( 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 .

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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 ) Cells were lysed and immunoblot performed with antibodies to CEBPB and 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 . 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 .

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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 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 .

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

(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 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 .

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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 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 .

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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 . 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 .

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

(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 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 .

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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 analysis performed using antibodies raised to CAMP and β - actin . Bottom , immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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

(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 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 .

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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 - 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 ) .

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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).


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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 animals 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 ) ( 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 .

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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 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 .

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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 staining in tumors from animals 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 IHC in two different cohorts of human prostatectomy samples . Representative pictures of benign and tumor 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 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 .

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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 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 .

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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 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 ) .

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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 and EGFR knockdown Raw 264 . 7 cells 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 and 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 and EGFR - / - HeLa cells 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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 ) 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 ) .

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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 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 ) .

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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 and 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 and EGFRfl + / - LysMCre + / + mice infected with HSV1 .

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


WT and EGFRfl + / - LysMCre + / + mice infected with HSV1 . HSV1 titers in the brains of the 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 and treated with gefitinib or DMSO before cGAMP transfection , 4h later STING was immunoprecipitated with anti - HA antibodies and 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 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 .

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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 ) 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 .

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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 cell lysates were immunoprecipitated with anti - STING antibody and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were transfected with cGAMP for 1h ; cell lysates were immunoprecipitated with anti - STING antibody and 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 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 .

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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 cell lysates were prepared at indicated time points after cGAMP transfection , immunoprecipitated with anti - EGFR antibodies and 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 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 .

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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 cells 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 cells were treated with cGAMP for the indicated time . Cell 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 cells were stimulated for 1h , cell lysates were immunoprecipitated with anti - STING antibody and 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 cells were treated with cGAMP for the indicated time . Cell lysates were subjected to Western blot using the 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 cells were transfected with cGAMP or Poly ( I : C ) ; after 3h , cell lysates were analyzed by Western blot using the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ; 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 ) .

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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 ) . 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

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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 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 .

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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 cells were pretreated with gefitinib and then treated with cGAMP . LC - MS / MS analysis 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 cells 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 . RNA was isolated from cells , expressing WT or Y245F STING , 3h after stimulation and subjected to qRT - PCR analysis ( 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 cells . Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the 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 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 .

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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 cells 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 cells .

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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 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 .

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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 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 ) .

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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 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 .

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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 and outer hair cell counts of Syne4 - / - organ of Corti at P8 , P10 , P12 , and 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 and Syne4 - / - organ of Corti . Top shows OHC plane and bottom shows IHC 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 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 .

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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 , 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 .

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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 Staining for FLAG at at P14 of the 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 Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope . Eight OHC were measured .

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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 the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining .

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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 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 .

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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 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 .

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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 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 .

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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 , and 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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and H3K9Acetyl were performed as in ( D ) . Only ' C ' position signals were showed and 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 and Pho - Pol II were performed in siNC and siCHAF1A J - Lat 10 . 6 cells . 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 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 .

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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 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 .

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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 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 .

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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 cells 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 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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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 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 .

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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 : 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 .

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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 : 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 .

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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.


The distribution of SUV39H2 ( E ) and MBD1 ( F ) in the 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 The distribution of SUMO paralogs in the absence of CHAF1A .

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


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 .

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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 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 .

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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 photographs 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 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 ) .

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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 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 ) .

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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 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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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 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 ) .

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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 and 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 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 ) .

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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 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 ) .

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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 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 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 and 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 and 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 , 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 ) .

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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 and plasmid - reporter transfection , determined by qRT - PCR . ( n = 7 / condition . ) This panel depicts data as means ± SEM .

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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 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 ) .

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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 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 ) .

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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).


E Expression of + 1 and + 2 ( CGG ) 100 NL - 3xF plasmid reporters with and without an AUG inserted 5 ' to the CGG repeat , with and without DDX3X knockdown . Black asterisks refer to comparisons between siDDX3X - and siEGFP - treated cells ; orange asterisks refer to comparisons between siDDX3X - treated cells expressing either + 1 or + 2 ( CGG ) 100 NL - 3xF and those expressing the respective AUG - driven variant ( two - way ANOVA with Tukey ' s multiple comparisons test ; n = 11 - 12 / 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 ) .

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E Expression of +1 and +2 (CGG)100 NL-3xF plasmid reporters with and without an AUG inserted 5' to the CGG repeat, with and without DDX3X knockdown. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing either +1 or +2 (CGG)100 NL-3xF and those expressing the respective AUG-driven variant (two-way ANOVA with Tukey's multiple comparisons test; n=11-12/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).