( d ) Splicing assay using the wild - type and mutant ACT1 - CUP1 reporters . RNAs isolated from the wild - type and the snr81Δ pusΔ strains ( indicated on the top ) , which carry the wild - type ACT1 - CUP1 reporter pre - mRNA or any of the mutant ACT1 - CUP1 reporter pre - mRNAs [ indicated , also see ( c ) ] , were used for splicing assay ( primer - extension analysis ) . The un - spliced pre - mRNA , lariat intermediate , and spliced mRNA are indicated . A primer complementary to U6 was also used ( as an internal control ) in the assay , and the U6 band is indicated as well . In addition , the growth phenotype of each strain ( in various concentrations of [ Cu2 + ] ) is also shown . Lanes 1 - 12 , lanes 13 and 14 , and lanes 15 and 16 are from separate gels .

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(d) Splicing assay using the wild-type and mutant ACT1-CUP1 reporters. RNAs isolated from the wild-type and the snr81Δ pusΔ strains (indicated on the top), which carry the wild-type ACT1-CUP1 reporter pre-mRNA or any of the mutant ACT1-CUP1 reporter pre-mRNAs [indicated, also see (c)], were used for splicing assay (primer-extension analysis). The un-spliced pre-mRNA, lariat intermediate, and spliced mRNA are indicated. A primer complementary to U6 was also used (as an internal control) in the assay, and the U6 band is indicated as well. In addition, the growth phenotype of each strain (in various concentrations of [Cu2+]) is also shown. Lanes 1-12, lanes 13 and 14, and lanes 15 and 16 are from separate gels.


( e ) Quantification of ACT1 - CUP1 mRNA levels . Spliced ACT1 - CUP1 mRNA levels were calculated relative to U6 in each lane . The quantification was based on three independent experiments .

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(e) Quantification of ACT1-CUP1 mRNA levels. Spliced ACT1-CUP1 mRNA levels were calculated relative to U6 in each lane. The quantification was based on three independent experiments.


( f ) Relative improvement / reduction for the mutant reporters . Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency ( mRNA / U6 ) of each mutant reporter to that of WT reporter ( set at 1 ) .

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(f) Relative improvement/reduction for the mutant reporters. Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency (mRNA/U6) of each mutant reporter to that of WT reporter (set at 1).


Figure 3 . Synthetic lethality assay . The Prp5 domain structure , and the mutations in some of the domains ( indicated ) are schematized at the top . Each of these PRP5 genes ( wild - type or mutants , plasmid - borne ) was used to replace the chromosomal PRP5 . The numbers in parentheses are the relative ATPase activities of the wild - type ( SAT ) Prp5 and some of the Prp5 mutants tested before ( Xu Query , 2007 ) . The U2 status , either containing Ψ42 and Ψ44 ( WT - U2 and snR81Δ pus1Δ with pΨ42 or Ψ44 ) or lacking Ψ42 and Ψ44 ( snR81Δ pus1Δ ) , is indicated ( on the right ) . Upon transformation with the plasmids carrying PRP5 genes ( wild - type or any of the mutants ) , cells were grown on the regular medium ( - FOA ) or on medium containing 5 - FOA ( + FOA ) .

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Figure 3. Synthetic lethality assay. The Prp5 domain structure, and the mutations in some of the domains (indicated) are schematized at the top. Each of these PRP5 genes (wild-type or mutants, plasmid-borne) was used to replace the chromosomal PRP5. The numbers in parentheses are the relative ATPase activities of the wild-type (SAT) Prp5 and some of the Prp5 mutants tested before (Xu Query, 2007). The U2 status, either containing Ψ42 and Ψ44 (WT-U2 and snR81Δ pus1Δ with pΨ42 or Ψ44) or lacking Ψ42 and Ψ44 (snR81Δ pus1Δ), is indicated (on the right). Upon transformation with the plasmids carrying PRP5 genes (wild-type or any of the mutants), cells were grown on the regular medium (-FOA) or on medium containing 5-FOA (+FOA).


( a ) Prp5 - U2 co - immunoprecipitation ( IP ) . Wild - type ( lanes 1 and 2 ) and snR81Δ pus1Δ ( lane 3 ) strains , each containing a FLAG - tagged PRP5 gene ( indicated on the top ) , were used for anti - FLAG IP in the absence ( lane 1 ) or presence ( lanes 2 and 3 ) of ATP . The precipitated Prp5 ( WT Prp5 - FLAG ) as well as co - precipitated U1 and U2 , detected by primer - extension , are indicated .

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(a) Prp5-U2 co-immunoprecipitation (IP). Wild-type (lanes 1 and 2) and snR81Δ pus1Δ (lane 3) strains, each containing a FLAG-tagged PRP5 gene (indicated on the top), were used for anti-FLAG IP in the absence (lane 1) or presence (lanes 2 and 3) of ATP. The precipitated Prp5 (WT Prp5-FLAG) as well as co-precipitated U1 and U2, detected by primer-extension, are indicated.


( b ) Quantification of co - precipitated U2 snRNA . For each lane [ the WT lane and the snr81Δ pus1Δ lane , corresponding to lane 2 and lane 3 of ( a ) , respectively ] , Prp5 - co - precipitated U2 was normalized against co - precipitated U1 [ ( intensity of the U2 band ) / ( intensity of the U1 band ) ] . The snr81Δ pus1Δ lane was then further normalized against the WT lane [ ( the value of normalized U2 from the snr81Δ pus1Δ lane ) / ( the value of normalized U2 from the WT lane ) ] . Quantification was based on three independent experiments .

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(b) Quantification of co-precipitated U2 snRNA. For each lane [the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 2 and lane 3 of (a), respectively], Prp5-co-precipitated U2 was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.


( c ) U2 expression level in wild - type and snR81Δ pus1Δ strains . Total RNA , isolated from the wild - type ( lane 1 ) or snR81Δ pus1Δ ( lane 2 ) strains , was used for primer - extension to measure the levels of U2 and U6 . The U2 and U6 bands are indicated .

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(c) U2 expression level in wild-type and snR81Δ pus1Δ strains. Total RNA, isolated from the wild-type (lane 1) or snR81Δ pus1Δ (lane 2) strains, was used for primer-extension to measure the levels of U2 and U6. The U2 and U6 bands are indicated.


d ) Depletion of Prp5 . Wild - type ( lanes 1 , 3 and 4 ) and snR81Δ pus1Δ ( lanes 2 and 5 ) cells containing a FLAG - tagged PRP5 were lysed , and anti - FLAG IP was performed at a high concentration of salt . Cell extracts , before ( lanes 1 and 2 ) , and after [ lanes 3 ( mock ) , 4 and 5 ] anti - FLAG IP , were used for western analysis using anti - FLAG . The Prp5 band is indicated .

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d) Depletion of Prp5. Wild-type (lanes 1, 3 and 4) and snR81Δ pus1Δ (lanes 2 and 5) cells containing a FLAG-tagged PRP5 were lysed, and anti-FLAG IP was performed at a high concentration of salt. Cell extracts, before (lanes 1 and 2), and after [lanes 3 (mock), 4 and 5] anti-FLAG IP, were used for western analysis using anti-FLAG. The Prp5 band is indicated.


( e ) IP of reconstituted Prp5 ( GAR ) - U2 . An equal amount of FLAG - tagged Prp5 ( GAR ) was added to the wild - type ( lane 1 ) and snR81Δ pus1Δ ( lane 2 ) cell extracts depleted of endogenous Prp5 [ see ( c ) ] . After the salt concentration was brought back to its original level , anti - FLAG IP was carried out . The Prp5 ( GAR ) band , detected by western , is indicated . The co - precipitated U1 and U2 bands , detected by primer - extension , are also indicated .

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(e) IP of reconstituted Prp5 (GAR)-U2. An equal amount of FLAG-tagged Prp5 (GAR) was added to the wild-type (lane 1) and snR81Δ pus1Δ (lane 2) cell extracts depleted of endogenous Prp5 [see (c)]. After the salt concentration was brought back to its original level, anti-FLAG IP was carried out. The Prp5 (GAR) band, detected by western, is indicated. The co-precipitated U1 and U2 bands, detected by primer-extension, are also indicated.


f ) Quantification of U2 that is co - precipitated with mutant Prp5 ( GAR ) . For each lane [ including the WT lane and the snr81Δ pus1Δ lane , corresponding to lane 1 and lane 2 of ( e ) , respectively ] , U2 co - precipitated with Prp5 ( GAR ) was normalized against co - precipitated U1 [ ( intensity of the U2 band ) / ( intensity of the U1 band ) ] . The snr81Δ pus1Δ lane was then further normalized against the WT lane [ ( the value of normalized U2 from the snr81Δ pus1Δ lane ) / ( the value of normalized U2 from the WT lane ) ] . Quantification was based on three independent experiments .

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f) Quantification of U2 that is co-precipitated with mutant Prp5 (GAR). For each lane [including the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 1 and lane 2 of (e), respectively], U2 co-precipitated with Prp5 (GAR) was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.


( g ) Prp5 - U2 - pre - mRNA co - IP . Reconstitution was carried out as described in ( d ) . Prior to IP , an equal amount of radiolabeled pre - mRNA was also added to each reaction . Anti - FLAG IP was then performed in the presence of ATP . The precipitated Prp5 ( WT and GAR ) band , detected by western , is indicated . The co - precipitated pre - mRNA , directly visualized after electrophoresis , is also indicated . The pre - mRNA input is also shown . In lanes 1 and 2 , wild - type and snR81Δ pus1Δ cell extracts , depleted of Prp5 , were reconstituted with the wild - type FLAG - Prp5 . In lanes 3 and 4 , wild - type and snR81Δ pus1Δ cell extracts , depleted of Prp5 , were reconstituted with mutant FLAG - Prp5 ( GAR ) .

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(g) Prp5-U2-pre-mRNA co-IP. Reconstitution was carried out as described in (d). Prior to IP, an equal amount of radiolabeled pre-mRNA was also added to each reaction. Anti-FLAG IP was then performed in the presence of ATP. The precipitated Prp5 (WT and GAR) band, detected by western, is indicated. The co-precipitated pre-mRNA, directly visualized after electrophoresis, is also indicated. The pre-mRNA input is also shown. In lanes 1 and 2, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with the wild-type FLAG-Prp5. In lanes 3 and 4, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with mutant FLAG-Prp5 (GAR).


( h ) Quantification of pre - mRNA co - precipitated with Prp5 and U2 . For each lane in ( g ) , pre - mRNA co - precipitated with Prp5 ( Wild - type or GAR ) was normalized against the Prp5 ( WT / GAR ) band in the same lane ( detected by Anti - FLAG ) [ ( intensity of pre - mRNA ) / ( intensity of WT / GAR Prp5 ) ] . Lanes 2 , 3 , and 4 ( see ( g ) ) were then further normalized against lane 1 ( see ( g ) ) [ ( the value of normalized pre - mRNA from lane 2 , 3 or 4 in ( g ) ) / ( the value of normalized pre - mRNA from lane 1 in ( g ) ) ] . The numbers at the bottom of ( h ) correspond to the lane numbers of ( g ) . Quantification was based on three independent experiments .

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(h) Quantification of pre-mRNA co-precipitated with Prp5 and U2. For each lane in (g), pre-mRNA co-precipitated with Prp5 (Wild-type or GAR) was normalized against the Prp5 (WT/GAR) band in the same lane (detected by Anti-FLAG) [(intensity of pre-mRNA) / (intensity of WT/GAR Prp5)]. Lanes 2,3,and 4 (see (g)) were then further normalized against lane 1 (see (g)) [(the value of normalized pre-mRNA from lane 2, 3 or 4 in (g)) / (the value of normalized pre-mRNA from lane 1 in (g))]. The numbers at the bottom of (h) correspond to the lane numbers of (g). Quantification was based on three independent experiments.


( a ) Prp5 ' s ATPase activity assay using synthetic U2 . The 5 ' sequence of S . cerevisiae U2 is shown ( top ) . The three ψs ( 35 , 42 and 44 ) as well as the branch site recognition sequence ( underlined ) are indicated . In the ATPase assay ( bottom ) , no RNA ( lane 3 ) , or an equal amount of LiCl - precipitated RNA ( lane 1 ) or tRNA ( lane 2 ) , a short U2 fragment ( nts 1 - 76 ) ( lane 4 ) , a fully pseudouridylated short U2 fragment ( nts 1 - 76 ) ( lane 5 ) , a long U2 fragment ( nts 1 - 120 ) ( lane 6 ) , or a fully pseudouridylated long U2 fragment ( nts 1 - 120 ) ( lane 7 ) , was used . The un - hydrolyzed ATP and hydrolyzed product ADP are indicated . Lanes 1 and 2 , lanes 3 - 5 , and lanes 6 and 7 are from separate gels .

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(a) Prp5's ATPase activity assay using synthetic U2. The 5' sequence of S. cerevisiae U2 is shown (top). The three ψs (35, 42 and 44) as well as the branch site recognition sequence (underlined) are indicated. In the ATPase assay (bottom), no RNA (lane 3), or an equal amount of LiCl-precipitated RNA (lane 1) or tRNA (lane 2), a short U2 fragment (nts 1-76) (lane 4), a fully pseudouridylated short U2 fragment (nts 1-76) (lane 5), a long U2 fragment (nts 1-120) (lane 6), or a fully pseudouridylated long U2 fragment (nts 1-120) (lane 7), was used. The un-hydrolyzed ATP and hydrolyzed product ADP are indicated. Lanes 1 and 2, lanes 3-5, and lanes 6 and 7 are from separate gels.


( b ) Quantification of Prp5 ' s ATPase activity activated by in vitro transcribed U2 . Prp5 ' s ATPase activity shown in ( a ) was calculated using the formula ( ADP ) / ( ADT + ATP ) . The numbers at the bottom of ( b ) correspond to the lane numbers of ( a ) . Quantification was based on three independent experiments .

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(b) Quantification of Prp5's ATPase activity activated by in vitro transcribed U2. Prp5's ATPase activity shown in (a) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (b) correspond to the lane numbers of (a). Quantification was based on three independent experiments.


( c ) U2 - Prp5 binding assay . FLAG - Prp5 and a radiolabeled short ( nts 1 - 76 ) ( lanes 1 and 2 ) or long ( nts 1 - 120 ) ( lanes 5 and 6 ) U2 fragment , or FLAG - Prp5 and a radiolabeled fully pseudouridylated short ( nts 1 - 76 ) ( lanes 3 and 4 ) or long ( nts 1 - 120 ) ( lanes 7 and 8 ) U2 fragment , were incubated under the conditions used for ATPase activity assay . Anti - FLAG IP was then carried out . Co - precipitated ( lanes 2 , 4 , 6 and 8 ) and un - precipitated ( lanes 1 , 3 , 5 and 7 ) U2 RNAs were analyzed by electrophoresis . The U2 band is indicated .

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(c) U2-Prp5 binding assay. FLAG-Prp5 and a radiolabeled short (nts 1-76) (lanes 1 and 2) or long (nts 1-120) (lanes 5 and 6) U2 fragment, or FLAG-Prp5 and a radiolabeled fully pseudouridylated short (nts 1-76) (lanes 3 and 4) or long (nts 1-120) (lanes 7 and 8) U2 fragment, were incubated under the conditions used for ATPase activity assay. Anti-FLAG IP was then carried out. Co-precipitated (lanes 2, 4, 6 and 8) and un-precipitated (lanes 1, 3, 5 and 7) U2 RNAs were analyzed by electrophoresis. The U2 band is indicated.


( d ) Quantification of Prp5 - U2 binding . Relative intensities of U2 bands shown in ( c ) were calculated [ setting the un - precipitated uridine - containing U2 ( lane 1 and 5 ) to 1 ] . The numbers at the bottom of ( d ) correspond to the lane numbers of ( c ) . Quantification was based on three independent experiments .

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(d) Quantification of Prp5-U2 binding. Relative intensities of U2 bands shown in (c) were calculated [setting the un-precipitated uridine-containing U2 (lane 1 and 5) to 1]. The numbers at the bottom of (d) correspond to the lane numbers of (c). Quantification was based on three independent experiments.


( e ) Prp5 ' s ATPase activity assay using cellular U2 . U2 RNA isolated from the wild - type strain ( lanes 2 and 4 ) or from the snR81Δ pus1Δ strain ( lanes 6 and 8 ) was used for the ATPase activity assay . In odd - numbered lanes , no RNA was added . Both the wild - type Prp5 ( lanes 1 - 4 ) and the mutant Prp5 ( GAR ) ( lanes 5 - 8 ) were tested .

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(e) Prp5's ATPase activity assay using cellular U2. U2 RNA isolated from the wild-type strain (lanes 2 and 4) or from the snR81Δ pus1Δ strain (lanes 6 and 8) was used for the ATPase activity assay. In odd-numbered lanes, no RNA was added. Both the wild-type Prp5 (lanes 1-4) and the mutant Prp5 (GAR) (lanes 5-8) were tested.


( f ) Quantification of Prp5 ' s ATPase activity activated by U2 from cells . Prp5 ' s ATPase activity shown in ( e ) was calculated using the formula ( ADP ) / ( ADT + ATP ) . The numbers at the bottom of ( f ) correspond to the lane numbers of ( e ) . Quantification was based on four independent experiments .

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(f) Quantification of Prp5's ATPase activity activated by U2 from cells. Prp5's ATPase activity shown in (e) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (f) correspond to the lane numbers of (e). Quantification was based on four independent experiments.


( g ) U2 level in the ATPase activity reactions . RNAs from lanes 2 and 4 in ( c ) were recovered , and U2 level was measured using primer - extension . Lanes 1 - 5 , a titration of U2 snRNA . Lane 6 , U2 from the wild - type strain [ lane 2 of ( c ) ] . Lane 7 , U2 from the snR81Δ pus1Δ strain [ lane 4 of ( c ) ] .

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(g) U2 level in the ATPase activity reactions. RNAs from lanes 2 and 4 in (c) were recovered, and U2 level was measured using primer-extension. Lanes 1-5, a titration of U2 snRNA. Lane 6, U2 from the wild-type strain [lane 2 of (c)]. Lane 7, U2 from the snR81Δ pus1Δ strain [lane 4 of (c)].


( a ) Native gel analysis of pre - splicing complexes . Pre - splicing complex assembly was carried out in the test tube with Prp5 - reconstituted cell extracts and labeled pre - mRNA . Lane 1 , Prp5 - depleted wild - type cell extract . Lane 2 , Prp5 - depleted wild - type cell extract , plus wild - type Prp5 . Lane 3 , Prp5 - depleted snR81Δ pus1Δ cell extract , plus wild - type Prp5 . Lane 4 , Prp5 - depleted wild - type cell extract , plus mutant Prp5 ( GAR ) . Lane 5 , Prp5 - depleted snR81Δ pus1Δ cell extract , plus mutant Prp5 ( GAR ) . The pre - splicing complexes A and B are indicated .

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(a) Native gel analysis of pre-splicing complexes. Pre-splicing complex assembly was carried out in the test tube with Prp5-reconstituted cell extracts and labeled pre-mRNA. Lane 1, Prp5-depleted wild-type cell extract. Lane 2, Prp5-depleted wild-type cell extract, plus wild-type Prp5. Lane 3, Prp5-depleted snR81Δ pus1Δ cell extract, plus wild-type Prp5. Lane 4, Prp5-depleted wild-type cell extract, plus mutant Prp5 (GAR). Lane 5, Prp5-depleted snR81Δ pus1Δ cell extract, plus mutant Prp5 (GAR). The pre-splicing complexes A and B are indicated.


( b ) DMS in vivo probing . After being exposed to DMS , yeast cells were lysed , total RNA collected , and primer - extension analysis carried out ( left panel ) . Lane 1 , cells that were not exposed to DMS . Lane 2 , wild - type cells exposed to DMS . Lane 3 , snR81Δ pus1Δ cells exposed to DMS . Lane 4 , snR81Δ pus1Δ cells , transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44 , and exposed to DMS . Lane 5 , snR81Δ pus1Δ cells , transformed with a plasmid carrying a control guide RNA gene with random guide sequences , and exposed to DMS . The nucleotides in parentheses are DMS - modified C and A residues that are one nucleotide after the actual primer - extension stops ( indicated by the arrows ) . A partial U2 sequence ( along with the BSL structure ) is shown ( right panel ) . DMS - modified adenosine and cytosine in the BSL are also indicated .

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(b) DMS in vivo probing. After being exposed to DMS, yeast cells were lysed, total RNA collected, and primer-extension analysis carried out (left panel). Lane 1, cells that were not exposed to DMS. Lane 2, wild-type cells exposed to DMS. Lane 3, snR81Δ pus1Δ cells exposed to DMS. Lane 4, snR81Δ pus1Δ cells, transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44, and exposed to DMS. Lane 5, snR81Δ pus1Δ cells, transformed with a plasmid carrying a control guide RNA gene with random guide sequences, and exposed to DMS. The nucleotides in parentheses are DMS-modified C and A residues that are one nucleotide after the actual primer-extension stops (indicated by the arrows). A partial U2 sequence (along with the BSL structure) is shown (right panel). DMS-modified adenosine and cytosine in the BSL are also indicated.


( c ) Residue accessibility quantification . Relative accessibility ( relative band intensity ) was quantified based on three independent experiments . The intensity of every band in each lane was normalized against the C46 band ( the least changed band ; compare lanes 2 - 5 in ( b ) ) in its respective lane [ ( intensity of a band ) / ( intensity of C46 in the same lane ) ] . Each C46 - normailzed band in lanes 3 - 5 ( refer to ( b ) ) was then further normalized against its respective counterpart in lane 2 [ ( the value of a C46 - normalized band in lanes 3 - 5 ) / ( the value of its counterpart in lane 2 ) ] . The error bars represent the standard deviations of the measurements .

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(c) Residue accessibility quantification. Relative accessibility (relative band intensity) was quantified based on three independent experiments. The intensity of every band in each lane was normalized against the C46 band (the least changed band; compare lanes 2-5 in (b)) in its respective lane [(intensity of a band) / (intensity of C46 in the same lane)]. Each C46-normailzed band in lanes 3-5 (refer to (b)) was then further normalized against its respective counterpart in lane 2 [(the value of a C46-normalized band in lanes 3-5) / (the value of its counterpart in lane 2)]. The error bars represent the standard deviations of the measurements.


( A ) Representative western blot of proteins pulled down by IP against Pum1 compared to IgG from wild - type mice brain without ( left ) and with ( right ) RNase treatment . In this panel , after IP - Pum1 , we immunoblotted for Pum1 ( positive control ) , Pum2 , Fmrp , Ago2 , Rbfox3 , Cnot1 , and

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blot of proteins pulled down by IP against Pum1 compared to IgG from wild-type mice brain without (left) and with (right) RNase treatment. In this panel, after IP-Pum1, we immunoblotted for Pum1 (positive control), Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10


( B ) Representative western blots with and without RNase . Molecular weights at the right are in kilodaltons ( kDa ) . IP against IgG was used as a negative control , and Input ( 1 % from the initial protein lysate as a loading control .

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(B) Representative western blots with and without RNase. Molecular weights at the right are in kilodaltons (kDa). IP against IgG was used as a negative control, and Input (1% from the initial protein lysate as a loading control.


( A - C ) Representative western blots of Pum1 , Pum2 , Fmrp , Ago2 , Rbfox3 , Cnot1 , and Mov10 in ( A ) cerebellum , ( B ) hippocampus , and ( C ) cortex in both male ( left panel ) and female ( right panel ) WT , Pum1 + / - , and Pum1 - / - mice . All the experiments were conducted with equal numbers of 10 - week - old male and female mice per genotype , for a total of at least 6 mice per genotype Graphs below show quantification for each protein by brain region , sex , and genotype . All data were normalized to Gapdh protein levels . P values were calculated by two - tailed Student ' s t test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . P1 indicates Pum1 .

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(A-C) Representative western blots of Pum1, Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10 in (A) cerebellum, (B) hippocampus, and (C) cortex in both male (left panel) and female (right panel) WT, Pum1+/-, and Pum1-/- mice. All the experiments were conducted with equal numbers of 10-week-old male and female mice per genotype, for a total of at least 6 mice per genotype Graphs below show quantification for each protein by brain region, sex, and genotype. All data were normalized to Gapdh protein levels. P values were calculated by two-tailed Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001. P1 indicates Pum1.


( D ) Heatmap showing 166 microRNAs from cerebella of Pum1 - / - male and female mice that were dysregulated ( fold change - 3 to + 3 ) relative to wild - type cerebellum . ( E ) Heatmap showing mRNA quantification by qPCR for 49 targets co - bound by a minimum of eight dysregulated miRNAs ( > 25 % change ) from panel D . Statistical significance and magnitude of dysregulation are illustrated for both male and

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(D) Heatmap showing 166 microRNAs from cerebella of Pum1-/- male and female mice that were dysregulated (fold change -3 to +3) relative to wild-type cerebellum. (E) Heatmap showing mRNA quantification by qPCR for 49 targets co-bound by a minimum of eight dysregulated miRNAs (>25% change) from panel D. Statistical significance and magnitude of dysregulation are illustrated for both male and female


( C ) GSEA analysis scores of Pum1 , Pum2 , Rbfox3 , and Ago2 HITS - CLIP data plotted against Fmrp HITS - CLIP data divided into 10 - percentile ranked bins shows the shared targets are among the top percentiles of targets for each protein .

(C) GSEA analysis scores of Pum1, Pum2, Rbfox3, and Ago2 HITS-CLIP data plotted against Fmrp HITS-CLIP data divided into 10-percentile ranked bins shows the shared targets are among the top percentiles of targets for each protein.


( A , B ) IP against PUM1 from Subject 1 ( S1 , R1147W ) ( A ) and Subject 2 ( S2 , T1035S ) ( B ) compared to their respective age - , sex - , and cell type - matched healthy controls confirm the interactions between PUM1 ( used here as a positive control ) , and PUM2 , FMRP , AGO2 , CNOT1 , and MOV10 . Input ( 1 % ) was used as a loading control and IP against IgG was used as a negative control . GAPDH was used here as loading control , see Methods for quantification .

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(A, B) IP against PUM1 from Subject 1 (S1, R1147W) (A) and Subject 2 (S2, T1035S) (B) compared to their respective age-, sex-, and cell type- matched healthy controls confirm the interactions between PUM1 (used here as a positive control), and PUM2, FMRP, AGO2, CNOT1, and MOV10. Input (1%) was used as a loading control and IP against IgG was used as a negative control. GAPDH was used here as loading control, see Methods for quantification.


( C , D ) Representative western blots ( C ) and relative quantification ( D ) of protein levels for PUM1 , PUM2 , FMRP , AGO2 , CNOT1 , and MOV10 in PADDAS patient - derived and PRCA patient - derived cells compared to their respective controls . Data were normalized to GAPDH protein levels .

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(C, D) Representative western blots (C) and relative quantification (D) of protein levels for PUM1, PUM2, FMRP, AGO2, CNOT1, and MOV10 in PADDAS patient-derived and PRCA patient-derived cells compared to their respective controls. Data were normalized to GAPDH protein levels.


( B ) Representative western blots ( left panel ) and relative quantifications ( right panel ) of PUM1 and its interactors ( AGO2 , CNOT1 , FMRP , and MOV10 ) in R1147W patient - derived cell lines after Myc - PUM1 - WT expression .

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(B) Representative western blots (left panel) and relative quantifications (right panel) of PUM1 and its interactors (AGO2, CNOT1, FMRP, and MOV10) in R1147W patient-derived cell lines after Myc-PUM1-WT expression.


( C ) mRNA quantification of the top 15 shared target genes from panel A in R1147W patient - derived cell lines after Myc - PUM1 - WT expression .

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(C) mRNA quantification of the top 15 shared target genes from panel A in R1147W patient-derived cell lines after Myc-PUM1-WT expression.


( C ) Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells . The color bar showed the relative RNA - seq signal ( Z - score of normalized FPKM ) .

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(C) Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells. The color bar showed the relative RNA-seq signal (Z-score of normalized FPKM).


Histological examinations of tumor types in ( F ) K versus KrasG12D ; NanogL / L ( KN ) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin ( H & E ) staining . Representative H & E images are shown . Scale bar , 500 μm ( top ) , 50 μm ( bottom ) .

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Histological examinations of tumor types in (F) K versus KrasG12D; NanogL/L (KN) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom).


( G ) qPCR analysis of stomach - restricted gene set between KL ADC and KLN mucinous tumor sections ( n = 3 or 4 per group ) . Results were shown in a box and whisker plot with the minimum value , first quartile , median , third quartile and maximum value . The box represented the 50 % of the central data , with a line inside represented the median . The significance was calculated by two - tailed unpaired Student ' s t - test with Welch ' s correction . * p = 0 . 0260 ( Hnf4α ) , * p = 0 . 0275 ( Vsig1 ) , * p = 0 . 0122 ( Gkn3 ) , * p = 0 . 0324 ( Ctse ) , * * p = 0 . 0084 ( Onecut2 ) .

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(G) qPCR analysis of stomach-restricted gene set between KL ADC and KLN mucinous tumor sections (n=3 or 4 per group). Results were shown in a box and whisker plot with the minimum value, first quartile, median, third quartile and maximum value. The box represented the 50% of the central data, with a line inside represented the median. The significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0260 (Hnf4α), *p=0.0275 (Vsig1), *p=0.0122 (Gkn3), *p=0.0324 (Ctse), **p=0.0084 (Onecut2).


( D ) Western blot for NANOG and TUBULIN in 7 paired human normal lungs ( N ) and ADC samples ( T ) and human ESCs . Mr , relative molecular mass . kDa , killoDalton .

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(D) Western blot for NANOG and TUBULIN in 7 paired human normal lungs (N) and ADC samples (T) and human ESCs. Mr, relative molecular mass. kDa, killoDalton.


( H ) Statistic analysis of LKB1 expression in ADC and IMA . The tumor tissues were analyzed by IHC staining and graded as low , medium , high expression . Significance was calculated by χ2 test for trend .

(H) Statistic analysis of LKB1 expression in ADC and IMA. The tumor tissues were analyzed by IHC staining and graded as low, medium, high expression. Significance was calculated by χ2 test for trend.


( I ) The expression grade of IHC staining of NANOG , NKX2 - 1 , LKB1 in 76 human IMA ( 1 sample / row ) .

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(I) The expression grade of IHC staining of NANOG, NKX2-1, LKB1 in 76 human IMA (1 sample/row).


( B - D ) H & E staining of tumors , IHC staining ( B ) and quantification of NICD ( C ) and HES1 ( D ) in KL ADC and KLN IMA . Representative images were shown . Scale bar , 50 μm . Results were shown as mean ± SD . The numbers of tumors analyzed over KL ADC and KLN IMA were 15 , 18 for NICD , 16 , 15 for HES1 . Significance was calculated by two - tailed unpaired Student ' s t - test with Welch ' s correction . * p = 0 . 0188 ( C ) , * * p = 0 . 0040 ( D ) .

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(B-D) H&E staining of tumors, IHC staining (B) and quantification of NICD (C) and HES1 (D) in KL ADC and KLN IMA. Representative images were shown. Scale bar, 50 μm. Results were shown as mean ± SD. The numbers of tumors analyzed over KL ADC and KLN IMA were 15, 18 for NICD, 16, 15 for HES1. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0188 (C), **p=0.0040 (D).


( G , H ) Statistical analysis of NICD ( G ) and HES1 ( H ) expression in lung sections of KLN mice following vehicle or LY - 411575 treatment . The numbers of tumors analyzed over vehicle and LY - 411575 groups were 22 , 19 for NICD , 22 , 22 for HES1 . Results were shown as mean ± SD . Significance was calculated by two - tailed unpaired Student ' s t - test with Welch ' s correction . * * * * p < 0 . 0001 .

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(G, H) Statistical analysis of NICD (G) and HES1 (H) expression in lung sections of KLN mice following vehicle or LY-411575 treatment. The numbers of tumors analyzed over vehicle and LY-411575 groups were 22, 19 for NICD, 22, 22 for HES1. Results were shown as mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. ****p<0.0001.


( I ) H & E and IHC staining of NICD and HES1 in lung tumor tissues of vehicle and LY - 411575 groups . Scale bar , 50 μm . ( J ) H & E , AB and PAS staining in lung tumors of vehicle and LY - 411575 groups . Scale bar , 50 μm .

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(I) H&E and IHC staining of NICD and HES1 in lung tumor tissues of vehicle and LY-411575 groups. Scale bar, 50 μm. (J) H&E, AB and PAS staining in lung tumors of vehicle and LY-411575 groups. Scale bar, 50 μm.


( I , J ) Statistical analysis of Ki67 ( I ) and CC3 ( J ) activity in ADC and IMA lung sections of vehicle and phenformin ( Phen ) groups . The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17 , 8 , 9 for Ki67 , 10 , 10 , 7 for CC3 . Results were shown as mean ± SEM . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons test . * * p = 0 . 0010 ( I ) , * * * p = 0 . 0009 ( I ) , * p = 0 . 0427 ( J ) .

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(I, J) Statistical analysis of Ki67 (I) and CC3 (J) activity in ADC and IMA lung sections of vehicle and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17, 8, 9 for Ki67, 10, 10, 7 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0010 (I), ***p=0.0009 (I), *p=0.0427(J).


( E , F ) Statistical analysis of Ki67 ( E ) and CC3 ( F ) reactivity in lung sections of KLN mice following vehicle , LY - 411575 treatment or a combinational therapy of phenformin and LY - 411575 . The numbers of tumors analyzed over vehicle , phenformin and combinational therapy groups were 20 , 30 , 13 for Ki67 , 14 , 11 , 9 for CC3 . Results were shown as mean ± SEM . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons test . * * p = 0 . 0014 ( E ) , * * * p = 0 . 0001 ( F ) .

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

(E, F) Statistical analysis of Ki67 (E) and CC3 (F) reactivity in lung sections of KLN mice following vehicle, LY-411575 treatment or a combinational therapy of phenformin and LY-411575. The numbers of tumors analyzed over vehicle, phenformin and combinational therapy groups were 20, 30, 13 for Ki67, 14, 11, 9 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0014 (E), ***p=0.0001 (F).


( A ) BI ‐ 1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5 - GFP to visualize the ER ( green ) . Then cells were stained with lysotracker ( red ) and observed with a confocal microscope . Nucleus was stained with Hoechst ( blue ) . Scale bar : 30 μm .

(A) BI‐1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5-GFP to visualize the ER (green). Then cells were stained with lysotracker (red) and observed with a confocal microscope. Nucleus was stained with Hoechst (blue). Scale bar: 30 μm.


( B ) BI ‐ 1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy . Acidic vesicles were visualized with a confocal microscope after lysotracker staining . Data represent the results of three independent experiments . Scale bar : 50 μm .

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(B) BI‐1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy. Acidic vesicles were visualized with a confocal microscope after lysotracker staining. Data represent the results of three independent experiments. Scale bar: 50 μm.


( C ) ( a ) Epifluorescence imaging of BI ‐ 1 KO cells stained with lysotracker ; ( b ) electron micrograph of the same field of cells shown in ( a ) ; ( c - e ) , magnification of lysotracker ‐ positive vesicles in BI ‐ 1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope ( c ) and the same field subsequently imaged by EM ( d ) . Overlapping images are presented ( e ) . Left panel : analysis of vesicular structures by EM with morphologies resembling early ( f ) , intermediate ( g ) and late ( h ) autophagy vesicles .

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(C) (a) Epifluorescence imaging of BI‐1 KO cells stained with lysotracker; (b) electron micrograph of the same field of cells shown in (a); (c-e), magnification of lysotracker‐positive vesicles in BI‐1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope (c) and the same field subsequently imaged by EM (d). Overlapping images are presented (e). Left panel: analysis of vesicular structures by EM with morphologies resembling early (f), intermediate (g) and late (h) autophagy vesicles.


( D ) Left panel : the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI ‐ 1 WT and KO MEFs cells at basal conditions ( NT ) or after exposure to EBSS for 3 h . Scale bar : left 15 μm and right 10 μm . Right panel : quantification of the number cells containing three or more LC3 ‐ positive vesicles ( N = 160 cells ) . Mean and standard deviation are presented ( N = 4 ) . Student ' s t ‐ test was used to analyse statistical significance , * * P0 . 001 and * P0 . 01 .

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(D) Left panel: the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI‐1 WT and KO MEFs cells at basal conditions (NT) or after exposure to EBSS for 3 h. Scale bar: left 15 μm and right 10 μm. Right panel: quantification of the number cells containing three or more LC3‐positive vesicles (N=160 cells). Mean and standard deviation are presented (N=4). Student's t‐test was used to analyse statistical significance, **P0.001 and *P0.01.


( E ) BI ‐ 1 WT and KO cells were transiently transfected with an expression vector for a monomeric ‐ tandem LC3 - RFP - GFP construct . After 24 h , cells were exposed to EBSS for 3 h . Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3 ‐ positive dots in the red and green channels using a confocal microscope . Scale bar : 10 μm . Right panel : quantification of the ratio between red and yellow dots per cell is presented . Mean and standard error of the analysis of 15 cells are shown .

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(E) BI‐1 WT and KO cells were transiently transfected with an expression vector for a monomeric‐tandem LC3-RFP-GFP construct. After 24 h, cells were exposed to EBSS for 3 h. Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3‐positive dots in the red and green channels using a confocal microscope. Scale bar: 10 μm. Right panel: quantification of the ratio between red and yellow dots per cell is presented. Mean and standard error of the analysis of 15 cells are shown.


( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS ( left panel ) or glucose / serum ‐ free RPMI media ( right panel ) for the indicated time points . Then , levels of LC3 were determined by western blot analysis . LC3 ‐ I and LC3 ‐ II forms are indicated . Hsp90 levels were assessed as loading control .

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(A) BI‐1 WT and KO MEFs were treated with EBSS (left panel) or glucose/serum‐free RPMI media (right panel) for the indicated time points. Then, levels of LC3 were determined by western blot analysis. LC3‐I and LC3‐II forms are indicated. Hsp90 levels were assessed as loading control.


( B ) Quantification of LC3 ‐ II levels relative to Hsp90 expression was performed in several experiments performed as presented in ( A ) .

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(B) Quantification of LC3‐II levels relative to Hsp90 expression was performed in several experiments performed as presented in (A).


( C ) Cells were pre ‐ treated with a cocktail of lysosomal inhibitors ( 200 nM bafilomycin A1 , 10 μg / ml pepstatin , and E64d ; left panel ) or 10 mM 3 ‐ methyladenine ( 3 ‐ MA ; right panel ) for 12 h and then exposed to starvation . LC3 levels monitored by western blot ( D ) and quantification of LC3 ‐ II levels relative to Hsp90 were performed in the experimental conditions described in ( C ) .

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

(C)Cells were pre‐treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3‐methyladenine (3‐MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3‐II levels relative to Hsp90 were performed in the experimental conditions described in (C).


( E ) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors ( Lys . Inh . ) in the presence of normal cell culture media . Right panel : quantification of independent experiments is presented .

O O O O O O O O O O O O O O O O O O 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) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors (Lys. Inh.) in the presence of normal cell culture media. Right panel: quantification of independent experiments is presented.


( F ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then levels of LC3 ‐ II were assessed over time by western blot after exposure to EBSS . Right panel : as control , the levels of BI ‐ 1 - GFP were monitored by western blot . Hsp90 levels were used as loading control . In ( B , D and E ) mean and standard deviation are presented . Two ‐ way ANOVA was applied to analyse statistical significance . In parenthesis , the number of independent experiments for each time point is indicated . Student ' s t ‐ test was also used in ( E ) to analyse the statistical significance between each time point ( * P0 . 001 ) . In ( B , D and E ) , normalization was performed as a ratio with the LC3 ‐ II / Hsp90 normalized levels from non ‐ treated BI ‐ 1 WT cells .

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(F) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then levels of LC3‐II were assessed over time by western blot after exposure to EBSS. Right panel: as control, the levels of BI‐1-GFP were monitored by western blot. Hsp90 levels were used as loading control. In (B, D and E) mean and standard deviation are presented. Two‐way ANOVA was applied to analyse statistical significance. In parenthesis, the number of independent experiments for each time point is indicated. Student's t‐test was also used in (E) to analyse the statistical significance between each time point (*P0.001). In (B, D and E), normalization was performed as a ratio with the LC3‐II/Hsp90 normalized levels from non‐treated BI‐1 WT cells.


( A ) Left panel : BI ‐ 1 WT and KO MEFs cells were incubated in EBSS , and then cell viability was monitored using the MTS assay . Right panel : a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h . Mean and standard deviation are presented of triplicates representative of three independent experiments .

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(A) Left panel: BI‐1 WT and KO MEFs cells were incubated in EBSS, and then cell viability was monitored using the MTS assay. Right panel: a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h. Mean and standard deviation are presented of triplicates representative of three independent experiments.


( B ) BI ‐ 1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h . Cell death was determined after propidium iodide ( PI ) staining and FACS analysis . In addition , cells were treated with 100 ng / ml tunicamycin ( Tm ) for 24 h . Mean and standard deviation are presented of one experiment performed in triplicates .

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

(B) BI‐1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h. Cell death was determined after propidium iodide (PI) staining and FACS analysis. In addition, cells were treated with 100 ng/ml tunicamycin (Tm) for 24 h. Mean and standard deviation are presented of one experiment performed in triplicates.


( C ) Cells were exposed to EBSS for 6 h or 1 μg / ml Tm for 2 h , and then replated in normal cell culture media . After 5 days , cell viability was monitored by staining with crystal violet . Data are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were exposed to EBSS for 6 h or 1 μg/ml Tm for 2 h, and then replated in normal cell culture media. After 5 days, cell viability was monitored by staining with crystal violet. Data are representative of three independent experiments.


( D ) BI ‐ 1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi ‐ 1 mRNA or control mRNA ( luciferase shRNA ) . Cell survival was measured after treatment of cells as described in ( B ) . Mean and standard deviation of an experiment made by triplicate , representative of two independent experiments .

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(D) BI‐1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi‐1 mRNA or control mRNA (luciferase shRNA). Cell survival was measured after treatment of cells as described in (B). Mean and standard deviation of an experiment made by triplicate, representative of two independent experiments.


( E ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then exposed to EBSS for indicated time points . Cell viability was monitored after PI staining by FACS . Mean and standard deviation are presented of triplicates representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then exposed to EBSS for indicated time points. Cell viability was monitored after PI staining by FACS. Mean and standard deviation are presented of triplicates representative of two independent experiments.


( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS for 2 h , and the levels of phospho ‐ p70S6k were determined by western blot analysis . Total p70S6k is also 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

(A) BI‐1 WT and KO MEFs were treated with EBSS for 2 h, and the levels of phospho‐p70S6k were determined by western blot analysis. Total p70S6k is also shown.


( B ) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125 . Mean and standard deviation are presented ( N = 3 ) . Student ' s t ‐ test was used to analyse statistical significance , * P0 . 001 and * * P0 . 0001 .

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

(B) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125. Mean and standard deviation are presented (N=3). Student's t‐test was used to analyse statistical significance, *P0.001 and **P0.0001.


( C ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points , in the presence or absence of 10 μM of the JNK inhibitor SP600125 . Levels of phosphorylation of JNK ( pJNK ) and LC3 ‐ II were determined by western blot . The levels of total JNK and Hsp90 are shown as control ( N = 4 ) . Image was assembled from cropped lanes of the same western blot analysis of the same gel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BI‐1 WT and KO cells were treated with EBSS for indicated time points, in the presence or absence of 10 μM of the JNK inhibitor SP600125. Levels of phosphorylation of JNK (pJNK) and LC3‐II were determined by western blot. The levels of total JNK and Hsp90 are shown as control (N=4). Image was assembled from cropped lanes of the same western blot analysis of the same gel.


( D ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125 , and the electrophoretic shift associated with BCL ‐ 2 phosphorylation was monitored by western blot .

O O 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

(D) BI‐1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125, and the electrophoretic shift associated with BCL‐2 phosphorylation was monitored by western blot.


( E ) BI ‐ 1 WT and KO cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . After 24 h , cells were treated with EBSS for 2 h or left untreated . The association of MYC ‐ tagged expressed Beclin ‐ 1 and BCL ‐ XL - FLAG was assessed by immunoprecipitation of Beclin ‐ 1 followed by western blot analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BI‐1 WT and KO cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. After 24 h, cells were treated with EBSS for 2 h or left untreated. The association of MYC‐tagged expressed Beclin‐1 and BCL‐XL-FLAG was assessed by immunoprecipitation of Beclin‐1 followed by western blot analysis.


( F ) 293T cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . Cell extracts were prepared in CHAPS buffer and Beclin ‐ 1 - MYC immunoprecipitated , and the possible co ‐ precipitation of BI ‐ 1 - HA , and BCL ‐ XL - FLAG was assessed by western blot analysis ( N = 3 ) . Asterisks indicate BI ‐ 1 oligomers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. Cell extracts were prepared in CHAPS buffer and Beclin‐1-MYC immunoprecipitated, and the possible co‐precipitation of BI‐1-HA, and BCL‐XL-FLAG was assessed by western blot analysis (N=3). Asterisks indicate BI‐1 oligomers.


( G ) ( G ) 293T cells were co ‐ transfected as described in ( F ) and BI ‐ 1 - HA was immunoprecipitated , and the possible co ‐ precipitation of Beclin ‐ 1 - MYC , and BCL ‐ XL - FLAG determined by western blot analysis ( N = 3 ) . Figure source data can be found with the Supplementary Information .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (G) 293T cells were co‐transfected as described in (F) and BI‐1-HA was immunoprecipitated, and the possible co‐precipitation of Beclin‐1-MYC, and BCL‐XL-FLAG determined by western blot analysis (N=3). Figure source data can be found with the Supplementary Information.


( A ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin ‐ 1 ( shBeclin ‐ 1 ) or ire1α ( shIRE1α ) mRNA or luciferase ( shLuc ) as control . The levels of LC3 , Beclin ‐ 1 , IRE1α and Hsp90 were monitored by western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BI‐1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin‐1 (shBeclin‐1) or ire1α (shIRE1α) mRNA or luciferase (shLuc) as control. The levels of LC3, Beclin‐1, IRE1α and Hsp90 were monitored by western blot.


( B ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors as described in A . Cells were exposed to EBSS for 3 h , and then LC3 levels were analysed by western blot . Image was assembled from cropped lanes of the same western blot analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BI‐1 KO MEFs were stably transduced with lentiviral vectors as described in A. Cells were exposed to EBSS for 3 h, and then LC3 levels were analysed by western blot. Image was assembled from cropped lanes of the same western blot analysis.


( C ) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shBeclin ‐ 1 cells . Quantification represents the visualization of at least 180 cells . Student ' s t ‐ test was used to analyse statistical significance . Mean and standard deviation are presented , * P0 . 001 , NS : non ‐ significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI‐1 KO/shLuc and BI‐1 KO/shBeclin‐1 cells. Quantification represents the visualization of at least 180 cells. Student's t‐test was used to analyse statistical significance. Mean and standard deviation are presented, *P0.001, NS: non‐significant.


( D ) LC3 was visualized and quantified in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shIRE1α cells described in ( B ) by immunofluorescence and confocal microscopy analysis .

O O O O O O O O O O O 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) LC3 was visualized and quantified in BI‐1 KO/shLuc and BI‐1 KO/shIRE1α cells described in (B) by immunofluorescence and confocal microscopy analysis.


( E ) BI ‐ 1 KO cells were transiently transfected with a TRAF2 dominant ‐ negative ( TRAF2 ‐ DN ) construct or empty vector ( mock ) , and after 48 h cells were stimulated with EBSS and the levels of LC3 ‐ II and Hsp90 were monitored by western blot . Right panel : quantification of relative LC3 ‐ II levels normalized with Hsp90 and non ‐ treated cells . Mean and standard deviation are presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BI‐1 KO cells were transiently transfected with a TRAF2 dominant‐negative (TRAF2‐DN) construct or empty vector (mock), and after 48 h cells were stimulated with EBSS and the levels of LC3‐II and Hsp90 were monitored by western blot. Right panel: quantification of relative LC3‐II levels normalized with Hsp90 and non‐treated cells. Mean and standard deviation are presented.


( F ) 293T cells were co ‐ transfected with expression vectors for HA ‐ tagged IRE1α ( IRE1 - HA ) , TRAF2 - FLAG , and MYC ‐ tagged BI ‐ 1 ( BI1 - MYC ) . After 48 h of transfection , HA ‐ tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot . Right panel : the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI ‐ 1 was quantified and normalized with the expression levels observed in the inputs . For comparison , the co ‐ IP signal observed in the absence of BI ‐ 1 was normalized as 100 % co ‐ IP in each independent experiment ( N = 3 ) . Mean and standard deviation are presented , * P0 . 05 . Figure source data can be found with the Supplementary Information .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co‐transfected with expression vectors for HA‐tagged IRE1α (IRE1-HA), TRAF2-FLAG, and MYC‐tagged BI‐1 (BI1-MYC). After 48 h of transfection, HA‐tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot. Right panel: the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI‐1 was quantified and normalized with the expression levels observed in the inputs. For comparison, the co‐IP signal observed in the absence of BI‐1 was normalized as 100% co‐IP in each independent experiment (N=3). Mean and standard deviation are presented, *P0.05. Figure source data can be found with the Supplementary Information.


( A ) The expression of dBI ‐ 1 was knocked down in Drosophila melanogaster . Then , relative expression levels of dBI ‐ 1 mRNA were monitored by semiquantitative PCR . Actin levels were monitored as control .

O O O O O O O O O O O O O O O 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) The expression of dBI‐1 was knocked down in Drosophila melanogaster. Then, relative expression levels of dBI‐1 mRNA were monitored by semiquantitative PCR. Actin levels were monitored as control.


( B ) ( B ) LC3 levels were monitored in control ( Da ‐ Gal4 > huLC3 : GFP ) or dBI ‐ 1 RNAi larvae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) under basal or fasting conditions . Then , huLC3 - GFP levels were analysed by western blot . In addition , dBI ‐ 1i larvae were treated with 100 μM SP600125 ( added in the growing media ) .

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

(B) (B) LC3 levels were monitored in control (Da‐Gal4>huLC3:GFP) or dBI‐1 RNAi larvae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) under basal or fasting conditions. Then, huLC3-GFP levels were analysed by western blot. In addition, dBI‐1i larvae were treated with 100 μM SP600125 (added in the growing media).


( C ) Left panel : the presence of LC3 ‐ positive vesicles ( white arrowheads ) was monitored by confocal microscopy in control ( Da ‐ Gal4 > huLC3 > GFP ) and dBI ‐ 1 knockdown pupae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) after 6 h of puparium formation . The organization of actin cytoskeleton was monitored by staining with phalloidin ( Ph , red ) . Nucleus was stained with Topro ( blue ) . Scale bar : left 20 μm and right 11 μm . Right panel : overexpression of dBI ‐ 1 ( dBI ‐ 1EY03662 ) delays salivary gland degradation . Overexpression of dBI ‐ 1 was confirmed by semiquantitative RT - PCR . Actin levels were monitored as loading control . Right panel : wild ‐ type control and dBI ‐ 1EY03662 pupae were analysed at 14 h after puparium formation . Superficial and internal confocal planes of the cells are presented . Scale bar : 40 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: the presence of LC3‐positive vesicles (white arrowheads) was monitored by confocal microscopy in control (Da‐Gal4>huLC3>GFP) and dBI‐1 knockdown pupae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) after 6 h of puparium formation. The organization of actin cytoskeleton was monitored by staining with phalloidin (Ph, red). Nucleus was stained with Topro (blue). Scale bar: left 20 μm and right 11 μm. Right panel: overexpression of dBI‐1 (dBI‐1EY03662) delays salivary gland degradation. Overexpression of dBI‐1 was confirmed by semiquantitative RT-PCR. Actin levels were monitored as loading control. Right panel: wild‐type control and dBI‐1EY03662 pupae were analysed at 14 h after puparium formation. Superficial and internal confocal planes of the cells are presented. Scale bar: 40 μm.


( D ) Wild ‐ type control and dBI ‐ 1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker ( red ) and Hoechst ( blue ) . Then , lysosomal content in the fat body was visualized by epifluorescence microscopy . The percentage of cells presenting lysotracker ‐ positive stain is indicated . Scale bar : 50 μm .

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(D) Wild‐type control and dBI‐1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker (red) and Hoechst (blue). Then, lysosomal content in the fat body was visualized by epifluorescence microscopy. The percentage of cells presenting lysotracker‐positive stain is indicated. Scale bar: 50 μm.


( E ) Control or dBI ‐ 1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days . In all , 100 individuals were monitored in each condition . Data represent mean and standard error ( N = 3 ) . Two ‐ way ANOVA was used to analyse statistical significance between groups .

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

(E) Control or dBI‐1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days. In all, 100 individuals were monitored in each condition. Data represent mean and standard error (N=3). Two‐way ANOVA was used to analyse statistical significance between groups.


( F ) Second instar dBI ‐ 1 RNAi or control larvae were grown in food supplemented with 25 μg / ml Tm dissolved in DMSO or 0 . 5 % DMSO as control . The number of individual reaching the adult fly stage was evaluated . Mean and standard error are presented ( N = 3 ) , * * P0 . 01 .

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(F) Second instar dBI‐1 RNAi or control larvae were grown in food supplemented with 25 μg/ml Tm dissolved in DMSO or 0.5% DMSO as control. The number of individual reaching the adult fly stage was evaluated. Mean and standard error are presented (N=3), **P0.01.


( A ) The levels of LC3 , Atg5 / Atg12 complex , Beclin ‐ 1 , Bcl ‐ 2 , Bcl ‐ XL , IRE1α and Hsp90 were monitored in liver protein extracts of bi ‐ 1 + / + and bi ‐ 1 − / − at 6 ‐ month ‐ old mice . Each well represents independent mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The levels of LC3, Atg5/Atg12 complex, Beclin‐1, Bcl‐2, Bcl‐XL, IRE1α and Hsp90 were monitored in liver protein extracts of bi‐1+/+ and bi‐1−/− at 6‐month‐old mice. Each well represents independent mice.


( B ) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver .

(B) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver.


( C ) bi ‐ 1 + / + and bi ‐ 1 − / − mice were injected with 50 ng / ml of tunicamycin by intraperitoneal injection ( N = 3 ) and then LC3 levels were monitored in liver protein extracts by western blot analysis . Right panel : quantification of relative LC3 ‐ II levels , * * P0 . 005 .

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

(C) bi‐1+/+ and bi‐1−/− mice were injected with 50 ng/ml of tunicamycin by intraperitoneal injection (N=3) and then LC3 levels were monitored in liver protein extracts by western blot analysis. Right panel: quantification of relative LC3‐II levels, **P0.005.


A Heatmap showing the unsupervised consensus clustering of TCGA melanomas ( n = 445 : 356 metastases and 89 primary melanomas ) . The identified clusters are indicated below the heatmap : cluster 1 ( n = 55 , red ) , cluster 2 ( n = 304 , blue ) and cluster 3 ( n = 86 , green ) . Type of melanoma samples ( primary or metastatic ) and of metastatic site ( subcutaneous , lymph node or distant ) within each cluster are indicated below and above the heatmap , respectively . B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas ( n = 89 ) using the 1 , 000 most variable expressed genes in the same dataset . The identified signature clearly divides the TCGA dataset in two groups : Primary - A ( n = 34 , red ) and Primary - B ( n = 55 , pink ) , as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap showing the unsupervised consensus clustering of TCGA melanomas (n=445: 356 metastases and 89 primary melanomas). The identified clusters are indicated below the heatmap: cluster 1 (n=55, red), cluster 2 (n=304, blue) and cluster 3 (n=86, green). Type of melanoma samples (primary or metastatic) and of metastatic site (subcutaneous, lymph node or distant) within each cluster are indicated below and above the heatmap, respectively. B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas (n=89) using the 1,000 most variable expressed genes in the same dataset. The identified signature clearly divides the TCGA dataset in two groups: Primary-A (n=34, red) and Primary-B (n=55, pink), as indicated.


C Boxplots showing the distributions of the normalized rank - sums of the up - regulated ( left panel ) and down - regulated ( right panel ) genes in the identified signature . Down - regulated genes in Primary - B melanomas better discriminate the two groups of primary melanoma samples ( p = 5 . 9e - 15 , Wilcoxon rank - sum test ) . Median , minimum , maximum , 25th , 75th whiskers of ranksum distribution are shown .

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C Boxplots showing the distributions of the normalized rank-sums of the up-regulated (left panel) and down-regulated (right panel) genes in the identified signature. Down-regulated genes in Primary-B melanomas better discriminate the two groups of primary melanoma samples (p=5.9e-15, Wilcoxon rank-sum test). Median, minimum, maximum, 25th, 75th whiskers of ranksum distribution are shown.


A Box and whiskers ( from 5th to 95th percentile ) plots depicting TINCR RNA levels in the TCGA SKCM RNA - seq dataset of melanoma samples . TINCR expression is higher in Primary A melanomas , and its reduction in Primary - B melanomas and all groups of metastatic samples is statistically significant ( Mann - Whitney test , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

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A Box and whiskers (from 5th to 95th percentile) plots depicting TINCR RNA levels in the TCGA SKCM RNA-seq dataset of melanoma samples. TINCR expression is higher in Primary A melanomas, and its reduction in Primary-B melanomas and all groups of metastatic samples is statistically significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.


B Scatter - plot showing correlation between the expression of TINCR RNA and normalized rank - sums s of the down - regulated ( upper panel ) and up - regulated ( lower panel ) genes in the primary melanoma signature . TINCR expression strongly correlates ( Spearman ' s correlation coefficient of 0 . 79 , Spearman rank p - value : * * p < 0 . 01 ) with the expression of the down - regulated genes .

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B Scatter-plot showing correlation between the expression of TINCR RNA and normalized rank-sums s of the down-regulated (upper panel) and up-regulated (lower panel) genes in the primary melanoma signature. TINCR expression strongly correlates (Spearman's correlation coefficient of 0.79, Spearman rank p-value: **p<0.01) with the expression of the down-regulated genes.


C Dot plot showing qRT - PCR analysis of TINCR expression in primary ( 8 ) and metastatic ( 8 ) melanoma samples from surgical specimens or patient - derived xenografts ( 4 primary and 8 metastatic PDXs ) . TINCR RNA levels relative to the L32P housekeeping gene are shown . Paired primary and metastasis PDXs are marked with an asterisk . The difference between primary and metastatic samples is significant ( Mann - Whitney test , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

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C Dot plot showing qRT-PCR analysis of TINCR expression in primary (8) and metastatic (8) melanoma samples from surgical specimens or patient-derived xenografts (4 primary and 8 metastatic PDXs). TINCR RNA levels relative to the L32P housekeeping gene are shown. Paired primary and metastasis PDXs are marked with an asterisk. The difference between primary and metastatic samples is significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.


A qRT - PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines , independently infected with two shRNA targeting TINCR expression ( shTINCR - 1 and - 2 ) . Cells transduced with shScamble ( shSCR ) were used as control . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) measured in three independent biological experiments are shown . In both cell lines TINCR knockdown efficiency is more than 70 % with the two shRNAs . Statistical analyses were performed using Student t - test ( * * : p < 0 . 01 ) .

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A qRT-PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines, independently infected with two shRNA targeting TINCR expression (shTINCR-1 and -2). Cells transduced with shScamble (shSCR) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) measured in three independent biological experiments are shown. In both cell lines TINCR knockdown efficiency is more than 70% with the two shRNAs. Statistical analyses were performed using Student t -test (**: p<0.01).


B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR - 1 , shTINCR - 2 or shSCR . Relative migration ( mean ± sd ) from three independent experiments is indicated . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) .

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B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR-1, shTINCR-2 or shSCR. Relative migration (mean±sd) from three independent experiments is indicated. Statistical analyses were performed using Student t -test (***: p<0.001).


C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter - Glo luminescent kit ( Promega ) . Cell growth from three biological replicates is indicated as proliferation rate relative to control cells ( mean ± sd ) . The difference between control and TINCR - silenced samples at all time points is not significant ( Student t - test ) .

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C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter-Glo luminescent kit (Promega). Cell growth from three biological replicates is indicated as proliferation rate relative to control cells (mean±sd). The difference between control and TINCR-silenced samples at all time points is not significant (Student t-test).


D Upper panel : in vivo growth of tumors derived from intradermal injection of shTINCR - 2 or shSCR WM902B cells in NSG mice . 600 , 000 WM902B cells per mouse were inoculated . Tumor volumes ( mean ± sd ) were measured 8 weeks after transplantation . Results from two independent experiments ( four animals per group ) are shown . Statistical analysis between control and TINCR - silenced groups was performed using the Student t - test ( * * = p < 0 . 01 ) . Lower panel : qRT - PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells ( 8 weeks after injection ) . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) from four biological replicates ( animals ) from both experiments are shown .

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D Upper panel: in vivo growth of tumors derived from intradermal injection of shTINCR-2 or shSCR WM902B cells in NSG mice. 600,000 WM902B cells per mouse were inoculated. Tumor volumes (mean±sd) were measured 8 weeks after transplantation. Results from two independent experiments (four animals per group) are shown. Statistical analysis between control and TINCR-silenced groups was performed using the Student t -test (**=p<0.01). Lower panel: qRT-PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells (8 weeks after injection). TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from four biological replicates (animals) from both experiments are shown.


E Drug - response curves showing increased resistance of shTINCR - WM902B cells after treatment with BRAF ( Vemurafenib ; from 10 to 10 , 000 nM ) and MEK ( Trametinib ; from 4 to 2 , 000 nM ) inhibitors . The shSCR - ( control ) and shTINCR - WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter - Glo luminescent kit ( Promega ) . Error bars represent s . e . m . of three biological replicates . The dose response curve was fit with nonlinear regression ( GraphPad Prism ) .

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E Drug-response curves showing increased resistance of shTINCR-WM902B cells after treatment with BRAF (Vemurafenib; from 10 to 10,000 nM) and MEK (Trametinib; from 4 to 2,000 nM) inhibitors. The shSCR- (control) and shTINCR-WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter-Glo luminescent kit (Promega). Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).


G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype . Total protein lysates were extracted from shSCR - and shTINCR - WM902B cells ( lane 1 and 2 ) or shSCR - and shTINCR - MMC70 cells ( lane 3 and 4 ) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies . Vinculin was used as loading control .

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G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype. Total protein lysates were extracted from shSCR- and shTINCR-WM902B cells (lane 1 and 2) or shSCR- and shTINCR-MMC70 cells (lane 3 and 4) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control.


A qRT - PCR analysis of TINCR expression ( left ) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full - length TINCR cDNA . PDX cells infected with empty vector ( Empty ) were used as control . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) from three biological replicates are shown . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) .

A qRT-PCR analysis of TINCR expression (left) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full-length TINCR cDNA. PDX cells infected with empty vector (Empty) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from three biological replicates are shown. Statistical analyses were performed using Student t -test (***: p<0.001).


B In vitro relative migration ( mean ± sd ) from three independent experiments ( biological replicates ) is shown in MM13 and MM2 TINCR overexpressing ( TINCR ) compared to control ( Empty ) cells . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) .

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B In vitro relative migration (mean±sd) from three independent experiments (biological replicates) is shown in MM13 and MM2 TINCR overexpressing (TINCR) compared to control (Empty) cells. Statistical analyses were performed using Student t -test (***: p<0.001).


C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points ( 1 - 5 days ) using CellTiter - Glo viability assay . Relative proliferation rate ( mean ± sd ) of three independent experiments are shown .

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C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points (1-5 days) using CellTiter-Glo viability assay. Relative proliferation rate (mean±sd) of three independent experiments are shown.


D Drug - response curves showing sensitization to MEK inhibitor Trametinib of MM13 ( NRAS - mutant PDX ) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells ( BRAF - mutant PDX ) . The Empty ( control ) and TINCR expressing MM13 cells were treated with increasing concentrations of drug ( ranging from 5 to 5000 nM ) and cell viability was assayed after 72 hours by CellTiter - Glo viability assay . Error bars represent s . e . m . of three biological replicates . The dose response curve was fit with nonlinear regression ( GraphPad Prism ) .

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D Drug-response curves showing sensitization to MEK inhibitor Trametinib of MM13 (NRAS-mutant PDX) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells (BRAF-mutant PDX). The Empty (control) and TINCR expressing MM13 cells were treated with increasing concentrations of drug (ranging from 5 to 5000 nM) and cell viability was assayed after 72 hours by CellTiter-Glo viability assay. Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).


E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2 , EGFR , AXL , PDGFRβ , SOX10 , and MITF in TINCR - overexpressing MM13 and MM2 cells . Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies . Vinculin was used as loading control . This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression ( 8 - 10 folds ) .

E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2, EGFR, AXL, PDGFRβ, SOX10, and MITF in TINCR-overexpressing MM13 and MM2 cells. Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control. This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression (8-10 folds).


F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice . Four animals ( 200 , 000 cells / mouse ) per group were used in each experiment . Tumor growth ( mean ± sd ) was monitored weekly and tumor volume ( mean ± sd ) measured at 4 weeks for primary tumors ( originated in the site of injection , left panel ) and at 8 weeks for spontaneous metastases which colonized lymph nodes ( right panel ) . The results of two independent experiments are shown . Statistical analyses of control and TINCR - overexpressing groups from both experiments were performed using Student t - test ( * * : p < 0 . 01 ) .

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F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice. Four animals (200,000 cells/mouse) per group were used in each experiment. Tumor growth (mean±sd) was monitored weekly and tumor volume (mean±sd) measured at 4 weeks for primary tumors (originated in the site of injection, left panel) and at 8 weeks for spontaneous metastases which colonized lymph nodes (right panel). The results of two independent experiments are shown. Statistical analyses of control and TINCR-overexpressing groups from both experiments were performed using Student t -test (**: p<0.01).


G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown . Hematoxylin - eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells . Metastasis formation was evaluated 8 weeks after transplantation into NSG mice . Pictures of representative lungs and livers infiltrated with metastases are shown in the inset . Scale bars : 200μm .

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G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown. Hematoxylin-eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells. Metastasis formation was evaluated 8 weeks after transplantation into NSG mice. Pictures of representative lungs and livers infiltrated with metastases are shown in the inset. Scale bars: 200μm.


A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells . Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies .

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A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells. Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies.


B Representative comparison of translational efficiency ( TE ) in control ( shSCR ) and shTINCR WM902B cells 72h post infection . The genes with statistically significant TE up - and downregulation ( FDR < 0 . 1 ) are highlighted in red and blue respectively . ATF4 is circled among the upregulated genes .

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B Representative comparison of translational efficiency (TE) in control (shSCR) and shTINCR WM902B cells 72h post infection. The genes with statistically significant TE up- and downregulation (FDR< 0.1) are highlighted in red and blue respectively. ATF4 is circled among the upregulated genes.


C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells . The plotted values are the log2 fold TE change values in each biological replicate ( rep1 and rep2 ) relative to control ( shSCR ) .

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C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells. The plotted values are the log2 fold TE change values in each biological replicate (rep1 and rep2) relative to control (shSCR).


D Bar - graph representing ribosome protected fragments ( RPF ) normalized coverage ( mean ± sd ) mapping to ATF4 CDS in control ( shSCR ) and TINCR ( shTINCR ) depleted WM902B cells from two biological replicates is shown .

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D Bar-graph representing ribosome protected fragments (RPF) normalized coverage (mean±sd) mapping to ATF4 CDS in control (shSCR) and TINCR (shTINCR) depleted WM902B cells from two biological replicates is shown.


E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells .

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E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells.


B Q - PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown . Enrichment relative to input ( mean ± sd ) from three independent pulldown experiments is shown . Statistical analyses were performed using Student t - test ( * * : p < 0 . 01 , * * * : p < 0 . 001 ) .

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B Q-PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown. Enrichment relative to input (mean±sd) from three independent pulldown experiments is shown. Statistical analyses were performed using Student t -test (**: p<0.01, ***: p<0.001).


C UCSC genome browser tracks of TINCR - RIA - seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells .

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C UCSC genome browser tracks of TINCR-RIA-seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells.


p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin - 3a ( Nutlin ) for 16 hours . Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin - treated p53 + / + , but not p53 - / - or p53EE / EE MEFs . For p53EE / EE MEFs three independent replicates are shown .

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p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin-3a (Nutlin) for 16 hours. Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin-treated p53+/+, but not p53-/- or p53EE/EE MEFs. For p53EE/EE MEFs three independent replicates are shown.


RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours . Shown are all Nutlin - regulated genes from the MSigDB gene set Hallmarks _ P53 _ Pathway with a mean log2FC ≥ 1 in p53 + / + cells . Shown are the z - transformed RNA expression values ( FPKM ) .

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RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours. Shown are all Nutlin-regulated genes from the MSigDB gene set Hallmarks_P53_Pathway with a mean log2FC≥1 in p53+/+ cells. Shown are the z-transformed RNA expression values (FPKM).