Figure 1 ( D and E ) qRT - PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence ( D ) or presence ( E ) of translation - blocking GFP MO . Bar charts show the average of three independent biological replicates . Error bars show standard deviation ( SD ) . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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Figure 1(D and E) qRT-PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence (D) or presence (E) of translation-blocking GFP MO. Bar charts show the average of three independent biological replicates. Error bars show standard deviation (SD). Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


Figure 3 ( B - E ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the codon effects measured by PACE ( y - axis ) . Each dot represents a single codon . Graphs for all codons ( B ) , codons for polar amino acids ( C ) , nonpolar amino acids ( D ) , and charged amino acids ( E ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test . ( F - I ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the tRNA levels ( y - axis , Each dot represents a single codon . Graphs for all codons ( F ) , codons for polar amino acids ( G ) , nonpolar amino acids ( H ) , and charged amino acids ( I ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test .

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

Figure 3(B-E) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the codon effects measured by PACE (y-axis). Each dot represents a single codon. Graphs for all codons (B), codons for polar amino acids (C), nonpolar amino acids (D), and charged amino acids (E) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.(F-I) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the tRNA levels (y-axis, Each dot represents a single codon. Graphs for all codons (F), codons for polar amino acids (G), nonpolar amino acids (H), and charged amino acids (I) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.


Figure 4 ( B ) Charged tRNA levels in mock - injected and AnsB - overexpressing embryos at 6 hpf . Total and charged tRNAs were measured by qRT - PCR coupled to the OXOPAP assay . The relative charged tRNA level in mock - injected embryos were set to one . Data information bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test . ( D ) Poly ( A ) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) . ( F ) Poly ( A ) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf . ( G ) Quantification of the PAT assay at 6 hpf in ( F ) .

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

Figure 4(B) Charged tRNA levels in mock-injected and AnsB-overexpressing embryos at 6 hpf. Total and charged tRNAs were measured by qRT-PCR coupled to the OXOPAP assay. The relative charged tRNA level in mock-injected embryos were set to one. Data information bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.(D) Poly(A) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).(F) Poly(A) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf. (G) Quantification of the PAT assay at 6 hpf in (F).


Figure 5 ( B ) Western blotting to detect the sfGFP levels at 6 hpf . Tubulin ( Tub ) was detected as a loading control . ( C ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos at 6 hpf relative to 2 hpf . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test . ( D ) Poly ( A ) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) . ( G ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf . ( H ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos , MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc - tagged full - length ( full ) or RING domain deleted ( ∆ RING ) Znf598 . mRNA levels at 6 hpf relative to 2 hpf are shown . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test . ( J ) Results of PACE in MZznf598 zebrafish embryos . Black circles show the relative stability of the reporter mRNAs ( averages of two biological replicates ) in MZznf598 embryos . Gray circles show the relative stability of reporter mRNAs in wild - type embryos The stability of a codon - tag reporter with a UGA stop codon was set to one . Error bars represent maximum and minimum data points . The relative effect of each codon on mRNA stability in wild - type embryos is shown as a color gradient from red ( destabilizing ) to blue ( stabilizing ) .

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

Figure 5(B) Western blotting to detect the sfGFP levels at 6 hpf. Tubulin (Tub) was detected as a loading control.(C) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos at 6 hpf relative to 2 hpf. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.(D) Poly(A) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).(G) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf. (H) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos, MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc-tagged full-length (full) or RING domain deleted (∆RING) Znf598. mRNA levels at 6 hpf relative to 2 hpf are shown. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.(J) Results of PACE in MZznf598 zebrafish embryos. Black circles show the relative stability of the reporter mRNAs (averages of two biological replicates) in MZznf598 embryos. Gray circles show the relative stability of reporter mRNAs in wild-type embryos The stability of a codon-tag reporter with a UGA stop codon was set to one. Error bars represent maximum and minimum data points. The relative effect of each codon on mRNA stability in wild-type embryos is shown as a color gradient from red (destabilizing) to blue (stabilizing).


Figure 6 ( B ) Results of the tandem ORF assay with Lys AAG × 8 and Lys AAA × 8 sequences . ( C ) Results of the tandem ORF assay with Gly × 8 and Pro × 8 sequences . ( D ) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags . ( E ) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities . Data information : normalized Fluc activity with no insert was set to one . All experiments were repeated three times , and the average Fluc activity is shown as bar charts . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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Figure 6(B) Results of the tandem ORF assay with Lys AAG×8 and Lys AAA×8 sequences. (C) Results of the tandem ORF assay with Gly×8 and Pro×8 sequences. (D) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags. (E) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities. Data information: normalized Fluc activity with no insert was set to one. All experiments were repeated three times, and the average Fluc activity is shown as bar charts. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


Figure 1X - ray analysis of 6 - months - old H2 - c - fosLTR / Egfrwa / + and H2 - c - fosLTR / Egfrwa / wa littermates . Scale bars : 1cmBone tumor number per mouse at 5 - 6 months of age ( n = 23 wa / + , 15 wa / wa ) Quantification of tumor size in tibiae at 5 - 6 months of age ( n = 23 wa / + , 15 wa / wa ) Alkaline Phosphatase ( ALP ) levels in the serum at endpoint ( age = 5 - 6 months ; n = 16 wa / + , 11 wa / wa ) X - ray analysis before ( age = 2 months ) and after ( age = 7 months ) vehicle or Erlotinib treatmentQuantification of tumor number during treatment ( n = 6 Vehicle , 5 Erlotinib ) Tumor size during treatment ( n = 6 Vehicle , 5 Erlotinib ) Analysis of serum ALP levels during treatment ( n = 5 ) PET summation images ( 0 - 90 min ) in horizontal ( upper panel ) and coronal view ( lower panel ) depicting [ 11C ] Erlotinib distribution in one H2 - c - fosLTR / Egfrwt mouse ( M125 ) . Anatomical structures are labelled with arrows ( T , tumor ; L , liver ; H , heart ; B , brain ) . Scale bars : 1cm Concentration - time curves of [ 11C ] Erlotinib in bone tumors in right scapula of three H2 - c - fosLTR / Egfrwt mice measured with PET . Broken line indicates threshold for in vitro effect of Erlotinib ( 1 µM )

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Figure 1X-ray analysis of 6-months-old H2-c-fosLTR/Egfrwa/+ and H2-c-fosLTR/Egfrwa/wa littermates. Scale bars: 1cmBone tumor number per mouse at 5-6 months of age (n=23 wa/+, 15 wa/wa)Quantification of tumor size in tibiae at 5-6 months of age (n=23 wa/+, 15 wa/wa)Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=16 wa/+, 11 wa/wa)X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatmentQuantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib)Tumor size during treatment (n=6 Vehicle, 5 Erlotinib)Analysis of serum ALP levels during treatment (n=5)PET summation images (0-90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [11C]Erlotinib distribution in one H2-c-fosLTR/Egfrwt mouse (M125). Anatomical structures are labelled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1cm Concentration-time curves of [11C]Erlotinib in bone tumors in right scapula of three H2-c-fosLTR/Egfrwt mice measured with PET. Broken line indicates threshold for in vitro effect of Erlotinib (1 µM)


Figure 2X - ray analysis of 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates . Scale bars : 1cmBone tumor number per mouse at 6 - 7 months of age ( n = 22 wt , 11 ∆ Ob ) Quantification of tumor size in tibiae ( n = 22 wt , 11 ∆ Ob ) ALP levels in the serum at 7 - month endpoint ( n = 17 wt , 8 ∆ Ob ) µPET / CT analysis of 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates Standardized uptake values ( SUV ) of the µPET - tracer Na [ 18F ] F in the pelvic osteosarcoma of 4 - and 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob mice . n = 6 wt , 4 ∆ Ob for 4 month time - point , n = 6 wt , 3 ∆ Ob for 7 month time -

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

Figure 2X-ray analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 1cmBone tumor number per mouse at 6-7 months of age (n=22 wt, 11 ∆Ob) Quantification of tumor size in tibiae (n=22 wt, 11 ∆Ob)ALP levels in the serum at 7-month endpoint (n=17 wt, 8 ∆Ob)µPET/CT analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates Standardized uptake values (SUV) of the µPET-tracer Na[18F]F in the pelvic osteosarcoma of 4- and 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice. n=6 wt, 4 ∆Ob for 4 month time-point, n=6 wt, 3 ∆Ob for 7 month time-point


Figure 3PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells ( for PCNA ) and as positive cells per mm² ( for cleaved Caspase 3 ) in OS from H2 - c - fosLTR / Egfrwt ( n = 6 ) and H2 - c - fosLTR / Egfr ∆ Ob ( n = 5 ) mice . Scale bars : 100µmEgfr , Ccnd1 , c - fos and transgenic c - fos ( c - fostg ) mRNA expression levels in tumors of H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob mice normalized to Tbp . n = 17 wt , 14 ∆ Ob ( Egfr , Ccnd1 ) , n = 16 wt , 13 ∆ Ob ( c - fos ) , n = 16 wt , 14 ∆ Ob ( c - fostgIHC staining and quantification showing pRSK2 ( n = 4 ) , pCREB ( n = 7 wt , 6 ∆ Ob ) and c - Fos ( n = 5 ) positive cells ( % ) in OS from H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates . Scale bars :

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

Figure 3PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm² (for cleaved Caspase 3) in OS from H2-c-fosLTR/Egfrwt (n=6) and H2-c-fosLTR/Egfr∆Ob (n=5) mice. Scale bars: 100µmEgfr, Ccnd1, c-fos and transgenic c-fos (c-fostg) mRNA expression levels in tumors of H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice normalized to Tbp. n=17 wt, 14 ∆Ob (Egfr, Ccnd1), n=16 wt, 13 ∆Ob (c-fos), n=16 wt, 14 ∆Ob (c-fostgIHC staining and quantification showing pRSK2 (n=4), pCREB (n=7 wt, 6∆Ob) and c-Fos (n=5) positive cells (%) in OS from H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 100µm


Figure 4Western Blot analysis of primary OS cells isolated from H2 ‑ c ‑ fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob micec - fos and transgenic c - fos ( c fostg ) mRNA expression levels in primary H2 - c - fosLTR OS cells after 4x in vitro passages , cultured under standard conditions ( n = 3 independent cell lines ) Western Blot analysis of H2 - c - fosLTR / Egfrwt OS cells treated for 24h with Erlotinibc - fos and c - fostg mRNA expression levels in H2 - c - fosLTR / Egfrwt OS cells treated for 24h with Erlotinib ( 10µM ) or DMSO as control ( n = 4 independent cell lines ) Western Blot analysis of starved H2 - c - fosLTR / Egfrwt OS cells , pre - treated with DMSO ( 1 : 1000 ) , Afatinib ( 5µM ) , GSK2233470 ( 10µM ) , Rapamycin ( 10nM ) or U0126 ( 10µM ) for 30 minutes and stimulated with EGF ( 50ng / ml ) as indicatedWestern Blot analysis of primary OS cells isolated from a p53f / f Rb1f / f Osx - Cre mouse after 24h Erlotinib treatmentc - fos mRNA expression levels in p53f / f Rb1f / f Osx - Cre OS cells treated for 24h with Erlotinib ( 10µM ) ( n = 3 ) Western Blot analysis of starved p53f / f Rb1f / f Osx - Cre OS cells , pre - treated with DMSO ( 1 : 1000 ) or Afatinib ( 5µM ) for 30 minutes and stimulated with EGF ( 50ng / ml ) as

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

Figure 4Western Blot analysis of primary OS cells isolated from H2‑c‑fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob micec-fos and transgenic c-fos (c fostg) mRNA expression levels in primary H2-c-fosLTR OS cells after 4x in vitro passages, cultured under standard conditions (n=3 independent cell lines)Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinibc-fos and c-fostg mRNA expression levels in H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib (10µM) or DMSO as control (n=4 independent cell lines)Western Blot analysis of starved H2-c-fosLTR/Egfrwt OS cells, pre-treated with DMSO (1:1000), Afatinib (5µM), GSK2233470 (10µM), Rapamycin (10nM) or U0126 (10µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicatedWestern Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatmentc-fos mRNA expression levels in p53f/f Rb1f/f Osx-Cre OS cells treated for 24h with Erlotinib (10µM) (n=3)Western Blot analysis of starved p53f/f Rb1f/f Osx-Cre OS cells, pre-treated with DMSO (1:1000) or Afatinib (5µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated


Figure 5X - ray analysis of 6 - months - old H2 - c - fosLTR and H2 - c - fosLTR / ColAREG littermates . Scale bars : 1cmBone tumor number per mouse at 5 - 6 months of age ( n = 22 wt , 13 ColAREG ) Quantification of tumor size in tibiae at 5 - 6 months of age ( n = 22 wt , 13 ColAREG ) Alkaline Phosphatase ( ALP ) levels in the serum at endpoint ( age = 5 - 6 months ; n = 29 wt , 19 ColAREG ) c - fos mRNA expression levels in osteosarcomas of H2 - c - fosLTR ( n = 11 ) and H2 - c - fosLTR / ColAREG mice ( n = 14 ) Western Blot analysis of bone tumor protein lysates from H2 - c - fosLTR and H2 - c - fosLTR / ColAREG

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Figure 5X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cmBone tumor number per mouse at 5-6 months of age (n=22 wt, 13 ColAREG)Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG)Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=29 wt, 19 ColAREG)c-fos mRNA expression levels in osteosarcomas of H2-c-fosLTR (n=11) and H2-c-fosLTR/ColAREG mice (n=14)Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice


Figure 6Representative images of OS biopsies stained with antibodies against EGFR and c ‑ Fos . Scale bars : 50µmKaplan - Meier survival curve comparing the survival of patients with EGFR and c - Fos double positive osteosarcomas against patients without co - expression of both proteins . ( n = 52 )

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Figure 6Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µmKaplan-Meier survival curve comparing the survival of patients with EGFR and c-Fos double positive osteosarcomas against patients without co-expression of both proteins. (n=52)


Figure 7Western Blot analysis showing EGFR and c - Fos protein levels in human OS cell lines cultured under standard conditionsCell viability of 143b or LM7 cells cultured for 24h in medium ( + 10 % FCS ) with DMSO ( 1 : 2000 ) or Erlotinib ( 10µM ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to DMSO ) Cell viability of 143b or LM7 cells cultured for 24h in medium ( + 0 . 5 % FCS ) containing 1xPBS ( contr . , 1 : 200 ) or EGF ( 50ng / ml ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to control ) 143b xenograft growth curve during therapeutic regime ( n = 8 vehicle , 6 Erlotinib ; two independent experiments ) 143b tumor weight at endpoint ( n = 8 Vehicle , 6 Erlotinib ; two independent experiments ) LM7 xenograft growth curve during therapeutic regime ( n = 7 Vehicle , 5 Erlotinib ; two independent experiments ) LM7 tumor weight at endpoint . n = 7 vehicle , 5 Erlotinib , two independent experimentIHC analysis of PCNA and cleaved Caspase 3 in 143b - derived tumors ( n = 4 ) . Scale bars : 50µm IHC analysis of pCREB and c - Fos in 143b - derived tumors . ( n = 5 for pCREB and n = 4 for c - Fos analysis ) . Scale bars : 50µmWestern Blot analysis of pCREB / CREB and p - c - Fos / c - Fos protein expression in lysates directly isolated from 143b xenografts at endpoint , after 14 days of vehicle or Erlotinib

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

Figure 7Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditionsCell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO)Cell viability of 143b or LM7 cells cultured for 24h in medium (+0.5% FCS) containing 1xPBS (contr., 1:200) or EGF (50ng/ml) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to control)143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments) 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments)LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments) LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experimentIHC analysis of PCNA and cleaved Caspase 3 in 143b-derived tumors (n=4). Scale bars: 50µm IHC analysis of pCREB and c-Fos in 143b-derived tumors. (n=5 for pCREB and n=4 for c-Fos analysis). Scale bars: 50µmWestern Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment


figf1 ( a ) Western blot analysis of HeLa cells treated with siRNA for 84 h . ( b ) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5 , ATG7 or NDP52 . GAPDH and total protein stained with Coomassie blue serve as loading controls . ( c ) Western blot analysis of HeLa cells cultured in Hank ' s buffered salt solution ( Starved ) or DMEM + 10 % FBS ( Serum ) . ( d ) Western blot analysis of HeLa cells treated ( 48 h ) with an inhibitor of mTORC1 ( RAP , 20 nM ) or control ( DMSO ) . ( e ) Western blot analysis comparing the levels of DICER in MDA - 231 , T47D and MDA - 435 cells . ( f ) Western blot analysis of MDA - 231 , T47D and MDA - 435 cells treated ( 48 h ) with inhibitors of mTORC1 ( RAP , 20 nM ) , mTORC1 and 2 ( PP242 , 100 nM ) , or DMSO ( control ) . ( g ) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h . ( h ) Western blot analysis of HeLa cells treated with BAF ( 200 nM , 12 h ) , RAP ( 20 nM , 24 h ) , or RAP and BAF . The results in a - h are representative of three experiments and use α - tubulin ( TUBA ) as a loading control . ( i ) Western blot analysis of DICER ( monoclonal antibody 13D6 ) or control ( EEF1A1 ) in HeLa cells treated with control siRNA or siRNA targeting DICER . ( j ) Confocal analysis of the signal from an anti - DICER monoclonal antibody ( clone 13D6 ) or an isotype control monoclonal antibody in HeLa cells . Scale bar , 10 μm . ( k , l ) RT - qPCR analysis of AGO1 , AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO , BAF or CQ ( k ) or siRNA for 84 h ( l ) . Error bars show s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

figf1(a) Western blot analysis of HeLa cells treated with siRNA for 84 h.(b) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5, ATG7 or NDP52. GAPDH and total protein stained with Coomassie blue serve as loading controls.(c) Western blot analysis of HeLa cells cultured in Hank's buffered salt solution (Starved) or DMEM+ 10% FBS (Serum).(d) Western blot analysis of HeLa cells treated (48 h) with an inhibitor of mTORC1 (RAP, 20 nM) or control (DMSO).(e) Western blot analysis comparing the levels of DICER in MDA-231, T47D and MDA-435 cells.(f) Western blot analysis of MDA-231, T47D and MDA-435 cells treated (48 h) with inhibitors of mTORC1 (RAP, 20 nM), mTORC1 and 2 (PP242, 100 nM), or DMSO (control).(g) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h.(h) Western blot analysis of HeLa cells treated with BAF (200 nM, 12 h), RAP (20 nM, 24 h), or RAP and BAF. The results in a-h are representative of three experiments and use α-tubulin (TUBA) as a loading control.(i) Western blot analysis of DICER (monoclonal antibody 13D6) or control (EEF1A1) in HeLa cells treated with control siRNA or siRNA targeting DICER.(j) Confocal analysis of the signal from an anti-DICER monoclonal antibody (clone 13D6) or an isotype control monoclonal antibody in HeLa cells. Scale bar, 10 μm.(k,l) RT-qPCR analysis of AGO1, AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO, BAF or CQ (k) or siRNA for 84 h (l). Error bars show s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


figf2 ( a ) Localization using confocal microscopy of endogenous DICER ( monoclonal antibody 13D6 ) and NDP52 in HeLa cells treated with RAP or control . The arrows highlight some examples of co - localization . The lower left insets show higher magnifications of the areas outlined in the main panel . Scale bars , 5 μm . ( b ) Quantification of DICER co - localization with NDP52 . Total fluorescence intensity of DICER co - localized with punctae ( > 0 . 2 μm3 ) of NDP52 was quantified with Volocity software in RAP - treated cells / control - treated cells over several Z - stacked microscope fields per experiment ( n = 4 , error bars s . e . m . ) . ( c ) Localization using confocal microscopy of endogenous DICER ( monoclonal antibody 13D6 ) and HcRed - LC3 in HeLa cells treated with BAF or control . The arrows highlight some examples of co - localization . Insets as in a . Scale bar , 5 μm . ( d ) Quantification of DICER co - localization with HcRed - LC3 was performed as for NDP52 in b . ( e ) Confocal microscopy analysis of GFP - DCP1A and HcRed - LC3wt in HeLa cells . Scale bar , 10 μm . ( f - i ) Localization of DICER ( monoclonal antibody 13D6 ) detected with anti - mouse IgG ( 10 nm gold beads ) by electron microscopy in CQ - ( 20 μM , 12 h ) treated HeLa cells . Mito , mitochondria ; MVB , multivesicular body . The black arrows highlight all gold beads . ( j ) Western blot analysis of fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( light AV ) and autophagolysosomes ( heavy AV ; ref . ) . HEK293T cells were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular body and lysosomal markers are indicated . Material that was pelleted by centrifugation at 100 , 000g ( 100 K pellet ) or that remained in solution after the 100 , 000g spin ( soluble ) was not added to the gradient . ( k ) Western blot analysis of UB and p62 in discontinuous Histodenz gradients ( as in j ) of HEK293T cells treated with control ( DMSO ) or CQ . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

figf2(a) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and NDP52 in HeLa cells treated with RAP or control. The arrows highlight some examples of co-localization. The lower left insets show higher magnifications of the areas outlined in the main panel. Scale bars, 5 μm. (b) Quantification of DICER co-localization with NDP52. Total fluorescence intensity of DICER co-localized with punctae (>0.2 μm3) of NDP52 was quantified with Volocity software in RAP-treated cells/control-treated cells over several Z-stacked microscope fields per experiment (n = 4, error bars s.e.m.).(c) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and HcRed-LC3 in HeLa cells treated with BAF or control. The arrows highlight some examples of co-localization. Insets as in a. Scale bar, 5 μm. (d) Quantification of DICER co-localization with HcRed-LC3 was performed as for NDP52 in b.(e) Confocal microscopy analysis of GFP-DCP1A and HcRed-LC3wt in HeLa cells. Scale bar, 10 μm.(f-i) Localization of DICER (monoclonal antibody 13D6) detected with anti-mouse IgG (10 nm gold beads) by electron microscopy in CQ- (20 μM, 12 h) treated HeLa cells. Mito, mitochondria; MVB, multivesicular body. The black arrows highlight all gold beads.(j) Western blot analysis of fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV; ref. ). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body and lysosomal markers are indicated. Material that was pelleted by centrifugation at 100,000g (100 K pellet) or that remained in solution after the 100,000g spin (soluble) was not added to the gradient.(k) Western blot analysis of UB and p62 in discontinuous Histodenz gradients (as in j) of HEK293T cells treated with control (DMSO) or CQ. Uncropped images of blots are shown in Supplementary Fig. S3.


figf3 ( a ) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz ( 15 % , 20 % , 24 % , 26 % ) described to enrich autophagosomes ( light AV ) and autophagolysosomes ( heavy AV ) . HEK293T cells were treated with CQ ( 20 μM , 16 h ) . Fractions enriched in multivesicular body ( MVB ) and lysosomal markers are indicated . ( b ) Western blot of GEMIN4 or loading control ( TUBA ) in HeLa cells treated with control siRNA or siRNA targeting ATG5 , ATG6 or ATG7 . ( c ) Western blot analysis of GEMIN4 or control ( TUBA ) in HeLa cells cultured in DMEM 10 % FBS ( Serum ) , Hank ' s buffered salt solution ( Starve ) for 2 h , or treated for 16 h with control ( DMSO ) , BAF ( 200 nM ) , RAP ( 20 nM ) , RAP + BAF , or PP242 ( 100 nM ) . ( d ) Top , western blot of GEMIN4 immunoprecipitates ( IP ) from HeLa cells . Bottom , western blot analysis of GEMIN4 in immunoprecipitates of anti - NDP52 , - p62 or control antibody from HeLa cells . ( e ) Western blot of DICER immunoprecipitates from HeLa cells . ( f ) Western blot of AGO2 immunoprecipitates from HeLa cells . ( g ) Western blot analysis of GEMIN4 , compared with loading controls ( DCP1A , CLN3 ) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4 . ( h ) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3 , GEMIN4 and ATG5 . Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells . ( i ) Western blot analysis with anti - UB monoclonal antibody ( mono - and polyubiquitylated proteins , FK2 clone ) in Flag immunoprecipitates performed under stringent denaturing conditions . Flag - AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet - inducible Flag - AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs . ( j ) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h . TUBA , α - tubulin loading control . ( k ) Top , RT - qPCR analysis of miR - 16 or let - 7a levels in AGO2 immunoprecipitates from DMSO - and BAF - treated HeLa cells . Error bars represent s . e . m . Bottom , a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

figf3(a) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body (MVB) and lysosomal markers are indicated.(b) Western blot of GEMIN4 or loading control (TUBA) in HeLa cells treated with control siRNA or siRNA targeting ATG5, ATG6 or ATG7.(c) Western blot analysis of GEMIN4 or control (TUBA) in HeLa cells cultured in DMEM 10% FBS (Serum), Hank's buffered salt solution (Starve) for 2 h, or treated for 16 h with control (DMSO), BAF (200 nM), RAP (20 nM), RAP+BAF, or PP242 (100 nM).(d) Top, western blot of GEMIN4 immunoprecipitates (IP) from HeLa cells. Bottom, western blot analysis of GEMIN4 in immunoprecipitates of anti-NDP52, -p62 or control antibody from HeLa cells.(e) Western blot of DICER immunoprecipitates from HeLa cells.(f) Western blot of AGO2 immunoprecipitates from HeLa cells.(g) Western blot analysis of GEMIN4, compared with loading controls (DCP1A, CLN3) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4.(h) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3, GEMIN4 and ATG5. Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells.(i) Western blot analysis with anti-UB monoclonal antibody (mono- and polyubiquitylated proteins, FK2 clone) in Flag immunoprecipitates performed under stringent denaturing conditions. Flag-AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet-inducible Flag-AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs.(j) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h. TUBA, α-tubulin loading control.(k) Top, RT-qPCR analysis of miR-16 or let-7a levels in AGO2 immunoprecipitates from DMSO- and BAF-treated HeLa cells. Error bars represent s.e.m. Bottom, a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments. Uncropped images of blots are shown in Supplementary Fig. S3.


figf4 ( a ) Northern blot of miR - 16 and let - 7a in HeLa cells treated with siRNA for 84 h . Levels of U6 , ethidium - bromide - stained total RNA on northern membrane ( loading ) and 28S and 18S ribosomal RNA on 1 % agarose gel are shown as controls for equal loading . ( b - d ) RT - qPCR analysis of let - 7a and miR - 16 ( b ) , pre - let - 7a and pre - miR - 16 ( c ) and let - 7a * and miR - 16 * ( d ) in HeLa cells depleted of NDP52 or ATG5 with siRNA ( 84 h ) . n = 3 independent experiments ; error bars represent s . e . m . ( e ) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let - 7a - let - 7a * labelled on the 3 ′ end of either let - 7 or let - 7a * with FITC . Cells were treated 16 h before with DMSO ( control ) , BAF ( 200 nm ) or geldanamycin ( 10 μM ) . Representative results of one experiment are shown . ( f ) Western blot analysis of AGO2 immunoprecipitates ( IP ) used in e . ( g ) Normalized results of four independent experiments ( n = 4 ) identical to e . Error bars represent s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

figf4(a) Northern blot of miR-16 and let-7a in HeLa cells treated with siRNA for 84 h. Levels of U6, ethidium-bromide-stained total RNA on northern membrane (loading) and 28S and 18S ribosomal RNA on 1% agarose gel are shown as controls for equal loading.(b-d) RT-qPCR analysis of let-7a and miR-16 (b), pre-let-7a and pre-miR-16 (c) and let-7a* and miR-16* (d) in HeLa cells depleted of NDP52 or ATG5 with siRNA (84 h). n = 3 independent experiments; error bars represent s.e.m.(e) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let-7a-let-7a* labelled on the 3′ end of either let-7 or let-7a* with FITC. Cells were treated 16 h before with DMSO (control), BAF (200 nm) or geldanamycin (10 μM). Representative results of one experiment are shown.(f) Western blot analysis of AGO2 immunoprecipitates (IP) used in e.(g) Normalized results of four independent experiments (n = 4) identical to e. Error bars represent s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


figf5 ( a ) Renilla luciferase expression controlled by an exogenous siRNA - siRNA * duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA ( black and grey bars correspond to two independent siRNAs ) . Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA - siRNA * duplex . n = 2 independent experiments . ( b ) Renilla luciferase expression controlled by let - 7 in HeLa cells depleted of NDP52 or ATG5 with siRNA ( black and grey bars are independent siRNAs ) . Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let - 7 binding sites . n = 3 independent experiments . The asterisks denote mutations in the let - 7 target sites of the reporter . ( c ) Western blot analysis of endogenous miRNA - repressed proteins ( RAS , HMGA2 and CDK6 are repressed by let - 7 ; CDK6 is also repressed by miR - 16 ( bottom panel ) ) or control ( TUBA , α - tubulin ) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were treated in parallel with nonspecific antagomir or let - 7 targeting antagomir . ( d ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with control antagomir or anti - let - 7 antagomir . ( e ) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were also treated with nonspecific antagomir or let - 7 targeting antagomir . ( f ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA . All error bars are s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

figf5(a) Renilla luciferase expression controlled by an exogenous siRNA-siRNA* duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA (black and grey bars correspond to two independent siRNAs). Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA-siRNA* duplex. n = 2 independent experiments.(b) Renilla luciferase expression controlled by let-7 in HeLa cells depleted of NDP52 or ATG5 with siRNA (black and grey bars are independent siRNAs). Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let-7 binding sites. n = 3 independent experiments. The asterisks denote mutations in the let-7 target sites of the reporter.(c) Western blot analysis of endogenous miRNA-repressed proteins (RAS, HMGA2 and CDK6 are repressed by let-7; CDK6 is also repressed by miR-16 (bottom panel)) or control (TUBA, α-tubulin) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were treated in parallel with nonspecific antagomir or let-7 targeting antagomir.(d) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with control antagomir or anti-let-7 antagomir.(e) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were also treated with nonspecific antagomir or let-7 targeting antagomir.(f) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA. All error bars are s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


Figure 1In vitro membrane pelleting assay comparing the TRAPPIII complex versus the core subunits . While both complexes exhibit a small amount of pelleting in the absence of synthetic liposomes , in their presence only the TRAPPIII complex is enriched in the membrane - bound fraction . ( S = supernatant , P = pellet ) Representative of n = 3 independent experiments . Quantification of D . Average % membrane binding between three assays is plotted , the error bars represent the 95 % confidence intervals . The value for each replicate is depicted . The negative values arise from subtraction of the average amount of protein that pellets in the absence of liposomes . Analyzed by unpaired , two - tailed Student ' s t - test with Welch ' s correction , * p = 0 . 0229 . n = 3 independent experiments .

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

Figure 1In vitro membrane pelleting assay comparing the TRAPPIII complex versus the core subunits. While both complexes exhibit a small amount of pelleting in the absence of synthetic liposomes, in their presence only the TRAPPIII complex is enriched in the membrane-bound fraction. (S=supernatant, P=pellet) Representative of n=3 independent experiments.   Quantification of D. Average % membrane binding between three assays is plotted, the error bars represent the 95% confidence intervals. The value for each replicate is depicted. The negative values arise from subtraction of the average amount of protein that pellets in the absence   of liposomes. Analyzed by unpaired, two-tailed Student's t-test with Welch's correction, * p = 0.0229. n=3 independent experiments.


Figure 4Representative Coomassie - stained gel of size exclusion chromatography fractions after purification of recombinant wild - type TRAPPIII complex . Representative of n = 3 independent experiments . As in A , using a Trs85 truncation containing only the final 198 amino acids . NOTE : Trs85 [ 501 - 698 ] is ~ 25kD in size and now migrates at the same size as some of the core subunits . The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct . Representative of n = 3 independent experiments . As in A / B , using a Trs85 mutant with a truncation of the loop from 575 - 603 . Trs85 [ ∆ 575 - 603 ] appears to dissociate from the core during chromatography , indicating a reduced affinity for the core subunits . Representative of n = 3 independent experiments . Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with the same Trs85 mutants . Representative of n = 3 independent experiments . ( WCE = whole cell extract , WT = wild - type , EV = empty vector ) Live cell fluorescence microscopy of Trs85 - mNeonGreen and mRFPmars - Ypt1 . Localization of Rab1 / Ypt1 to punctate structures is indicative of its activation by TRAPPIII . Scale bar is 2 microns . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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

Figure 4Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex. Representative of n=3 independent experiments.   As in A, using a Trs85 truncation containing only the final 198 amino acids. NOTE: Trs85[501-698] is ~25kD in size and now migrates at the same size as some of the core subunits. The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct. Representative of n=3 independent experiments.   As in A/B, using a Trs85 mutant with a truncation of the loop from 575-603. Trs85[∆575-603] appears to dissociate from the core during chromatography, indicating a reduced affinity for the core subunits. Representative of n=3 independent experiments.   Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)Live cell fluorescence microscopy of Trs85-mNeonGreen and mRFPmars-Ypt1. Localization of Rab1/Ypt1 to punctate structures is indicative of its activation by TRAPPIII. Scale bar is 2 microns. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


Figure 5E . Quantification of the colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents the MOC for a cropped image containing 2 - 9 cells . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons test , * * * * p < 0 . 0001 . F . Quantification of the colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . The sequence of wild - type Rab1 / Ypt1 HVD is in black , the substituted residues are colored to correspond with their location on the graph . The " CIM " motif is required for enzymatic prenylation of the Rab , but these chimeras are anchored to the plasma membrane independently of prenylation , via Fis1 - protein fusion . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents the MOC for a cropped image containing 2 - 9 cells . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons test , but significance indicators are only present for each substitution mutant compared to " wild - type " Ypt31HVDYpt1 : vs . 181TTQ p = 0 . 9924 , vs . 184KKE p = 0 . 9997 , vs . 187DKGN * p = 0 . 0145 , vs . 191VNL , 194KGQS , 198LTNT * * * * p < 0 . 0001 , ns = not significant .

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

Figure 5E. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, **** p < 0.0001. F. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. The sequence of wild-type Rab1/Ypt1 HVD is in black, the substituted residues are colored to correspond with their location on the graph. The "CIM" motif is required for enzymatic prenylation of the Rab, but these chimeras are anchored to the plasma membrane independently of prenylation, via Fis1-protein fusion. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, but significance indicators are only present for each substitution mutant compared to "wild-type" Ypt31HVDYpt1: vs. 181TTQ p = 0.9924, vs. 184KKE p = 0.9997, vs. 187DKGN * p = 0.0145, vs. 191VNL, 194KGQS, 198LTNT **** p < 0.0001, ns = not significant.


Figure 6Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with the same Trs85 substitution mutants . Representative of n = 3 independent experiments . ( WCE = whole cell extract , WT = wild - type , EV = empty vector ) Live cell fluorescence microscopy of Trs85 - mNeonGreen substitution mutants and mRFPmars - Ypt1 . Scale bar = 2 μm . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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

Figure 6Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 substitution mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)Live cell fluorescence microscopy of Trs85-mNeonGreen substitution mutants and mRFPmars-Ypt1. Scale bar = 2 μm. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


Figure 7GFP - Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix ( membrane binding ) substitutions . With functional Trs85 , there is an accumulation of free GFP . Samples are from pre - rapamycin treatment ( - ) and 1hr after addition of rapamycin ( + ) . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector ) Quantification of A . The average of at least three replicates is plotted with error bars representing the 95 % confidence interval . The percent of free GFP for wild - type Trs85 is compared to each mutant using an unpaired , two - tailed Student ' s t - test with Welch ' s correction : vs . EV * * p = 0 . 0022 , vs . R618E , R619E , R620E , K621E * * * p = 0 . 0008 , vs . R618A , R619A , R620A , K621A * * * p = 0 . 0006 , vs . 501 - 698 * * * p = 0 . 0003 , vs . 4A p = 0 . 3295 , vs . 7A * * * p = 0 . 0008 , vs . 4E * * * p = 0 . 0005 , ns = not significant . ( WT = wild - type , EV = empty vector )

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

Figure 7GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions. With functional Trs85, there is an accumulation of free GFP. Samples are from pre-rapamycin treatment (-) and 1hr after addition of rapamycin (+). Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)   Quantification of A. The average of at least three replicates is plotted with error bars representing the 95% confidence interval. The percent of free GFP for wild-type Trs85 is compared to each mutant using an unpaired, two-tailed Student's t-test with Welch's correction: vs. EV ** p =   0.0022, vs. R618E, R619E, R620E, K621E *** p = 0.0008, vs. R618A, R619A, R620A, K621A *** p = 0.0006, vs. 501-698 *** p = 0.0003, vs. 4A p = 0.3295, vs. 7A *** p = 0.0008, vs. 4E *** p = 0.0005, ns = not significant. (WT = wild-type, EV = empty vector)


figf1 ( a ) Activation of the gB - specific CD8 + T cell hybridoma ( which expresses β - galactosidase as an indicator of T cell activation ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , then incubated for 12 h at 37 ° C with the hybridoma . A595 , absorbance at 595 nm . b ) Activation of the hybridoma as described in a , with the addition of dimethyl sulfoxide ( DMSO ; negative control ) , bafilomycin A ( Baf ) , brefeldin A ( BFA ) or MG - 132 at 2 h after macrophage infection . ( c ) Activation of the hybridoma as described in a , with the addition of bafilomycin A at 2 h after macrophage infection . ( d ) Immunofluorescence microscopy of gB ( blue ) and LAMP - 1 ( red ) in HSV - 1 - infected macrophages ; pink indicates colocalization . Original magnification , × 40 . ( e ) CD8 + T cell - stimulatory capacity ( as described in a ) of uninfected macrophages ( Mock ) , of BMA ( BMA - HSV ) or J774 ( J774 - HSV ) macrophages infected for 8 h with HSV - 1 , and of cocultures of J774 macrophages ( H - 2d ) infected for 1 h with HSV - 1 , then mixed with uninfected BMA ( H - 2b ) macrophages at a ratio of 1 : 1 and cultured together for 8 h ( J774 - HSV + BMA ) .

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

figf1(a) Activation of the gB-specific CD8+ T cell hybridoma (which expresses β-galactosidase as an indicator of T cell activation) by macrophages infected for various times (horizontal axis) with HSV-1, then incubated for 12 h at 37 °C with the hybridoma. A595, absorbance at 595 nm.b) Activation of the hybridoma as described in a, with the addition of dimethyl sulfoxide (DMSO; negative control), bafilomycin A (Baf), brefeldin A (BFA) or MG-132 at 2 h after macrophage infection.(c) Activation of the hybridoma as described in a, with the addition of bafilomycin A at 2 h after macrophage infection.(d) Immunofluorescence microscopy of gB (blue) and LAMP-1 (red) in HSV-1-infected macrophages; pink indicates colocalization. Original magnification, × 40.(e) CD8+ T cell-stimulatory capacity (as described in a) of uninfected macrophages (Mock), of BMA (BMA-HSV) or J774 (J774-HSV) macrophages infected for 8 h with HSV-1, and of cocultures of J774 macrophages (H-2d) infected for 1 h with HSV-1, then mixed with uninfected BMA (H-2b) macrophages at a ratio of 1:1 and cultured together for 8 h (J774-HSV + BMA).


figf2 ( a ) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2 - 8 h ( left margin ) with HSV - 1 . Original magnification , × 20 . ( b ) Activation of the gB - specific CD8 + T cell hybridoma ( described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , with ( DMEM + 3 - MA ) or without ( DMEM ) the addition of 3 - methyladenine 2 h after infection , then incubated for 12 h at 37 ° C with the hybridoma . ( c ) Activation of the hybridoma ( as described in b ) by macrophages transfected for 60 h with control ( Ctrl ) siRNA or Atg5 - specific siRNA , then infected for 8 h or 12 h with HSV - 1 . ( d ) Immunoblot analysis of Atg - 5 in siRNA - treated macrophages . ( e ) Activation of the hybridoma ( as described in b ) by macrophages infected with HSV - 1 and incubated at 37 ° C ( Basal ) , incubated for 12 h at 39 ° C before being infected with HSV - 1 ( heat shock ( HS ) ) , or treated with rapamycin during HSV - 1 infection ( Rapa ) , with ( + Baf ) or without the addition of bafilmycin A at 2 h after infection . ( f ) Activation of the hybridoma ( as described in b ) by macrophages transfected for 60 h with control siRNA or Atg5 - specific siRNA , then infected for 8 h with HSV - 1 , with ( + ) or without ( − ) the addition of rapamycin at 2 h after infection . Results in b , c , e , f are normalized to results obtained for CD8 + T cells stimulated with macrophages infected for 8 h at 37 ° C without further treatment ( b , e ) or infected macrophages treated with control siRNA ( c , f ) and are presented in arbitrary units . Data are representative of three independent experiments ( a , d ) or are from three independent experiments ( mean and s . e . m . of triplicate samples ; b , c , e , f ) .

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

figf2(a) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2-8 h (left margin) with HSV-1. Original magnification, × 20.(b) Activation of the gB-specific CD8+ T cell hybridoma (described in Fig. 1a) by macrophages infected for various times (horizontal axis) with HSV-1, with (DMEM + 3-MA) or without (DMEM) the addition of 3-methyladenine 2 h after infection, then incubated for 12 h at 37 °C with the hybridoma.(c) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control (Ctrl) siRNA or Atg5-specific siRNA, then infected for 8 h or 12 h with HSV-1.(d) Immunoblot analysis of Atg-5 in siRNA-treated macrophages. (e) Activation of the hybridoma (as described in b) by macrophages infected with HSV-1 and incubated at 37 °C (Basal), incubated for 12 h at 39 °C before being infected with HSV-1 (heat shock (HS)), or treated with rapamycin during HSV-1 infection (Rapa), with (+ Baf) or without the addition of bafilmycin A at 2 h after infection.  (f) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control siRNA or Atg5-specific siRNA, then infected for 8 h with HSV-1, with (+) or without (−) the addition of rapamycin at 2 h after infection. Results in b,c,e,f are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 8 h at 37 °C without further treatment (b,e) or infected macrophages treated with control siRNA (c,f) and are presented in arbitrary units. Data are representative of three independent experiments (a,d) or are from three independent experiments (mean and s.e.m. of triplicate samples; b,c,e,f).


figf3 ( a - c ) Immunofluorescence microscopy of uninfected macrophages incubated at 37 ° C ( control ; a ) , subjected to mild heat shock ( b ) or treated with rapamycin ( c ) , then stained with anti - LC3 . d ) Immunofluorescence microscopy of the expression of LC3 , gB and GFP ( VP26 ) by macrophages infected for various times ( left margin ) with HSV - 1 . White indicates colocalization . ( e ) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h ( left margin ) with wild - type HSV - 1 ( WT ) or HSV - 1 lacking ICP34 . 5 ( Δ34 . 5 ) . Blue , staining of nuclei with DAPI ( 4 , 6 - diamidino - 2 - phenylindole ) . Original magnification , × 100 ( a - c ) or × 63 ( d , e ) . Results in a - e are representative of three independent experiments . ( f ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with wild - type HSV - 1 strain 17 + ( WT 17 + ) or Δ34 . 5 HSV - 1 . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) . ( g , h ) Immunoblot analysis ( IB ; g ) and flow cytometry ( h ) of the expression of HSV - 1 proteins ( g ) and gB ( h ) in macrophages infected for various times ( above lanes ( g ) or plots ( h ) ) with wild - type or Δ34 . 5 HSV - 1 . Ctrl , control ( uninfected BMA macrophages ; h ) . Data are representative of two ( g ) or three ( h ) independent experiments . ( i ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( below graph ) with wild - type or Δ34 . 5 HSV - 1 , with ( + ) or without ( - ) the addition of bafilomycin A at 2 h after infection . Results in f , i are normalized to results obtained for CD8 + T cells stimulated with macrophages infected for 6 h with wild - type virus ( f ) or with infected macrophages incubated without bafilomycin ( i ) and are presented in arbitrary units . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

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

figf3 (a-c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3. d) Immunofluorescence microscopy of the expression of LC3, gB and GFP (VP26) by macrophages infected for various times (left margin) with HSV-1. White indicates colocalization. (e) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h (left margin) with wild-type HSV-1 (WT) or HSV-1 lacking ICP34.5 (Δ34.5). Blue, staining of nuclei with DAPI (4,6-diamidino-2-phenylindole). Original magnification, × 100 (a-c) or × 63 (d,e). Results in a-e are representative of three independent experiments. (f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (horizontal axis) with wild-type HSV-1 strain 17+ (WT 17+) or Δ34.5 HSV-1. Data are from three independent experiments (mean and s.e.m. of triplicate samples). (g,h) Immunoblot analysis (IB; g) and flow cytometry (h) of the expression of HSV-1 proteins (g) and gB (h) in macrophages infected for various times (above lanes (g) or plots (h)) with wild-type or Δ34.5 HSV-1. Ctrl, control (uninfected BMA macrophages; h). Data are representative of two (g) or three (h) independent experiments.  (i) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (below graph) with wild-type or Δ34.5 HSV-1, with (+) or without (-) the addition of bafilomycin A at 2 h after infection. Results in f,i are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 6 h with wild-type virus (f) or with infected macrophages incubated without bafilomycin (i) and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


figf4 Electron microscopy of macrophages 10 h after infection with HSV - 1 . ( a ) Arrows indicate membrane - coiled structures emerging from the nucleus of an infected cell . ( b - d ) Four - layered membrane structures formed by coiling of the nuclear membrane . ( e , f ) Glucose - 6 - phosphatase ( black deposits ) on autophagosome - like structures emerging from the nuclear envelope or free in the cytoplasm , and viral capsids in the cytoplasm engulfed in the lumen of an autophagosome - like compartment . N , nucleus ; VP , viral particles . Scale bars , 1 μm ( a ) , 0 . 25 μm ( b - d , f ) or 0 . 4 μm ( e ) . Images are representative of three independent experiments with at least 100 cell profiles in each .

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

figf4 Electron microscopy of macrophages 10 h after infection with HSV-1. (a) Arrows indicate membrane-coiled structures emerging from the nucleus of an infected cell. (b-d) Four-layered membrane structures formed by coiling of the nuclear membrane. (e,f) Glucose-6-phosphatase (black deposits) on autophagosome-like structures emerging from the nuclear envelope or free in the cytoplasm, and viral capsids in the cytoplasm engulfed in the lumen of an autophagosome-like compartment. N, nucleus; VP, viral particles. Scale bars, 1 μm (a), 0.25 μm (b-d,f) or 0.4 μm (e). Images are representative of three independent experiments with at least 100 cell profiles in each.


figf5 Immunoelectron microscopy of macrophages 10 h after infection with HSV - 1 . ( a - c ) Accumulation of LC3 ( a , b ) and gB ( c ) . ( d ) Fusion of four - layered membrane structures and lysosomes preloaded with bovine serum albumin - gold ( BSA - gold ; black dots ) . Original magnification , × 54 , 800 ( a , b ) , × 69 , 000 ( c ) or × 38 , 000 ( d ) . Images are representative of three ( a - c ) or two ( d ) independent experiments .

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figf5 Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a-c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin-gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a-c) or two (d) independent experiments.


figf6 ( a ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages exposed to DMSO ( negative control ) , mild heat shock , IL - 1β or IFN - γ . ( b ) Dansylcadaverin staining of untreated macrophages ( Basal ) or macrophages exposed to mild heat shock , IFN - γ or IL - 1β and infected for 8 h with wild - type HSV - 1 , normalized to the signal obtained in basal conditions and presented in arbitrary units . ( c - f ) Activation of the gB - specific CD8 + T cell hybridoma ( as described in Fig . 1a ) by macrophages incubated at 37 ° C ( c ) or exposed to mild heat shock ( d ) , IL - 1β ( e ) or IFN - γ ( f ) and infected for 8 h with wild - type HSV - 1 with the addition of DMSO ( negative control ) , 3 - methyladenine , bafilomycin , brefeldin A or MG - 132 at 2 h after infection . Results in a , c - f are normalized to results obtained for CD8 + T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units . Data are from three independent experiments ( mean and s . e . m . of triplicate samples ) .

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figf6 (a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages exposed to DMSO (negative control), mild heat shock, IL-1β or IFN-γ.  (b) Dansylcadaverin staining of untreated macrophages (Basal) or macrophages exposed to mild heat shock, IFN-γ or IL-1β and infected for 8 h with wild-type HSV-1, normalized to the signal obtained in basal conditions and presented in arbitrary units.  (c-f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages incubated at 37 °C (c) or exposed to mild heat shock (d), IL-1β (e) or IFN-γ (f) and infected for 8 h with wild-type HSV-1 with the addition of DMSO (negative control), 3-methyladenine, bafilomycin, brefeldin A or MG-132 at 2 h after infection. Results in a,c-f are normalized to results obtained for CD8+ T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


Figure 1Viability curve of cells following 24h of recombinant Pol II expression induction by removal of tetracycline ( Tet - Off system ) and treatment with α - amanitin ( black arrow - head ) . Untransfected Raji ( WT : black ) is shown for reference . For all experiments presented in this article , cells are collected after 24 hours of α - amanitin treatment for CTD - ∆ 5 and YFFF mutants expressing cells since CTD - ∆ 5 is unstable after 48h ( see panel C ) . Error bars representing top and bottom values are based on the average of 3 independent experiments . Western blot analysis of the recombinant RPB1 expression ( rWT , YFFF , and ∆ 5 ) assessed by ( HA ) - tag expression after 24 or 48 hours of α - amanitin treatment following 24h of induction . Untransfected Raji extracts are shown for comparison ( WT ) . Total RPB1 levels are represented by the F12 western blot . Tubulin was used as a loading control . Volcano plot of differentially expressed genes in chromatin associated RNA - seq ( ChrRNA ) data sets : protein coding genes UP ( purple ) and DOWN ( pink ) regulated in B relative to A after 24h hours α - amanitin treatment ( n = 22810 ) . Experiments were done in biological duplicates . Threshold : log2 fold change ≥ 3 , p - value < 0 . 05 .

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Figure 1Viability curve of cells following 24h of recombinant Pol II expression induction by removal of tetracycline (Tet-Off system) and treatment with α-amanitin (black arrow-head). Untransfected Raji (WT: black) is shown for reference. For all experiments presented in this article, cells are collected after 24 hours of α-amanitin treatment for CTD-∆5 and YFFF mutants expressing cells since CTD-∆5 is unstable after 48h (see panel C). Error bars representing top and bottom values are based on the average of 3 independent experiments.Western blot analysis of the recombinant RPB1 expression (rWT, YFFF, and ∆5) assessed by (HA)-tag expression after 24 or 48 hours of α-amanitin treatment following 24h of induction. Untransfected Raji extracts are shown for comparison (WT). Total RPB1 levels are represented by the F12 western blot. Tubulin was used as a loading control.Volcano plot of differentially expressed genes in chromatin associated RNA-seq (ChrRNA) data sets: protein coding genes UP (purple) and DOWN (pink) regulated in B relative to A after 24h hours α-amanitin treatment (n=22810). Experiments were done in biological duplicates. Threshold: log2 fold change ≥ 3, p-value < 0.05.


Figure 2Example of ChrRNA read - through phenotype at the 5 ' ( anti - sense ) and 3 ' ( sense ) ends in CTD - ∆ 5 at the GBE1 locus . Boxplots of ChrRNA read - through ( RT ) indexes based on signal over 20kb upstream ( anti - sense ) and 20kb downstream ( sense ) regions of coding genes in rWT ( dotted grey line ) and CTD - ∆ 5 mutant ( blue ) . Units are asinh transformed . Experiments were done in biological duplicates . Boxplots represent minimal and maximal values , first and third quartiles with median value as central band . P - values were calculated using two - sided Wilcoxon tests .

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Figure 2Example of ChrRNA read-through phenotype at the 5' (anti-sense) and 3' (sense) ends in CTD-∆5 at the GBE1 locus.Boxplots of ChrRNA read-through (RT) indexes based on signal over 20kb upstream (anti-sense) and 20kb downstream (sense) regions of coding genes in rWT (dotted grey line) and CTD-∆5 mutant (blue). Units are asinh transformed. Experiments were done in biological duplicates. Boxplots represent minimal and maximal values, first and third quartiles with median value as central band. P-values were calculated using two-sided Wilcoxon tests.


Figure 3Example of Pol - HA ChIP - seq showing global spreading of Pol II along the genome accompanied by a loss of Pol - II accumulation on coding genes in CTD - ∆ 5 ( orange ) compared to rWT ( black ) and their associated ChrRNA - signal . Average metagene profiles comparing Pol - II , H3K4me3 and H3K4me1 signals on active promoters ( top ) and active enhancers ( bottom ) in rWT and CTD - ∆ 5 . Experiments were done in biological duplicates however only the sample with the best signal to noise ratio was used for subsequent analysis . P - values were calculated using two - sided Wilcoxon tests . Example of Pol - HA , H3K4me3 and H3K4me1 Chip - seq signals in rWT and CTD - ∆ 5 illustrating the metagene profiles in figure 3B . Top : Light green rectangles highlight putative active enhancers in rWT .

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Figure 3Example of Pol-HA ChIP-seq showing global spreading of Pol II along the genome accompanied by a loss of Pol-II accumulation on coding genes in CTD-∆5 (orange) compared to rWT (black) and their associated ChrRNA-signal.Average metagene profiles comparing Pol-II, H3K4me3 and H3K4me1 signals on active promoters (top) and active enhancers (bottom) in rWT and CTD-∆5. Experiments were done in biological duplicates however only the sample with the best signal to noise ratio was used for subsequent analysis. P-values were calculated using two-sided Wilcoxon tests.Example of Pol-HA, H3K4me3 and H3K4me1 Chip-seq signals in rWT and CTD-∆5 illustrating the metagene profiles in figure 3B. Top: Light green rectangles highlight putative active enhancers in rWT.


Figure 4Example showing ChrRNA - seq ( top ) and its associated PolyA RNA - seq signals ( bottom ) in rWT and CTD - ∆ 5 . Boxplots of PolyA read - through ( RT ) indexes based on signal over 20kb upstream ( anti - sense ) and 20kb downstream ( sense ) regions of coding genes in rWT ( dotted grey line ) and CTD - ∆ 5 mutant ( blue ) . Units are asinh transformed . Experiments were done in biological duplicates . Boxplots represent minimal and maximal values , first and third quartiles with median value as central band . P - values were calculated using two - sided Wilcoxon tests .

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Figure 4Example showing ChrRNA-seq (top) and its associated PolyA RNA-seq signals (bottom) in rWT and CTD-∆5.Boxplots of PolyA read-through (RT) indexes based on signal over 20kb upstream (anti-sense) and 20kb downstream (sense) regions of coding genes in rWT (dotted grey line) and CTD-∆5 mutant (blue). Units are asinh transformed. Experiments were done in biological duplicates. Boxplots represent minimal and maximal values, first and third quartiles with median value as central band. P-values were calculated using two-sided Wilcoxon tests.


Figure 5Volcano plot comparing the Pol II interactome in rWT and CTD - ∆ 5 mutant . Left : Proteins lost in CTD - ∆ 5 , Right : Proteins enriched in CTD - ∆ 5 . Highlighted are the subunits of the Mediator ( purple ) and Integrator complexes ( orange ) CTD phosphatases scaffolds ( pink ) , Ubiquitin ligases ( light blue ) and SOSS complex subunits ( red ) . In black are highlighted some chosen interactors gained by the CTD - ∆ 5 Threshold : log2 fold change ≥ 4 ; p - value < 0 . 05 . Data based on three independent biological replicates . The table in the figure sums up the proteins that do not change significantly ; dotted lines connected to the table represent the region where these proteins are found in the

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Figure 5Volcano plot comparing the Pol II interactome in rWT and CTD-∆5 mutant. Left: Proteins lost in CTD-∆5, Right: Proteins enriched in CTD-∆5. Highlighted are the subunits of the Mediator (purple) and Integrator complexes (orange) CTD phosphatases scaffolds (pink), Ubiquitin ligases (light blue) and SOSS complex subunits (red). In black are highlighted some chosen interactors gained by the CTD-∆5 Threshold: log2 fold change ≥ 4; p-value < 0.05. Data based on three independent biological replicates. The table in the figure sums up the proteins that do not change significantly; dotted lines connected to the table represent the region where these proteins are found in the volcano


Figure 6Example of CTD - ∆ 5 polymerases transcribing through TAD borders marked by CTCF sites ( highlighted in light purple ) . HiC map on top represents the rWT interactions shown in the zoom in panel A . Purple boxes represent TADs called in human B cells CTCF ChIP - seq in rWT and CTD - ∆ 5 are both shown in black and their associated ChrRNA - seq signal are also shown .

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Figure 6Example of CTD-∆5 polymerases transcribing through TAD borders marked by CTCF sites (highlighted in light purple). HiC map on top represents the rWT interactions shown in the zoom in panel A. Purple boxes represent TADs called in human B cells CTCF ChIP-seq in rWT and CTD-∆5 are both shown in black and their associated ChrRNA-seq signal are also shown.


Figure 2Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of Ca2 + monitored at 280 nm absorption wavelength . Size exclusion chromatography ( SEC ) results for MICU2 _ WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength .

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Figure 2Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.


Figure 3MICU2 _ WT ( A ) , MICU2 _ K172A ( B ) were pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + . MICU2 _ EF1mut was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + ( C ) , while MICU2 _ EF2mut was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( D ) . MICU2 _ R352A was not pulled - down by GST - MICU1 in the absence of 2 mM Ca2 + ( E ) , while MICU2 _ E329A was pulled - down by GST - MICU1 both in the absence and presence of 2 mM Ca2 + ( F ) . MICU2 _ D185A was partially pulled - down by GST - MICU1 in the presence of Ca2 + , while they showed a strong interaction in the absence of 2 mM Ca2 + . MICU2 _ E196K was not pulled - down by GST - MICU1 in the presence of 2 mM Ca2 + , while a strong interaction was observed in the absence of 2 mM Ca2 + .

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Figure 3MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+. MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D). MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F). MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+. MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.


Figure 4GST - MICU1 _ EF1mut failed to pull down MICU2 _ WT in the absence of 2 mM Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A & H385A failed to pull - down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . C GST - MICU1 _ E242K failed to pull - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ D231A partly pulled - down MICU2 _ WT in the absence of Ca2 + , whereas they displayed a strong interaction in the presence of 2 mM Ca2 + . GST - MICU1 _ F383A failed to pull down MICU2 _ WT in the presence of Ca2 + , whereas they displayed a strong interaction in the absence of 2 mM Ca2 + . GST - MICU1 _ H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2 + . ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA ( G ) and CaCl2 ( H ) .

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Figure 4GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA (G) and CaCl2 (H).


Figure 1 ( A ) Anti - NANP5 and anti - C - CSP serum IgG . Circles represent ELISA area under the curve ( AUC ) values for individual donors ( n = 12 ) . ( B ) Ten most frequent Ig heavy and light chain V gene pairs in PfCSP - reactive memory B cells ( MBCs ; n = 1172 ) . somatic hypermutation ( SHM ) count ( E ) in IGHV3 - 21 and IGHV3 - 33 genes from MBCs ( F ) NANP10 ( left ) and C - CSP ( right ) binding of FL - CSP - reactive mAbs ( n = 177 ) . Circles represent ELISA AUC values for each mAb .

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Figure 1(A) Anti-NANP5 and anti-C-CSP serum IgG. Circles represent ELISA area under the curve (AUC) values for individual donors (n=12). (B) Ten most frequent Ig heavy and light chain V gene pairs in PfCSP-reactive memory B cells (MBCs; n=1172).somatic hypermutation (SHM) count (E) in IGHV3-21 and IGHV3-33 genes from MBCs(F) NANP10 (left) and C-CSP (right) binding of FL-CSP-reactive mAbs (n=177). Circles represent ELISA AUC values for each mAb.


Figure 2 ( C ) VH SHM load of VH3 - 21 and non - VH3 - 21 mAbs . mAbs with unmutated VH are highlighted in green . ( D ) Amino acid ( aa ) VH replacement ( red bars ) and silent ( black bars ) SHM in VH3 - 21 mAbs ( n = 113 ) . FWR , framework region ; CDR , complementarity - determining region .

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Figure 2(C) VH SHM load of VH3-21 and non-VH3-21 mAbs. mAbs with unmutated VH are highlighted in green.(D) Amino acid (aa) VH replacement (red bars) and silent (black bars) SHM in VH3-21 mAbs (n=113). FWR, framework region; CDR, complementarity-determining region.


Figure 3 ( D ) C - CSP reactivity of the α - TSR - specific mAb 1710 in a blocking ELISA with C - CSP - specific VH3 - 21 ( n = 21 ; upper panel ) or non - VH3 - 21 ( n = 17 ; lower panel ) mAbs with the indicated SPR affinities . mAb 1710 binding without blocking ( blue ) and after self - blocking ( gold ) is shown for comparison . ( E ) C - CSP reactivity of mAb 1512 in a blocking ELISA with mAbs ( with the indicated SPR affinities ) that do not block C - CSP binding of mAb 1710 ( open circles ; D ) . mAb 1512 binding without blocking ( purple ) and after self - blocking ( magenta ) is shown for comparison . ( F ) ELISA cross - reactivity with Pf - 7G8 FL - CSP of CSP - specific mAbs that block ( left ) or do not block ( right ) mAb 1710 binding to NF54 C - CSP .

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Figure 3(D) C-CSP reactivity of the α-TSR-specific mAb 1710 in a blocking ELISA with C-CSP-specific VH3-21 (n=21; upper panel) or non-VH3-21 (n=17; lower panel) mAbs with the indicated SPR affinities. mAb 1710 binding without blocking (blue) and after self-blocking (gold) is shown for comparison.(E) C-CSP reactivity of mAb 1512 in a blocking ELISA with mAbs (with the indicated SPR affinities) that do not block C-CSP binding of mAb 1710 (open circles; D). mAb 1512 binding without blocking (purple) and after self-blocking (magenta) is shown for comparison.(F) ELISA cross-reactivity with Pf-7G8 FL-CSP of CSP-specific mAbs that block (left) or do not block (right) mAb 1710 binding to NF54 C-CSP.