( E ) Quantification of northern blot analysis for pre - tRNATyrGUA ( left ) and tRNATyrGUA ( right ) after one hour and 24 hours respectively in HBEC30 cells upon exposure to oxidative stress ( 200μM H2O2 ) as in ( A ) ( n = 6 ) .

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(E) Quantification of northern blot analysis for pre-tRNATyrGUA (left) and tRNATyrGUA (right) after one hour and 24 hours respectively in HBEC30 cells upon exposure to oxidative stress (200μM H2O2) as in (A) (n=6).


( A ) Growth curves of MCF10A cells exposed to oxidative stress ( 200μM H2O2 ) relative to control cells ( n = 3 ) . Two - way ANOVA was used to test for significance .

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(A) Growth curves of MCF10A cells exposed to oxidative stress (200μM H2O2) relative to control cells (n=3). Two-way ANOVA was used to test for significance.


( B ) Northern blot of MCF10A cells expressing a control short - hairpin RNA or a hairpin targeting tRNATyrGUA .

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(B) Northern blot of MCF10A cells expressing a control short-hairpin RNA or a hairpin targeting tRNATyrGUA.


( C ) Western blot of MCF10A expressing a control short - hairpin RNA or a hairpin targeting the tyrosine - tRNA synthetase , YARS ( red arrow ) . HSC70 was used as a loading control .

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(C) Western blot of MCF10A expressing a control short-hairpin RNA or a hairpin targeting the tyrosine-tRNA synthetase, YARS (red arrow). HSC70 was used as a loading control.


( D ) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin ( n = 3 ) . Two - way ANOVA was used to test for significance .

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(D) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin (n=3). Two-way ANOVA was used to test for significance.


( E ) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector ( n = 3 ) . A one - tailed Mann - Whitney test was used to test for significance at day 3 .

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(E) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector (n=3). A one-tailed Mann-Whitney test was used to test for significance at day 3.


( A ) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2 - fold change of 0 . 5 or more . This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion .

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(A) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2-fold change of 0.5 or more. This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion.


( C ) Quantitative western blot validation depicting abundances of protein targets ( EPCAM , SCD , and USP3 ) identified from ( A ) . HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA . ( D ) Quantification of western blot analysis in ( C ) ( n = 4 ) . A one - tailed Mann - Whitney test was used to test for statistical significance .

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(C) Quantitative western blot validation depicting abundances of protein targets (EPCAM, SCD, and USP3) identified from (A). HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA. (D) Quantification of western blot analysis in (C) (n=4). A one-tailed Mann-Whitney test was used to test for statistical significance.


( E ) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM , SCD , or USP3 . Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments . Two - way ANOVA was used to test for significance ( n = 3 ) .

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(E) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM, SCD, or USP3. Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments. Two-way ANOVA was used to test for significance (n=3).


( A ) Quantitative western blot EPCAM , SCD , and USP3 in MCF10A cells 24 hours after treatment with H2O2 ( 200μM ) . HSC70 was used as a loading control . ( B ) Quantification of western results in ( A ) ( n = 9 ) . A one - tailed Mann - Whitney test was used to test for statistical significance .

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(A) Quantitative western blot EPCAM, SCD, and USP3 in MCF10A cells 24 hours after treatment with H2O2 (200μM). HSC70 was used as a loading control. (B) Quantification of western results in (A) (n=9). A one-tailed Mann-Whitney test was used to test for statistical significance.


( D ) Quantitative western blot for the Myc - tag and Luciferase ( top ) with normalized fluorescent intensities ( bottom ) are shown ( n = 3 ) .

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(D) Quantitative western blot for the Myc-tag and Luciferase (top) with normalized fluorescent intensities (bottom) are shown (n=3).


( E ) Ribosome occupancy of 20 - 22nt RPFs in tRNATyrGUA - depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA - depleted cells using a Wilcoxon test .

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(E) Ribosome occupancy of 20-22nt RPFs in tRNATyrGUA-depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA-depleted cells using a Wilcoxon test.


( A - B ) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A ( A ) ( n = 4 ) and in HBEC30 ( B ) ( n = 6 ) with mean ± s . e . m shown for each cell line .

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(A-B) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A (A) (n=4) and in HBEC30 (B) (n=6) with mean ± s.e.m shown for each cell line.


( C ) Volcano plot of mass spectrometry results from a synthetic 5 ' - biotinylated tRFTyrGUA co - precipitation experiment with cell lysate . Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples .

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(C) Volcano plot of mass spectrometry results from a synthetic 5'-biotinylated tRFTyrGUA co-precipitation experiment with cell lysate. Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples.


( D ) Western blot validation of mass spectrometry results for three of the top hits in ( A ) , showing co - precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence ( Scr ) .

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(D) Western blot validation of mass spectrometry results for three of the top hits in (A), showing co-precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence (Scr).


( B ) IGV plots from an hnRNPA1 HITS - CLIP ( Huelga et al . , 2012 ) reveals interactions with tRFTyrGUA .

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(B) IGV plots from an hnRNPA1 HITS-CLIP (Huelga et al., 2012) reveals interactions with tRFTyrGUA.


( C ) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion . SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA . ( D ) Similar to the IGV plots shown in ( C ) , but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion .

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(C) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion. SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA. (D) Similar to the IGV plots shown in (C), but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion.


( E ) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3 ' UTR hnRNPA1 CLIP binding ( Huelga et al . , 2012 ) . Transfection of tRFTyrGUA led to a significant right - shift in the expression levels of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was measured using the Kolmogorov - Smirnov test . ( F ) Cumulative distribution as in ( E ) . Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left - shift in mRNA stability of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was assessed using the Kolmogorov - Smirnov test .

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(E) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3' UTR hnRNPA1 CLIP binding (Huelga et al., 2012). Transfection of tRFTyrGUA led to a significant right-shift in the expression levels of 3' UTR bound hnRNPA1 transcripts. Statistical significance was measured using the Kolmogorov-Smirnov test. (F) Cumulative distribution as in (E). Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left-shift in mRNA stability of 3'UTR bound hnRNPA1 transcripts. Statistical significance was assessed using the Kolmogorov-Smirnov test.


( H ) UMAP maps color - coded for transcript counts of genes enriched in subsets of the analyzed cells . The scale bars represent color coded sequencing depth - corrected log transformed transcript counts .

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(H) UMAP maps color-coded for transcript counts of genes enriched in subsets of the analyzed cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.


( H ) Representative immunofluorescence images of Iba1 ( green ) and APOE ( red ) on cortical sections from SPF and GF mice . Nuclei were stained with DAPI ( blue ) . Scale bar : 50 μm . White arrowheads indicate APOE + / Iba1 + microglia and arrows show APOE - / Iba1 + microglia . Quantification of APOE + / Iba1 + microglia . Each symbol represents one mouse . At least three slides were examined per individual mouse . Data are presented as mean ± s . e . m . Significant differences were determined by unpaired t - test and marked with asterisks ( * P < 0 . 05 ) . Data are representative of two independent experiments .

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(H) Representative immunofluorescence images of Iba1 (green) and APOE (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. White arrowheads indicate APOE+/Iba1+ microglia and arrows show APOE-/Iba1+ microglia. Quantification of APOE+/Iba1+ microglia. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by unpaired t-test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in the choroid plexus of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Scale bar = 50 µm . ( I - O ) Quantification of stromal CPMΦ . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . Significant differences were evaluated by unpaired t - test or one - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of at least two independent experiments .

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(H) Representative Iba1+ immunohistochemistry in the choroid plexus of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Scale bar = 50 µm. (I-O) Quantification of stromal CPMΦ. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. Significant differences were evaluated by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of at least two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in the perivascular space of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Arrows indicate Iba1 + perivascular macrophages . Scale bar = 50 µm . ( I - O ) Quantification thereof . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . No significant differences were determined by unpaired t - test or one - way ANOVA followed by Tukey ' s post - hoc comparison test . Data are representative of at least two independent experiments .

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(H) Representative Iba1+ immunohistochemistry in the perivascular space of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ perivascular macrophages. Scale bar = 50 µm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in the leptomeninges of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Arrows indicate Iba1 + meningeal macrophages . Scale bar = 50 μm . ( I - O ) Quantification thereof . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . No significant differences were determined by unpaired t - test or one - way ANOVA followed by Tukey ' s post - hoc comparison test . Data are representative of at least two independent experiments .

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(H) Representative Iba1+ immunohistochemistry in the leptomeninges of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ meningeal macrophages. Scale bar = 50 μm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.


( A ) Iba1 immunohistochemistry depicting cortical microglia , meningeal macrophages , perivascular macrophages and choroid plexus macrophages 4 days after i . c . challenge with LCMV or vehicle controls . Arrows indicate Iba1 + meningeal or perivascular macrophages , respectively . ( B ) Quantification of Iba1 + microglia , choroid plexus macrophages , meningeal macrophages and perivascular macrophages . Each symbol represents one mouse . Three to four sections per mouse were examined . Significant differences were evaluated by two - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

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(A) Iba1 immunohistochemistry depicting cortical microglia, meningeal macrophages, perivascular macrophages and choroid plexus macrophages 4 days after i.c. challenge with LCMV or vehicle controls. Arrows indicate Iba1+ meningeal or perivascular macrophages, respectively. (B) Quantification of Iba1+ microglia, choroid plexus macrophages, meningeal macrophages and perivascular macrophages. Each symbol represents one mouse. Three to four sections per mouse were examined. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( C , D ) Representative immunofluorescence images ( left ) of ( C ) Iba1 ( green ) and KI67 ( red ) or ( D ) CD74 ( red ) on cortical sections from SPF and GF mice . Nuclei were stained with DAPI ( blue ) . Scale bars : 25 μm . White arrows indicate KI67 + / Iba1 + or CD74 + / Iba1 + macrophages and arrowheads show KI67 - / Iba1 + or CD74 - / Iba1 + macrophages . Quantification thereof ( right ) . Each symbol represents one mouse . At least three slides were examined per individual mouse . Data are presented as mean ± s . e . m . Significant differences were evaluated by two - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

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(C, D) Representative immunofluorescence images (left) of (C) Iba1 (green) and KI67 (red) or (D) CD74 (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bars: 25 μm. White arrows indicate KI67+/Iba1+ or CD74+/Iba1+ macrophages and arrowheads show KI67-/Iba1+ or CD74-/Iba1+ macrophages. Quantification thereof (right). Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( A - C ) Representative immunofluorescence images visualizing ( A ) cortical mMΦ , ( B ) cpMΦ located in the lateral ventricle and ( C ) hippocampal pvMΦ from SPF ( left ) , GF ( middle ) and ABX - treated 5xFAD mice ( right ) . Iba1 ( green ) and Aβ ( 6E10 , red ) . Nuclei were stained with DAPI ( blue ) . White arrowheads indicate Iba1 + Aβ + MΦ and white arrows show Iba1 + Aβ - MΦ . Scale bar : 50 μm .

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(A-C) Representative immunofluorescence images visualizing (A) cortical mMΦ, (B) cpMΦ located in the lateral ventricle and (C) hippocampal pvMΦ from SPF (left), GF (middle) and ABX-treated 5xFAD mice (right). Iba1 (green) and Aβ (6E10, red). Nuclei were stained with DAPI (blue). White arrowheads indicate Iba1+Aβ+ MΦ and white arrows show Iba1+Aβ- MΦ. Scale bar: 50 μm.


( D ) Quantification of the number of 6E10 + Aβ - plaques per mm2 . Each symbol represents one mouse . Means ± s . e . m . are indicated . Significant differences were evaluated by one - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * * * P < 0 . 001 ) . Data are representative of four independent experiments .

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(D) Quantification of the number of 6E10+ Aβ-plaques per mm2. Each symbol represents one mouse. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (***P < 0.001). Data are representative of four independent experiments.


( E - G ) Quantification of Iba1 + ( E ) mMΦ , ( F ) cpMΦ and ( G ) pvMΦ per mm2 . Each symbol represents one mouse . At least three slides were examined per individual mouse . Data are presented as mean ± s . e . m . Significant differences were determined by two - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

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(E-G) Quantification of Iba1+ (E) mMΦ, (F) cpMΦ and (G) pvMΦ per mm2. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( H - J ) Percentage of Iba1 + Aβ + ( H ) cortical mMΦ , ( I ) cpMΦ and ( J ) hippocampal pvMΦ of SPF , GF and ABX - treated 5xFAD mice . Means ± s . e . m . are indicated . Significant differences were evaluated by one - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 ) . Data are representative of two independent experiments .

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(H-J) Percentage of Iba1+Aβ+ (H) cortical mMΦ, (I) cpMΦ and (J) hippocampal pvMΦ of SPF, GF and ABX-treated 5xFAD mice. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.


B RT - PCR on cytoplasmic ( C ) and nuclear ( N ) extracts showing the subcellular localization of lnc - SMaRT . RNA was isolated from C2C12 cells after two days of differentiation . GAPDH mRNA and pre - mRNA ( pre - GAPDH ) were used respectively as cytoplasmic and nuclear controls . Representative results from three independent experiments are shown .

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B RT-PCR on cytoplasmic (C) and nuclear (N) extracts showing the subcellular localization of lnc-SMaRT. RNA was isolated from C2C12 cells after two days of differentiation. GAPDH mRNA and pre-mRNA (pre-GAPDH) were used respectively as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown.


C qPCR analysis of lnc - SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points . The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as the mean ± s . e . m . of three biological replicates ( dots ) .

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C qPCR analysis of lnc-SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots).


D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs ( si - SCR ) or with two different siRNAs against lnc - SMaRT ( si - SMaRT - 1 , si - SMaRT - 2 ) and induced to differentiate for two days . The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as the mean ± s . e . m . of three biological replicates ( dots ) . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * * P < 0 . 001 .

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

D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs (si-SCR) or with two different siRNAs against lnc-SMaRT (si-SMaRT-1, si-SMaRT-2) and induced to differentiate for two days. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


E Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI staining ( in blue ) C2C12 murine myoblasts transfected with either control ( si - SCR ) or lnc - SMaRT siRNAs ( si - SMaRT - 1 , si - SMaRT - 2 ) fixed after at two days of differentiation . Histograms represent MHC - positive mononucleated cells / total MHC - positive cells ratio and fusion index quantification ( F . I . ) . At least 5 randomly chosen microscope fields of two independent biological samples were analyzed ( n > 600 cells for each field ) . Data are presented as the mean . of the two biological replicates ( dots ) .

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

E Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).


A Gene expression heatmap showing the hierarchical clustering of protein - coding genes whose expression is altered upon lnc - SMaRT depletion . Expression values , calculated as RPKMs , were log2 - transformed and mean centered . Only genes with an average RPKM between 10 and 1000 were plotted .

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

A Gene expression heatmap showing the hierarchical clustering of protein-coding genes whose expression is altered upon lnc-SMaRT depletion. Expression values, calculated as RPKMs, were log2-transformed and mean centered. Only genes with an average RPKM between 10 and 1000 were plotted.


B Volcano plot describing the differential gene expression between si - SCR and si - SMaRT treated C2C12 samples . For each gene , log2 of fold - change versus - log10 of the unadjusted p - value is plotted . Points in red represent differentially expressed genes ( FDR adjusted p - value < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plot describing the differential gene expression between si-SCR and si-SMaRT treated C2C12 samples. For each gene, log2 of fold-change versus -log10 of the unadjusted p-value is plotted. Points in red represent differentially expressed genes (FDR adjusted p-value < 0.05).


D Western blot on protein extract from control and lnc - SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation . CASP3 antibody was used . ACTN was used as loading control . Representative results from three independent experiments are shown .

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

D Western blot on protein extract from control and lnc-SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation. CASP3 antibody was used. ACTN was used as loading control. Representative results from three independent experiments are shown.


A Localization on the lnc - SMaRT transcript of the two sets of biotinylated probes ( Set # 1 and Set # 2 ) used for lnc - SMaRT pull - down experiment .

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

A Localization on the lnc-SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc-SMaRT pull-down experiment.


B Left panel : qPCR analysis of lnc - SMaRT enrichment in the RNA pull - down performed in C2C12 cells at day 2 of differentiation ( DM2 ) ; Set # 1 and Set # 2 probes were used against lnc - SMaRT together with a control set of probes against LacZ mRNA ( LacZ ) . Data are expressed in percentage of input and presented as the mean ± s . e . m . of three biological replicates ( dots ) . Right panel : Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc - SMaRT pull - down ; GAPDH was used as the negative control . Representative results from three independent experiments are shown .

B Left panel: qPCR analysis of lnc-SMaRT enrichment in the RNA pull-down performed in C2C12 cells at day 2 of differentiation (DM2); Set#1 and Set#2 probes were used against lnc-SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots). Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc-SMaRT pull-down; GAPDH was used as the negative control. Representative results from three independent experiments are shown.


C Upper panel : Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation . Input sample ( IN ) accounts for 2 . 5 % of the extract . Representative results from three independent experiments are shown . Lower panel : qPCR analysis of lnc - SMaRT enrichment in DHX36 RIP - derived RNA extracts . WBP4 was used as positive control [ 31 ] , while Neat1 ( a lncRNA expressed at comparable level of lnc - SMaRT according to the RNA - sequencing ) and Rps7 were used as negative controls . Data are expessed as percentage of input normalized on IgG control , and presented as the mean ± s . e . m . of three biological replicates ( dots ) .

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

C Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown. Lower panel: qPCR analysis of lnc-SMaRT enrichment in DHX36 RIP-derived RNA extracts. WBP4 was used as positive control [31], while Neat1 (a lncRNA expressed at comparable level of lnc-SMaRT according to the RNA-sequencing) and Rps7 were used as negative controls. Data are expessed as percentage of input normalized on IgG control, and presented as the mean ± s.e.m. of three biological replicates (dots).


D RT - PCR validation of Mlx - α , Mlx - β and MLX - γ mRNA enrichment upon the lnc - SMaRT pull - down performed with Set # 1 and Set # 2 probes ; a control set of probes against LacZ mRNA ( LacZ ) was used as negative control . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown .

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

D RT-PCR validation of Mlx-α, Mlx-β and MLX-γ mRNA enrichment upon the lnc-SMaRT pull-down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.


E RT - PCR analysis of Mlx - α , Mlx - β and Mlx - γ mRNA enrichment in DHX36 RIP - derived RNA extracts in samples treated with control siRNA ( siSCR , upper panel ) or siRNA against lnc - SMaRT ( siSMaRT , lower panel ) . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown .

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

E RT-PCR analysis of Mlx-α, Mlx-β and Mlx-γ mRNA enrichment in DHX36 RIP-derived RNA extracts in samples treated with control siRNA (siSCR, upper panel) or siRNA against lnc-SMaRT (siSMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.


F Upper panel : Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG - tagged MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . FLAG - tag has been inserted at the N - terminus of MLX protein isoforms . HPRT was used as loading control . Representative results from three independent experiments are shown . Middle panel : Quantification of FLAG - tagged protein levels normalized on HPRT signals . Data are expressed presented as the mean ± s . d . of three biological replicates ( dots ) . Statistical analysis was performed with two - way ANOVA followed by Bonferroni ' s multiple comparisons test . * * * P < 0 . 001 . Lower panel : RT - PCR analysis of lnc - SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged - MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . GAPDH was used as loading control . Representative results from three independent experiments are shown .

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

F Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG-tagged MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). FLAG-tag has been inserted at the N-terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown. Middle panel: Quantification of FLAG-tagged protein levels normalized on HPRT signals. Data are expressed presented as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two-way ANOVA followed by Bonferroni's multiple comparisons test. ***P < 0.001. Lower panel: RT-PCR analysis of lnc-SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged-MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). GAPDH was used as loading control. Representative results from three independent experiments are shown.


G Upper panel : Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector ( CTRL ) or with a plasmid overexpressing FLAG - tagged MLX - γ ( MLX - γ ) in control conditions ( si - SCR ) or in knockdown of DHX36 ( si - DHX36 ) . ACTININ was used as loading control . Representative results from three independent experiments are shown . Lower panel : RT - PCR analysis of Mlx - γ expression on the described RNA extracts . GAPDH was used as loading control . Representative results from three independent experiments are shown .

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

G Upper panel: Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector (CTRL) or with a plasmid overexpressing FLAG-tagged MLX-γ (MLX-γ) in control conditions (si-SCR) or in knockdown of DHX36 (si-DHX36). ACTININ was used as loading control. Representative results from three independent experiments are shown. Lower panel: RT-PCR analysis of Mlx-γ expression on the described RNA extracts. GAPDH was used as loading control. Representative results from three independent experiments are shown.


A Immunofluorescence for total MLX protein detection ( red signal ) performed on C2C12 cells treated with siRNAs against Mlx - γ ( si - Mlx - γ ) , lnc - SMaRT ( si - SMaRT ) , DHX36 ( si - DHX36 ) or with a control siRNA ( si - SCR ) . Cells were fixed two days after the switch to differentiation medium . Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment . The inserts on the topside of the images sketch the MLX signal peaks in the nuclei ( white stars ) to highlight the variation of MLX staining in each condition . Dashed lines indicate the edge of the nucleus .

A Immunofluorescence for total MLX protein detection (red signal) performed on C2C12 cells treated with siRNAs against Mlx-γ (si-Mlx-γ), lnc-SMaRT (si-SMaRT), DHX36 (si-DHX36) or with a control siRNA (si-SCR). Cells were fixed two days after the switch to differentiation medium. Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment. The inserts on the topside of the images sketch the MLX signal peaks in the nuclei (white stars) to highlight the variation of MLX staining in each condition. Dashed lines indicate the edge of the nucleus.


B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear ( Nuc ) and cytoplasmic ( Cyt ) compartment in the indicated conditions . About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates ( dots ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear (Nuc) and cytoplasmic (Cyt) compartment in the indicated conditions. About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates (dots).


A Left panel : schematic representation of the luciferase constructs produced for Mlx . Mlx 5 ' - and 3 ' UTR - interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region ( RLuc - Mlx 5 ' - 3 ' ) and mutant derivatives devoid of the 5 ' interacting region ( RLuc - Mlx - 3 ' ) or the 3 ' UTR interacting region ( RLuc - Mlx - 5 ' ) were obtained . These constructs were co - transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx 5 ' - 3 ' , RLuc - Mlx - 3 ' , RLuc - Mlx - 5 ' ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * P < 0 . 05 .

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

A Left panel: schematic representation of the luciferase constructs produced for Mlx. Mlx 5'- and 3'UTR-interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region (RLuc-Mlx 5'-3') and mutant derivatives devoid of the 5' interacting region (RLuc-Mlx-3') or the 3' UTR interacting region (RLuc-Mlx-5') were obtained. These constructs were co-transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc-SMaRT (+) or with a control vector (-). Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx 5'-3', RLuc-Mlx-3', RLuc-Mlx-5') set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P < 0.05.


B The RLuc - Mlx - 5 ' construct was co - transfected in N2a cells together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) and treated with control siRNA ( + ) or siRNA against DHX36 ( - ) . Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to RLuc - Mlx - 5 ' vector set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * P < 0 . 01 , * * * P < 0 . 001 .

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B The RLuc-Mlx-5' construct was co-transfected in N2a cells together with plasmids expressing lnc-SMaRT (+) or with a control vector (-) and treated with control siRNA (+) or siRNA against DHX36 (-). Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to RLuc-Mlx-5' vector set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P < 0.01, ***P < 0.001.


C Left panel : schematic representation of the luciferase constructs used . The RLuc - Mlx - 5 ' construct and its derived mutants ( RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) were transfected in N2a cells in growth conditions together with lnc - SMaRT - expressing plasmids ( lnc - SMaRT ) or with its derived mutant ( lnc - SMaRTmut ) in the indicated combinations . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx - 5 ' , RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * * P < 0 . 001 .

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

C Left panel: schematic representation of the luciferase constructs used. The RLuc-Mlx-5' construct and its derived mutants (RLuc-Mlxmut30, RLuc-Mlx Δ75) were transfected in N2a cells in growth conditions together with lnc-SMaRT-expressing plasmids (lnc-SMaRT) or with its derived mutant (lnc-SMaRTmut) in the indicated combinations. Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx-5', RLuc-Mlxmut30, RLuc-Mlx Δ75) set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


A Upper panel : schematic representation of the predicted base pairing between lnc - SMaRT ( region A ) and Mlx γ mRNA ; the predicted G - 4 structure is boxed in red while the synthetic oligos sequences are underlined in red . Lower panel : Lane 1 and 2 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA ( SYBR GOLD , indicated as SYBR in the figure ) . Lane 3 and 4 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 5 and 6 : 200 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX ( NMM ) . Lane 7 and 8 : 5 pmol of Mlx oligo alone ( 7 ) or in combination with 5pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 9 and 10 : 200 pmol of Mlx oligo alone ( 7 ) or in combination with 200 pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX . Asterisk ( * ) indicates the second NMM - positive band obtained upon SMaRT and γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown .

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

A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.


B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel . Oligos were treated as described . ° C = oligos were heated ( + ) or were not heated ( - ) at 100 ° C for 10 min and loaded after slow cooling . KCl = oligos were incubated ( + ) or were not ( - ) in a buffer containing 100mM KCl ( for G - quadruplex stabilization ) . The gel was stained with total RNA staining ( SYBR GOLD ) . Data information : Representative gels from at least three independent experiments are shown .

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

B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel. Oligos were treated as described. °C = oligos were heated (+) or were not heated (-) at 100 °C for 10 min and loaded after slow cooling. KCl= oligos were incubated (+) or were not (-) in a buffer containing 100mM KCl (for G-quadruplex stabilization). The gel was stained with total RNA staining (SYBR GOLD). Data information: Representative gels from at least three independent experiments are shown.


C 200 pmol of indicated oligos treated as in ( B ) were loaded on a native polyacrylamide gel with the selective G - quadruplex staining N - methyl mesoporphyrin IX . Asterisk ( * ) indicates the second NMM - positive band obtained upon SMaRT and γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown .

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

C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.


C ) ykt6ts pho8 ∆ 60 cells containing an empty plasmid , 3HA - Ykt6 or 3HA - tagged Ykt6 mutant variants as indicated were analyzed as in A ) .

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C) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6 or 3HA-tagged Ykt6 mutant variants as indicated were analyzed as in A).


D ) ykt6ts pho8 ∆ 60 cells containing an empty plasmid , 3HA - Ykt6 , or ADH1 promoter driven 3HA - Ykt6 wild type or 3HA - tagged Ykt6 mutant variants as indicated were grown and Pho8 ∆ 60 activity was monitored as described in A ) .

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D) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6, or ADH1 promoter driven 3HA-Ykt6 wild type or 3HA-tagged Ykt6 mutant variants as indicated were grown and Pho8∆60 activity was monitored as described in A).


E ) Ykt6 levels were analyzed from the strains shown in D ) and corresponding cell extracts were analyzed by anti - HA and anti - Pgk1 Western blotting .

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E) Ykt6 levels were analyzed from the strains shown in D) and corresponding cell extracts were analyzed by anti-HA and anti-Pgk1 Western blotting.


A ) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC . Cells were lysed and the total extract ( T ) separated into a cytoplasmic ( S ) and a 100 ' 000x g membrane pellet ( P ) fraction . Fractions were analyzed by anti - HA , anti - Tom70 and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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A) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC. Cells were lysed and the total extract (T) separated into a cytoplasmic (S) and a 100'000x g membrane pellet (P) fraction. Fractions were analyzed by anti-HA, anti-Tom70 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


B ) vam3 ∆ or ykt6ts vam3 ∆ cells containing the indicated centromeric plasmids were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ° C for 1 hour followed by 1 hour starvation in SD - N at 37 ºC . After cell lysis , samples were subjected to proteinase K ( ProtK ) and Triton X - 100 ( TX100 ) treatment as indicated and analyzed by anti - Ape1 Western blotting . Ape1 * : proteinase K - resistant fragment of Ape1 . One representative experiment out of three biological replicates is shown .

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B) vam3∆ or ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown.


C ) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid , 3HA - Ykt6 wild type , or 3HA - tagged Ykt6 mutant variants . Cells were grown at the permissive temperature ( 23 ºC ) to late exponential phase in selective medium , shifted to the restrictive temperature ( 37 ° C ) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting . Samples were analyzed by anti - CPY and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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C) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


D ) Exponentially growing ykt6ts cells at permissive temperature ( 23 ºC ) containing centromeric plasmids as indicated were shifted to the restrictive temperature ( 37 ºC ) for 1 hour before starvation for 4 hours in SD - N . Trichloroacetic acid extracts were prepared . Lipidation of Atg8 was analyzed on a 15 % SDS - PAGE containing 6 M urea by anti - Atg8 and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


A and B ) GFP - Atg8 atg1 ∆ , GFP - Atg8 atg1 ∆ vam3 ∆ , GFP - Atg8 wild type , GFP - Atg8 vam3 ∆ , GFP - Atg8 ykt6ts or GFP - Atg8 ykt6ts vam3 ∆ cell containing centromeric plasmids as indicated were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD - N . Representative microscopy images are shown in A ) . GFP - Atg8 puncta per cell were quantified from 3 biological replicates ( n ≥ 100 cells ) and the mean was plotted in B ) . Error bars are SD . Scale bar : 5 µm .

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A and B) GFP-Atg8 atg1∆, GFP-Atg8 atg1∆ vam3∆, GFP-Atg8 wild type, GFP-Atg8 vam3∆, GFP-Atg8 ykt6ts or GFP-Atg8 ykt6ts vam3∆ cell containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD-N. Representative microscopy images are shown in A). GFP-Atg8 puncta per cell were quantified from 3 biological replicates (n≥100 cells) and the mean was plotted in B). Error bars are SD. Scale bar: 5 µm.


Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ cells containing centromeric plasmids as indicated . Cells were starved for 16 hours at the permissive temperature ( 23 ° C ) to allow formation of mature , closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ cells grown under rich conditions at 30 ° C . Fusion reactions were incubated at the restrictive temperature ( 30 ° C ) for 2 hours . Representative images are shown in C ) .

Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. Representative images are shown in C).


Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ cells containing centromeric plasmids as indicated . Cells were starved for 16 hours at the permissive temperature ( 23 ° C ) to allow formation of mature , closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ cells grown under rich conditions at 30 ° C . Fusion reactions were incubated at the restrictive temperature ( 30 ° C ) for 2 hours . The graph in D ) shows the mean from at least three independent biological experiments . Error bars represent the standard deviation .

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Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. The graph in D) shows the mean from at least three independent biological experiments. Error bars represent the standard deviation.


The indicated strains were grown to late exponential phase at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ° C for 1 hour followed by starvation in SD - N for 1 hour at 37 ºC . 3HA - Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated ( IP ) and analyzed by anti - HA Western blotting . Co - precipitating proteins were analyzed by anti - Vam3 and anti - Vti1 Western blotting . One representative experiment out of three biological replicates is shown .

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

The indicated strains were grown to late exponential phase at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by starvation in SD-N for 1 hour at 37 ºC. 3HA-Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated (IP) and analyzed by anti-HA Western blotting. Co-precipitating proteins were analyzed by anti-Vam3 and anti-Vti1 Western blotting. One representative experiment out of three biological replicates is shown.


X - ray analysis of 6 - months - old H2 - c - fosLTR / Egfrwa / + and H2 - c - fosLTR / Egfrwa / wa littermates . Scale bars :

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X-ray analysis of 6-months-old H2-c-fosLTR/Egfrwa/+ and H2-c-fosLTR/Egfrwa/wa littermates. Scale bars: 1cm


Bone tumor number per mouse at 5 - 6 months of age ( n = 23 wa / + , 15 wa / wa )

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Bone 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 )

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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 )

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

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X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatment


Quantification of tumor number during treatment ( n = 6 Vehicle , 5 Erlotinib )

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Quantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib)


Tumor size during treatment ( n = 6 Vehicle , 5 Erlotinib )

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Tumor size during treatment (n=6 Vehicle, 5 Erlotinib)


Analysis of serum ALP levels during treatment ( n = 5 )

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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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)


X - ray analysis of 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates . Scale bars :

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X-ray analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 1cm


Bone 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 )

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Bone 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 )

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 -

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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


PCNA 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 :

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PCNA 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: 100µm


Egfr , 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 -

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Egfr, 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-fostg


IHC 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 :

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IHC 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


Western Blot analysis of primary OS cells isolated from H2 ‑ c ‑ fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob

Western Blot analysis of primary OS cells isolated from H2‑c‑fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice


c - 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 )

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c-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

Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib


c - 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 )

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c-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

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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 indicated


Western Blot analysis of primary OS cells isolated from a p53f / f Rb1f / f Osx - Cre mouse after 24h Erlotinib

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Western Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatment


c - fos mRNA expression levels in p53f / f Rb1f / f Osx - Cre OS cells treated for 24h with Erlotinib ( 10µM ) ( n = 3 )

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c-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

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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


X - ray analysis of 6 - months - old H2 - c - fosLTR and H2 - c - fosLTR / ColAREG littermates . Scale bars :

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X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cm


Bone tumor number per mouse at 5 - 6 months of age ( n = 22 wt , 13 ColAREG )

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Bone 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 )

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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 )

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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 )

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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

Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice


Representative images of OS biopsies stained with antibodies against EGFR and c ‑ Fos . Scale bars :

Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µm


Kaplan - 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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Kaplan-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)


Western Blot analysis showing EGFR and c - Fos protein levels in human OS cell lines cultured under standard

Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditions


Cell 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 )

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Cell 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 )

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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 )

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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

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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 experiment


IHC 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 :

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IHC 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: 50µm


Western 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

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Western 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


( B ) Automatic cluster - mapping of the temperature - dependent splicing changes in CHX , based on changes in PSI ( percent spliced in ) . Categories ( right ) were manually assigned .

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(B) Automatic cluster-mapping of the temperature-dependent splicing changes in CHX, based on changes in PSI (percent spliced in). Categories (right) were manually assigned.


( C ) Examples for cold - induced ( blue ) or heat - induced ( red ) AS - decay via exon inclusion or exon skipping . In each example , on top a simplified exon - intron structure is given , PTCs and the length of frameshift inducing exons are highlighted . Predicted splicing changes were validated by radioactive RT - PCR with primers binding to the surrounding constitutive exons . Box plots show median PSI of validation PCRs of four different samples from at least two different mice , whiskers show min to max . Individual data points are shown as circles .

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(C) Examples for cold-induced (blue) or heat-induced (red) AS-decay via exon inclusion or exon skipping. In each example, on top a simplified exon-intron structure is given, PTCs and the length of frameshift inducing exons are highlighted. Predicted splicing changes were validated by radioactive RT-PCR with primers binding to the surrounding constitutive exons. Box plots show median PSI of validation PCRs of four different samples from at least two different mice, whiskers show min to max. Individual data points are shown as circles.


( A ) Temperature - dependent poison events in Hnrnpdl ( left , see also Figure 1C ) , Hnrnph3 ( middle ) and Cirbp ( right ) . For each target , on top a simplified exon - intron structure is given and below Sashimi plots show the distribution of raw sequencing reads . Exon - Exon junction reads are indicated by the numbers connecting the exons . Below , sequence conservation across placental species is indicated . Hnrnpdl exhibits a heat - included exon that leads to a PTC , Hnrnph3 a cold - skipped exon that leads to a frameshift , and in Cirbp , heat leads to inclusion of an alternative transcript end , which leads to formation of a PTC . ( B ) For each gene from A , the triplicate normalized read counts in DMSO ( left y - axis , green ) and the percentage of the poison isoform in CHX ( right y - axis , orange ) are plotted at the two temperatures . Line represents mean PSI .

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(A) Temperature-dependent poison events in Hnrnpdl (left, see also Figure 1C), Hnrnph3 (middle) and Cirbp (right). For each target, on top a simplified exon-intron structure is given and below Sashimi plots show the distribution of raw sequencing reads. Exon-Exon junction reads are indicated by the numbers connecting the exons. Below, sequence conservation across placental species is indicated. Hnrnpdl exhibits a heat-included exon that leads to a PTC, Hnrnph3 a cold-skipped exon that leads to a frameshift, and in Cirbp, heat leads to inclusion of an alternative transcript end, which leads to formation of a PTC. (B) For each gene from A, the triplicate normalized read counts in DMSO (left y-axis, green) and the percentage of the poison isoform in CHX (right y-axis, orange) are plotted at the two temperatures. Line represents mean PSI.


( A ) Temperature - dependent poison events in Hnrnpdl ( left , see also Figure 1C ) , Hnrnph3 ( middle ) and Cirbp ( right ) . For each target , on top a simplified exon - intron structure is given and below Sashimi plots show the distribution of raw sequencing reads . Exon - Exon junction reads are indicated by the numbers connecting the exons . Below , sequence conservation across placental species is indicated . Hnrnpdl exhibits a heat - included exon that leads to a PTC , Hnrnph3 a cold - skipped exon that leads to a frameshift , and in Cirbp , heat leads to inclusion of an alternative transcript end , which leads to formation of a PTC .

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(A) Temperature-dependent poison events in Hnrnpdl (left, see also Figure 1C), Hnrnph3 (middle) and Cirbp (right). For each target, on top a simplified exon-intron structure is given and below Sashimi plots show the distribution of raw sequencing reads. Exon-Exon junction reads are indicated by the numbers connecting the exons. Below, sequence conservation across placental species is indicated. Hnrnpdl exhibits a heat-included exon that leads to a PTC, Hnrnph3 a cold-skipped exon that leads to a frameshift, and in Cirbp, heat leads to inclusion of an alternative transcript end, which leads to formation of a PTC.


( C ) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature - dependent AS - decay skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform between the CHX samples . Top shows the GE change between the DMSO samples and bottom between the CHX samples . R² and P ( deviation from zero slope ) are indicated . N = 24 .

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(C) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature-dependent AS-decay skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform between the CHX samples. Top shows the GE change between the DMSO samples and bottom between the CHX samples. R² and P (deviation from zero slope) are indicated. N=24.


( E ) Splicing reporters , containing only an intron ( GFP ctrl , # 1 ) or additionally temperature - dependent exons with ~ 1kB surrounding introns . Representative radioactive RT - PCRs ( bottom ) confirm cold - induced inclusion of Ythdf3 exon 7 ( # 2 ) and human Synerg exon 21 ( # 3 ) after 24 hours at the indicated temperatures . Representative images are derived from three independent gels . See SourceDataForFigure2E for uncropped versions of these gels .

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(E) Splicing reporters, containing only an intron (GFP ctrl, #1) or additionally temperature-dependent exons with ~1kB surrounding introns. Representative radioactive RT-PCRs (bottom) confirm cold-induced inclusion of Ythdf3 exon 7 (#2) and human Synerg exon 21 (#3) after 24 hours at the indicated temperatures. Representative images are derived from three independent gels. See SourceDataForFigure2E for uncropped versions of these gels.


( F - G ) GFP expression was quantified using FACS and is shown relative to GFP ctrl . In ( F ) transfected cells were incubated at the indicated temperatures for 24 hours ( n = 3 , mean + / - SD ) . In ( G ) cells were incubated directly after transfection for 12 hours at 39 ° C ( blue ) or 33 ° C ( red ) and then shifted for 2 , 4 , 8 and 12 hours ( n = 3 , mean + / - SD ) .

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(F-G) GFP expression was quantified using FACS and is shown relative to GFP ctrl. In (F) transfected cells were incubated at the indicated temperatures for 24 hours (n=3, mean +/- SD). In (G) cells were incubated directly after transfection for 12 hours at 39°C (blue) or 33°C (red) and then shifted for 2, 4, 8 and 12 hours (n=3, mean +/- SD).


( A ) SR protein specific temperature - dependent AS - decay . Sashimi plots are shown for heat - induced poison exon inclusion for Srsf2 ( left ) and cold - induced poison isoform formation for Srsf10 ( right ) . Quantification of GE and AS are presented in the center ( as in Figure 2B ) , lines represent mean .

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(A) SR protein specific temperature-dependent AS-decay. Sashimi plots are shown for heat-induced poison exon inclusion for Srsf2 (left) and cold-induced poison isoform formation for Srsf10 (right). Quantification of GE and AS are presented in the center (as in Figure 2B), lines represent mean.


( B ) Poison exon inclusion for Srsf2 ( left ) and Srsf10 ( right ) in a 24h temperature - rhythm in human cells . Hek293 cells were pre - entrained with square - wave temperature cycles ( 12h 34 ° C / 12h 38 ° C ) for 48h . For the last 24h , cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT - PCR ( n = 3 , mean ± SD ) . White area : 34 ° C ; Red area : 38 ° C . In Hek293 cells , inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target .

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(B) Poison exon inclusion for Srsf2 (left) and Srsf10 (right) in a 24h temperature-rhythm in human cells. Hek293 cells were pre-entrained with square-wave temperature cycles (12h 34°C/ 12h 38°C) for 48h. For the last 24h, cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT-PCR (n=3, mean ± SD). White area: 34°C; Red area: 38°C. In Hek293 cells, inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target.


( C ) AS - decay of human targets at 34 ° C ( blue ) and 38 ° C ( red ) was investigated after a square - wave temperature regime ( see B ) . RNAs were harvested after 56 and 68 hours , respectively ( n = 3 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA and is indicated by asterisks : adjusted p values : not significant ( ns ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . Individual data points are shown as circles .

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(C) AS-decay of human targets at 34°C (blue) and 38°C (red) was investigated after a square-wave temperature regime (see B). RNAs were harvested after 56 and 68 hours, respectively (n=3, mean ± SD). Statistical significance was determined by 2-way ANOVA and is indicated by asterisks: adjusted p values: not significant (ns), **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.


( E ) Quantification of Srsf2 AS - decay in hamster , rabbit and chicken cells ( n = 2 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . Individual data points are shown as circles .

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(E) Quantification of Srsf2 AS-decay in hamster, rabbit and chicken cells (n=2, mean ± SD). Statistical significance was determined by 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.


Rhythmic Srsf2 productive mRNA levels ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h ,

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Rhythmic Srsf2 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h,


Rhythmic Srsf10 productive mRNA levels ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated ( ∆ E3 A and B ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * * p < 0 . 01 , * * * * p < 0 . 0001 .

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Rhythmic Srsf10 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated (∆E3 A and B). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: **p<0.01, ****p<0.0001.


Comparing temperature - induced expression of the core clock components Bmal1 in WT and Srsf10 ∆ E3 cells ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± SEM .

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Comparing temperature-induced expression of the core clock components Bmal1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.


Comparing temperature - induced expression of the core clock components Per1 in WT and Srsf10 ∆ E3 cells ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± SEM .

Comparing temperature-induced expression of the core clock components Per1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.


( A ) Normalized GE values of plant SR proteins in a 48 - hour time course . In the first day , plants were kept at 20 ° C ( red ) and shifted to 4 ° C ( blue ) for the second day . Plants were under a 12 - hour dark / light regimen ( gray / yellow bars ) . Warm - and cold - expressed genes are indicated .

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(A) Normalized GE values of plant SR proteins in a 48-hour time course. In the first day, plants were kept at 20°C (red) and shifted to 4°C (blue) for the second day. Plants were under a 12-hour dark/light regimen (gray/yellow bars). Warm- and cold-expressed genes are indicated.


( C ) Sashimi plots as shown in Figure 2A ( without conservation score ) for decay - inducing AS events upon cold ( At - SCL33 , left ) or warm temperature ( At - SR34 , right ) . Data shown represent , from top to bottom , warm - dark , warm - light , cold - dark and cold - light conditions as indicated on the right of the plots . ( D ) For validation , A . thaliana were incubated for 3 days either at 20 ° C or 4 ° C and RNA was investigated for gene expression ( green , left y - axis ) and poison isoform formation ( orange , right y - axis ) . Gene expression of At - SCL33 ( left ) or At - SR34 ( right ) is shown relative to Ipp2 . Lines represent mean , individual data points are shown .

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(C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots. (D) For validation, A. thaliana were incubated for 3 days either at 20°C or 4°C and RNA was investigated for gene expression (green, left y-axis) and poison isoform formation (orange, right y-axis). Gene expression of At-SCL33 (left) or At-SR34 (right) is shown relative to Ipp2. Lines represent mean, individual data points are shown.


( C ) Sashimi plots as shown in Figure 2A ( without conservation score ) for decay - inducing AS events upon cold ( At - SCL33 , left ) or warm temperature ( At - SR34 , right ) . Data shown represent , from top to bottom , warm - dark , warm - light , cold - dark and cold - light conditions as indicated on the right of the plots .

(C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots.


( E ) Correlation of poison isoform inclusion and GE levels for temperature - dependent poison skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform . Decay targets were identified by using data from CHX - treated plants . R² and P ( deviation from zero slope ) are indicated . N = 80

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(E) Correlation of poison isoform inclusion and GE levels for temperature-dependent poison skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform. Decay targets were identified by using data from CHX-treated plants. R² and P(deviation from zero slope) are indicated. N=80