D). Only 'C' position signals were showed and normalized to Input.


M ChIP assays with antibodies against CDK9 Pho - Pol II were performed in siNC siCHAF1A J - Lat 10 . 6 . ChIP signals in each group were normalized to Input .

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M ChIP assays with antibodies against CDK9 and Pho-Pol II were performed in siNC and siCHAF1A J-Lat 10.6 cells. ChIP signals in each group were normalized to Input.


siRNAs targeting each CAF - 1 subunits corresponding enriched genes were transfected into TZM - bl lines respectively . fold change of luciferase expression of each groups was normalized to siNC .

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siRNAs targeting each CAF-1 subunits and corresponding enriched genes were transfected into TZM-bl cell lines respectively. The fold change of luciferase expression of each groups was normalized to siNC.


C , D ImmunoFISH of CAF - 1 bodies HIV - 1 genomic within J - Lat 10 . 6 . Naïve were treated with DMSO . Activated were acquired by treating with TNFα .

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C, D ImmunoFISH images of CAF-1 bodies and HIV-1 genomic DNA within J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were acquired by treating with TNFα.


E RFP - tagged CHAF1A GFP - tagged PML component SP100 were co - overexpressed in HEK293T imaged by SIM . F GFP - tagged CHAF1A RFP - tagged BRD4 were co - overexpressed in HEK293T imaged by SIM .

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E RFP-tagged CHAF1A and GFP-tagged PML body component SP100 were co-overexpressed in HEK293T cells and imaged by SIM. F GFP-tagged CHAF1A and RFP-tagged BRD4 were co-overexpressed in HEK293T cells and imaged by SIM.


SUV39H1 , HP1α , DNMT1 , MBD1 , RING1B , EZH2 were co - overexpressed with CHAF1A in HEK293T respectively . SIM imaging was performed for each combination .

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SUV39H1, HP1α, DNMT1, MBD1, RING1B, EZH2 were co-overexpressed with CHAF1A in HEK293T cells respectively. SIM imaging was performed for each combination.


E GFP - tagged CHAF1A - N - NLS CHAF1A - C were imaged with RFP - tagged CHAF1A respectively . N : CHAF1A N - terminal . NLS : localization signal of CHAF1A , which was presented within C - terminal . C : CHAF1A C - terminal .

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E GFP-tagged CHAF1A-N-NLS and CHAF1A-C were imaged with RFP-tagged CHAF1A respectively. N: CHAF1A N-terminal. NLS: nuclear localization signal of CHAF1A, which was presented within C-terminal. C: CHAF1A C-terminal.


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F Upper panel showed WT-CHAF1A bodies. Lower panel showed dLLPS-CHAF1A which was CHAF1A-Q34A-I116A-K395D-R402D-K409D.


G - I GFP - tagged CHAF1A - IDR CHAF1A - mIDR were purified in vitro . Ten micromole of each was incubated with gradient droplet formation buffer . droplets of CHAF1A - IDR appeared in NaCl of 125 mM or lower ( G ) . No CHAF1A - mIDR droplet was detected within any NaCl ( I ) . volumes of CHAF1A - IDR droplets increased with increase of ( H ) . Left subpanels within each panel represented of droplets in different buffers . Right subpanels within each panel represented statistical results of droplets areas numbers .

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G-I GFP-tagged CHAF1A-IDR and CHAF1A-mIDR were purified in vitro. Ten micromole of each protein was incubated with gradient droplet formation buffer. The droplets of CHAF1A-IDR appeared in NaCl concentration of 125 mM or lower (G). No CHAF1A-mIDR droplet was detected within any NaCl concentration (I). The volumes of CHAF1A-IDR droplets increased with the increase of protein concentration (H). Left subpanels within each panel represented images of protein droplets in different buffers. Right subpanels within each panel represented statistical analysis results of droplets areas and numbers.


B First panel : co - localization of PCNA CHAF1A . Second panel : distribution of PCNA in absence of endogenous CHAF1A . Third panel : distribution of PCNA in presence of CHAF1A - dPIP1 mutant absence of endogenous CHAF1A . Fourth panel : co - localization of PCNA over - expressed DNMT1 in absence of endogenous CHAF1A .

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B First panel: the co-localization of PCNA and CHAF1A. Second panel: the distribution of PCNA in the absence of endogenous CHAF1A. Third panel: the distribution of PCNA in the presence of CHAF1A-dPIP1 mutant and the absence of endogenous CHAF1A. Fourth panel: the co-localization of PCNA and over-expressed DNMT1 in the absence of endogenous CHAF1A.


C Upper two panels : distribution of HP1α in presence or absence of CHAF1A . Lower two panels : distribution of HP1α in presence of CHAF1A - dHP1BD or CHAF1A - DS2 - dHP1BD . Both of lower two panels were in absence of endogenous CHAF1A .

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C Upper two panels: the distribution of HP1α in the presence or absence of CHAF1A. Lower two panels: the distribution of HP1α in the presence of CHAF1A-dHP1BD or CHAF1A-DS2-dHP1BD. Both images of lower two panels were in the absence of endogenous CHAF1A.


distribution of SUV39H2 ( E ) MBD1 ( F ) in presence or absence of CHAF1A .

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The distribution of SUV39H2 (E) and MBD1 (F) in the presence or absence of CHAF1A.


G distribution of SUMO paralogs in absence of CHAF1A .

G The distribution of SUMO paralogs in the absence of CHAF1A.


A Flow cytometry figures of CAF - 1 rescue experiment . First figure : Wildtype J - Lat 10 . 6 . Second figure : CHAF1A knock - out J - Lat 10 . 6 . Third figure : CHAF1A - KO J - Lat 10 . 6 was re - introduced with wildtype CHAF1A . Fourth figure : CHAF1A - KO J - Lat 10 , 6 was re - introduced with dLLPS - CHAF1A which harbored five mutations . percentages of GFP - positive were labeled in top right corner of each figure .

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A Flow cytometry figures of CAF-1 body rescue experiment. First figure: Wildtype J-Lat 10.6. Second figure: CHAF1A knock-out J-Lat 10.6. Third figure: CHAF1A-KO J-Lat 10.6 was re-introduced with wildtype CHAF1A. Fourth figure: CHAF1A-KO J-Lat 10,6 was re-introduced with dLLPS-CHAF1A which harbored five mutations. The percentages of GFP-positive cells were labeled in the top right corner of each figure.


D amounts of intracellular HIV - 1 RNAs of latently infected CD4 + T which were isolated from PBMCs of HIV - 1 - infected individuals . αCD3 / αCD28 / IL - 2 treatment was performed as positive control . shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A . shluc lentiviruses treatment was performed as negative control . SAHA was added as LRA supplement .

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D The amounts of intracellular HIV-1 RNAs of latently infected primary CD4+ T cells which were isolated from PBMCs of HIV-1-infected individuals. αCD3/αCD28/IL-2 treatment was performed as positive control. shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A. shluc lentiviruses treatment was performed as negative control. SAHA was added as LRA supplement.


A Representative of fly eyes expressing ( CGG ) 90 - EGFP under a GMR - GAL4 driver , with various belle disruptions .

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A Representative photographs of fly eyes expressing (CGG)90-EGFP under a GMR-GAL4 driver, with various belle disruptions.


B Quantitation of GMR - GAL4 , ( CGG ) 90 - EGFP eye with belle disruptions ( Mann - Whitney U with Bonferonni corrections for multiple comparisons ; n = 35 - 77 / genotype ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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B Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with belle disruptions (Mann-Whitney U test with Bonferonni corrections for multiple comparisons; n=35-77/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


Longevity assays of ( CGG ) 90 - EGFP ; Tub5 - GS ( Log - rank Mantel - Cox with Bonferroni corrections for multiple comparisons ; n = 110 - 219 / genotype ) flies with belle knockdown . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


Longevity assays of ( CGG ) 90 - EGFP ; ElaV - GS ( n = 147 - 299 / genotype ) flies with belle knockdown . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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Longevity assays of (CGG)90-EGFP; ElaV-GS (n=147-299/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


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F Quantitation of FMRpolyG - EGFP band density , normalized to β tubulin band density , from blots in E ( Student ' s t ; n = 4 - 5 / genotype ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


G Abundance of ( CGG ) 90 - EGFP mRNA normalized to RPL32 mRNA , following belle knockdown , determined by qRT - PCR ( n = 8 / genotype ) .

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G Abundance of (CGG)90-EGFP mRNA normalized to RPL32 mRNA, following belle knockdown, determined by qRT-PCR (n=8/genotype).


H Western blot of AUG - driven EGFP in EGFP ; Tub5 - GS flies with without belle knockdown .

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H Western blot of AUG-driven EGFP in EGFP; Tub5-GS flies with and without belle knockdown.


I Quantitation of EGFP band density , normalized to β tubulin band density , from blot in H ( n = 4 / genotype ) .

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I Quantitation of EGFP band density, normalized to β tubulin band density, from blot in H (n=4/genotype).


A Dose - response curves showing effects of two independent anti - DDX3X siRNAs on expression of AUG - NL - 3xF ( top ) ( CGG ) 100 + 1 NL - 3xF ( bottom ) reporters . Plasmid - reporters were transfected into HeLa 24 hours after knockdown , reporter expression was quantified by luminescence . Nanoluciferase ( NL ) luminescence has been normalized to luminescence from firefly luciferase ( FF ) , which was co - transfected , in order to control for transfection variability . Asterisks refer to comparisons between anti - DDX3X siRNAs siRNAs against EGFP ( siEGFP ; two - way ANOVA with Dunnett ' s multiple comparisons ; n = 12 / condition ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

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A Dose-response curves showing the effects of two independent anti-DDX3X siRNAs on the expression of AUG-NL-3xF (top) and (CGG)100 +1 NL-3xF (bottom) reporters. Plasmid-based reporters were transfected into HeLa cells 24 hours after knockdown, and reporter expression was quantified by luminescence. Nanoluciferase (NL) luminescence has been normalized to luminescence from firefly luciferase (FF), which was co-transfected, in order to control for transfection variability. Asterisks refer to comparisons between anti-DDX3X siRNAs and siRNAs against EGFP (siEGFP; two-way ANOVA with Dunnett's multiple comparisons test; n=12/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


( CGG ) n + 1 NL - 3xF expression ( normalized to FF ) with without DDX3X knockdown across a range of CGG repeat sizes . Black asterisks refer to comparisons between siDDX3X - siEGFP - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing AUG - NL - 3xF those expressing a different reporter ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 17 - 30 / condition ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

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(CGG)n +1 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


( CGG ) n + 2 NL - 3xF expression ( normalized to FF ) with without DDX3X knockdown across a range of CGG repeat sizes . Black asterisks refer to comparisons between siDDX3X - siEGFP - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing AUG - NL - 3xF those expressing a different reporter ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 17 - 30 / condition ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

(CGG)n +2 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


Western blots of FMRpolyG - NL - 3xF products with without DDX3X knockdown across a range of repeat sizes .

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Western blots of FMRpolyG-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.


Western blots of FMRpolyA - NL - 3xF products with without DDX3X knockdown across a range of repeat sizes .

Western blots of FMRpolyA-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.


A Expression of in vitro transcribed AUG , + 1 ( CGG ) 100 , + 2 ( CGG ) 100 NL - 3xF RNAs following DDX3X knockdown in HeLa , expressed as NL luminescence normalized to FF luminescence . ( Student ' s t with Bonferroni corrections for multiple comparisons ; n = 21 / condition ) . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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A Expression of in vitro transcribed AUG, +1 (CGG)100, and +2 (CGG)100 NL-3xF RNAs following DDX3X knockdown in HeLa cells, expressed as NL luminescence normalized to FF luminescence. (Student's t test with Bonferroni corrections for multiple comparisons; n=21/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


B Abundance of reporter mRNAs following DDX3X knockdown plasmid - reporter transfection , determined by qRT - PCR . ( n = 7 / condition . ) This panel depicts data as means ± .

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B Abundance of reporter mRNAs following DDX3X knockdown and plasmid-reporter transfection, determined by qRT-PCR. (n=7/condition.) This panel depicts data as means ± SEM.


C Expression of AUG - NL - 3xF + 1 ( CGG ) 100 NL - 3xF in in vitro translation extracts , collected from HeLa treated with siRNAs against EGFP or DDX3X ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 4 / condition ) . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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C Expression of AUG-NL-3xF and +1 (CGG)100 NL-3xF in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X (two-way ANOVA with Tukey's multiple comparisons test; n=4/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


D Enrichment of HSPA1A + 1 ( CGG ) 100 NL - 3xF mRNA following anti - DDX3X RIP , relative to incubation with isotype control IgG . MALAT , in contrast , is not enriched ( Student ' s t , n = 3 ) .

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

D Enrichment of HSPA1A and +1 (CGG)100 NL-3xF mRNA following anti-DDX3X RIP, relative to incubation with isotype control IgG. MALAT RNA, in contrast, is not enriched (Student's t test, n=3).