E Gli1 / DNA binding . Double ‐ stranded oligonucleotide containing the canonical GliBS sequence ( 5 ′ - TTGCCTACCTGGGTGGTCTCTCCACTT - 3 ′ ) or mutated GliBS sequence used as control ( 5 ′ - TTGCCTACCTCCCACTTCTCTCCACTT - 3 ′ ) was used as probe ( P ) in EMSA experiments . The assay was performed using recombinant GST ‐ Gli1ZF ‐ WT ( Gli1 zinc finger fragment : aa 242 - 424 ) , GST ‐ Gli1ZF ‐ K350A and GST ‐ Gli1ZF ‐ K340A . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF mutants bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding ( as described in ) . * P 0 . 05 versus Gli1 WT .

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E Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′-TTGCCTACCTGGGTGGTCTCTCCACTT-3′) or mutated GliBS sequence used as control (5′-TTGCCTACCTCCCACTTCTCTCCACTT-3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242-424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.


A Inhibition of Gli1 ‐ induced transcription in transfected HEK293T cells . HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61 . Treatment time was 24 h , and control cells were treated with DMSO only .

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A Inhibition of Gli1‐induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only.


B GlaB chemical structure with numbering scheme for NMR analysis .

B GlaB chemical structure with numbering scheme for NMR analysis.


C Inhibition of Gli1 ‐ induced transcription in transfected Smo − / − MEF cells . Smo − / − MEF cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control .

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C Inhibition of Gli1‐induced transcription in transfected Smo−/−MEF cells. Smo−/−MEF cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control.


D The graphs show the Hh target gene expression levels in Ptch1 − / − MEFs treated for 48 h with GlaB and DMSO as a control . mRNA levels were determined by quantitative real ‐ time PCR ( qRT - PCR ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Pfkfb3 gene was used as a negative control .

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D The graphs show the Hh target gene expression levels in Ptch1−/−MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real‐time PCR (qRT-PCR) normalized to endogenous control (β2‐microglobulin and HPRT). Pfkfb3 gene was used as a negative control.


F SuFu − / − MEFs were treated for 48 h with GlaB and DMSO as a control . Gli1 and Ptch1mRNA levels were determined by qRT - PCR normalized to β2 ‐ microglobulin and HPRT expression . Pfkfb3 gene was used as a negative control .

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F SuFu−/−MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1mRNA levels were determined by qRT-PCR normalized to β2‐microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control.


Promoter occupancy of Gli1 is prevented by GlaB treatment . MEF WT cells were transfected with Flag ‐ tagged Gli1 or empty vectors , and chromatin immunoprecipitation ( ChIP ) was carried out . qRT - PCR was performed using primers encompassing the Gli ‐ BS of mouse Ptch1 promoter ( right , schematic representation ) . Results are indicated as fold difference , relative to empty ( pcDNA3 ) control .

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Promoter occupancy of Gli1 is prevented by GlaB treatment. MEF WT cells were transfected with Flag‐tagged Gli1 or empty vectors, and chromatin immunoprecipitation (ChIP) was carried out. qRT-PCR was performed using primers encompassing the Gli‐BS of mouse Ptch1 promoter (right, schematic representation). Results are indicated as fold difference, relative to empty (pcDNA3) control.


H Ptch1 mRNA expression levels ( left panel ) were determined by qRT - PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 ( siGli1 / 2 ) or a non ‐ specific control siRNA ( siCtr ) and treated for 24 h with GlaB or DMSO as a control . ( right panel ) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT - PCR in Daoy cells transfected with siGli1 / 2 or siCtr . Results are expressed as fold repression relative to control , and data were normalized to GAPDH and HPRT expression .

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H Ptch1 mRNA expression levels (left panel) were determined by qRT-PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non‐specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT-PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression.


A The predicted binding mode of GlaB ( green sticks ) to Gli1ZF ( blue cartoons ) . Residues highlighted by the single ‐ point mutation study to impact on Gli1 binding to DNA are shown as blue sticks ; K340 and K350 of the GlaB binding site are colored magenta . GlaB protons highlighted by the NMR study are shown as orange spheres .

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A The predicted binding mode of GlaB (green sticks) to Gli1ZF (blue cartoons). Residues highlighted by the single‐point mutation study to impact on Gli1 binding to DNA are shown as blue sticks; K340 and K350 of the GlaB binding site are colored magenta. GlaB protons highlighted by the NMR study are shown as orange spheres.


B Detail of GlaB binding to Gli1ZF ‐ WT , Gli1ZF ‐ K340A , GLI1ZF ‐ K350A , GLI1ZF ‐ K340A / K350A mutants and respective theoretical affinity values . Gli1 is shown as blue transparent surface ; K340 and K350 are colored magenta . GlaB is shown as green sticks .

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B Detail of GlaB binding to Gli1ZF‐WT, Gli1ZF‐K340A, GLI1ZF‐K350A, GLI1ZF‐K340A/K350A mutants and respective theoretical affinity values. Gli1 is shown as blue transparent surface; K340 and K350 are colored magenta. GlaB is shown as green sticks.


C Inhibition of Gli1 / DNA binding by GlaB . EMSA using recombinant GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A in the presence of different concentrations of GlaB or with DMSO only . The shifted complex is competed with a 50 × excess of cold probe . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding in absence of GlaB . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A + GlaB versus Gli1 WT + GlaB .

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C Inhibition of Gli1/DNA binding by GlaB. EMSA using recombinant GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A in the presence of different concentrations of GlaB or with DMSO only. The shifted complex is competed with a 50× excess of cold probe. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding in absence of GlaB. *P 0.05 versus DMSO; **P 0.05 K340A + GlaB versus Gli1 WT + GlaB.


D HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty vector ) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB . Treatment time was 24 h , and control cells were treated with DMSO only . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A versus Gli1 WT .

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D HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty vector) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB. Treatment time was 24 h, and control cells were treated with DMSO only. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO; **P 0.05 K340A versus Gli1 WT.


A qRT - PCR shows Hh target mRNA expression levels determined in 6 ‐ day ‐ old mouse cerebellar progenitors after s . c . injections of GlaB .

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A qRT-PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.c. injections of GlaB.


B Representative EGLHE staining of 6 ‐ day ‐ old mousecerebellum after s . c . injections of GlaB ( upper panel ) ; immunohistochemistry shows Gli1 ( middle panel ) and Ki67 stainings ( lower panel ) . In all images , the scale bar represents 50 μm .

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B Representative EGLHE staining of 6‐day‐old mousecerebellum after s.c. injections of GlaB (upper panel); immunohistochemistry shows Gli1 (middle panel) and Ki67 stainings (lower panel). In all images, the scale bar represents 50 μm.


C The graph shows the percentage of EGL thickness reduction in GlaB ‐ treated mouse cerebella in comparison with vehicle mouse cerebella . Data show the mean ± SD of cerebella ( n = 10 ) for each treatment . * P 0 . 05 versus Ctr .

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C The graph shows the percentage of EGL thickness reduction in GlaB‐treated mouse cerebella in comparison with vehicle mouse cerebella. Data show the mean ± SD of cerebella (n = 10) for each treatment. *P 0.05 versus Ctr.


D BrdU assay in GCPs . Cerebellargranule cell progenitors ( GCPs ) isolated from 4 ‐ day ‐ old mice were treated with Shh ( recombinant mouseSonic Hedgehog , amino ‐ terminal peptide , ShhN , 3 μg / ml ) and with different concentrations of GlaB , as indicated , for 48 h . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample . Shown is the mean of three independent experiments . Error bars indicate SD . * P 0 . 05 Shh versus DMSO ; * * P 0 . 05 Shh + GlaB versus Shh .

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D BrdU assay in GCPs. Cerebellargranule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouseSonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh.


E qRT - PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB . In all qRT - PCR experiments , the results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . * P 0 . 05 versus Ctr .

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E qRT-PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB. In all qRT-PCR experiments, the results were normalized to endogenous control (β2‐microglobulin and HPRT). *P 0.05 versus Ctr.


F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB .

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F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB.


A , B Ex vivo cell cultures from Ptch1 + / − mice MBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , a trypan blue count was performed ( A ) to determine the growth rate of viable cells .

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A, B Ex vivo cell cultures from Ptch1+/−mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells.


A , B Ex vivo cell cultures from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , Gli1 mRNA expression levels were determined by qRT - PCR ( B ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) .

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A, B Ex vivo cell cultures from Ptch1+/−miceMBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, Gli1 mRNA expression levels were determined by qRT-PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( C ) Suspension of single MB ‐ SCs isolated from Ptch1 + / − mice . MBs were cultured in stem cell medium to allow the formation of primary neurospheres . Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only . After 7 days of treatment , the number of secondary neurospheres derived from a known number of single cells was counted . The self ‐ renewal MB ‐ SCs ' capability is expressed as percentage of neurosphere ‐ forming cells ( left ) . Representative bright field images of tumor neurospheres after GlaB treatment are also shown ( right ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . qRT - PCR analysis show Hh , proliferation and stemness target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . Western blot analysis show Hh , proliferation and stemness target protein expression levels .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels.


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( F ) BrdU assay in MB ‐ SCs treated with GlaB ( 5 μM ) for 24 or 48 h and plated on polylysinated chamber slides . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.


G TUNEL assay in MB ‐ SCs . MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) and compared to DMSO ‐ treated sample . Bottom panel shows a Western blot of caspase ‐ 3 in GlaB ‐ treated versus control cells .

G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.


A Change of tumor volume during GlaB or vehicle treatment period ( 18 days ) .

A Change of tumor volume during GlaB or vehicle treatment period (18 days).


B Representative flank allografts average volumes . Error bars indicate SD . * P 0 . 05 .

B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05.


C Representative Masson ' s trichromestaining of tumors ; immunohistochemistry of Gli1 , Ki67 and TUNEL stainings . Scale bars represent 35 μm for Masson ' s trichromestaining , 25 μm for Gli1 and TUNEL , and 10 μm for Ki67 stainings .

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C Representative Masson's trichromestaining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichromestaining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings.


D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in ( C ) . Error bars indicate SD . * P 0 . 05 .

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D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05.


E qRT - PCR analysis show Hh and proliferation target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


F Western blot analysis show Hh and proliferation target protein expression levels . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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F Western blot analysis show Hh and proliferation target protein expression levels. Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


A Representative images ( low and high magnification ) of HE ( upper panel ) and Ki67immunohistochemistry staining ( lower panel ) of a human Daoy MB cell ‐ derived orthotopic tumor in NOD / SCIDmicecerebella after i . p . injections of GlaB . Scale bars represent 500 or 200 μm for HE staining , and 150 μm or 50 μm for Ki67 staining . B Representative tumor average volumes at the end of treatment . Error bars indicate SD . * P 0 . 05 . C Quantification of Ki67 staining from immunohistochemistry shown in ( A ) . Error bars indicate SD . * P 0 . 05 .

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A Representative images (low and high magnification) of HE (upper panel) and Ki67immunohistochemistry staining (lower panel) of a human Daoy MB cell‐derived orthotopic tumor in NOD/SCIDmicecerebella after i.p. injections of GlaB. Scale bars represent 500 or 200 μm for HE staining, and 150 μm or 50 μm for Ki67 staining.B Representative tumor average volumes at the end of treatment. Error bars indicate SD. *P 0.05.C Quantification of Ki67 staining from immunohistochemistry shown in (A). Error bars indicate SD. *P 0.05.


A , B ASZ001 BCC cells were treated with GlaB ( 5 μM ) or DMSO only . After the indicated times , a trypan blue count was performed to determine ( A ) the growth rate and ( B ) the percentage of cell death . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO .

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A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.


C Gli1 mRNA expression levels were determined by qRT - PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times . Results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO .

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C Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( D ) Change of tumor volume during GlaB or vehicle treatment period ( 18 days ) .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (D) Change of tumor volume during GlaB or vehicle treatment period (18 days).


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( E ) Representative flank allografts average volumes ( upper panel ) .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (E) Representative flank allografts average volumes (upper panel).


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( F ) HE and immunohistochemistry staining of Gli1 , Ki67 and TUNEL of allograft tumor samples . Scale bars represent 50 μm for HE , 25 μm for Gli1 , 25 μm for Ki67 and 30 μm for TUNEL stainings . ( G ) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (F) HE and immunohistochemistry staining of Gli1, Ki67 and TUNEL of allograft tumor samples. Scale bars represent 50 μm for HE, 25 μm for Gli1, 25 μm for Ki67 and 30 μm for TUNEL stainings. (G) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (F)


D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( H ) qRT - PCR of Hh and proliferation target mRNA expression levels . Results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Shown is the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (H) qRT-PCR of Hh and proliferation target mRNA expression levels. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Shown is the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


Autophagy is activated in dying nurse cells during late oogenesis in D . melanogaster . ( A - C ) Confocal micrographs of egg chambers of flies expressing the UASp - GFP - mCherry - DrAtg8a transgene exclusively in the germline ( genotype : UASp - GFP - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . Expression of UASp - GFP - mCherry - DrAtg8a exhibits punctate staining pattern ( arrows ) . Note that during developmental stages early 12 ( A ) and late 12 ( B ) , the punctate dots are yellow ( merge ) . In late stage 13 ( C ) , large red dots are evident , indicating the presence of acidic compartments ( autolysosomes ; arrows ) . The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 ( not observed during late stage 12 ) .

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

Autophagy is activated in dying nurse cells during late oogenesis in D. melanogaster. (A-C) Confocal micrographs of egg chambers of flies expressing the UASp-GFP-mCherry-DrAtg8a transgene exclusively in the germline (genotype: UASp-GFP-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). Expression of UASp-GFP-mCherry-DrAtg8a exhibits punctate staining pattern (arrows). Note that during developmental stages early 12 (A) and late 12 (B), the punctate dots are yellow (merge). In late stage 13 (C), large red dots are evident, indicating the presence of acidic compartments (autolysosomes; arrows). The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 (not observed during late stage 12).


( D and E ) Transmission electron micrographs of nurse cells in stage 12 ( D ) and in stage 13 ( E ) egg chambers . Autophagosomes ( inset in D ) and autolysosomes ( arrow in E ) are evident in the remaining nurse cell cytoplasm ( arrow in D indicates actin bundles ) . Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus ( NCN ) , providing evidence for nuclear

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E) Transmission electron micrographs of nurse cells in stage 12 (D) and in stage 13 (E) egg chambers. Autophagosomes (inset in D) and autolysosomes (arrow in E) are evident in the remaining nurse cell cytoplasm (arrow in D indicates actin bundles). Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus (NCN), providing evidence for nuclear autophagy


( F ) Nurse cell of a late stage 13 egg chamber expressing UASp - mCherry - DrAtg8a exclusively in the germline ( genotype : UASp - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . mCherry - DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus ( arrows and insets ) . Hoechst staining ( blue ) was performed to visualize the nuclei . Nurse cells are outlined with a white line . FC , follicle cell ; FCN , follicle cell nucleus ; OC , oocyte . Bars : ( A - C ) 10 µm ; ( D - F ) 1 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

(F) Nurse cell of a late stage 13 egg chamber expressing UASp-mCherry-DrAtg8a exclusively in the germline (genotype: UASp-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). mCherry-DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus (arrows and insets). Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. FC, follicle cell; FCN, follicle cell nucleus; OC, oocyte. Bars: (A-C) 10 µm; (D-F) 1 µm.


Genetic inhibition of autophagy in the germline prevents DNA fragmentation . Confocal micrographs of stage 13 / 14 egg chambers after TUNEL staining . ( A ) Wild - type ( wt ) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA ( positive TUNEL staining ; arrows ) . ( B ) Wild - type stage 14 egg chambers do not contain nurse cells . Arrows point to the respiratory appendages ( RA ) . ( C - E ) Stage 14 atg1 − / − ( C ) , atg13 − / − ( D ) , and vps34 − / − ( E ) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA ( negative TUNEL staining ; arrows ) . Nurse cells are outlined with a white line . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . Bars , 50 µm .

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 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 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

Genetic inhibition of autophagy in the germline prevents DNA fragmentation. Confocal micrographs of stage 13/14 egg chambers after TUNEL staining. (A) Wild-type (wt) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA (positive TUNEL staining; arrows). (B) Wild-type stage 14 egg chambers do not contain nurse cells. Arrows point to the respiratory appendages (RA). (C-E) Stage 14 atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA (negative TUNEL staining; arrows). Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase - 3 . Confocal micrographs of stage 13 / 14 egg chambers stained for cleaved caspase - 3 ( casp - 3 ) . ( A ) Wild - type ( wt ) late stage 13 egg chamber showing cleaved caspase - 3 staining in the degenerating nurse cell cluster . ( B ) In wild - type stage 14 egg chamber , nurse cells are completely degraded ( arrows ) . ( C - E ) atg1 − / − ( C ) , atg13 − / − ( D ) , and vps34 − / − ( E ) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase - 3 and contain persisting nurse cell nuclei . Nurse cells are outlined with a white line . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . ( F ) Quantification of mean intensity of cleaved caspase - 3 staining observed in the nurse cells of stage 12 - 14 egg chambers in wild - type and germline autophagy mutant egg chambers . Wild type ( WT ) : three independent experiments , n = 30 egg chambers ; atg1 − / − GLCs : four independent experiments , n = 30 egg chambers ; atg13 − / − GLCs : four independent experiments , n = 30 egg chambers ; vps34 − / − GLCs : three independent experiments , n = 30 egg chambers . Data are presented as mean ± SD . Difference was significant with P < 0 . 001 for all values versus wild type . Bars , 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase-3. Confocal micrographs of stage 13/14 egg chambers stained for cleaved caspase-3(casp-3). (A) Wild-type (wt) late stage 13 egg chamber showing cleaved caspase-3 staining in the degenerating nurse cell cluster. (B) In wild-type stage 14 egg chamber, nurse cells are completely degraded (arrows). (C-E) atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase-3 and contain persisting nurse cell nuclei. Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. (F) Quantification of mean intensity of cleaved caspase-3 staining observed in the nurse cells of stage 12-14 egg chambers in wild-type and germline autophagy mutant egg chambers. Wild type (WT): three independent experiments, n = 30 egg chambers; atg1−/− GLCs: four independent experiments, n = 30 egg chambers; atg13−/− GLCs: four independent experiments, n = 30 egg chambers; vps34−/− GLCs: three independent experiments, n = 30 egg chambers. Data are presented as mean ± SD. Difference was significant with P < 0.001 for all values versus wild type. Bars, 20 µm.


dBruce colocalizes with the autophagic marker Atg8a - GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants . Confocal micrographs of late stage egg chambers expressing Atg8a - GFP and stained for dBruce . ( A - C ) Stage 10 ( A ) , early stage 12 ( B ) , and late stage 12 ( C ) . dBruce colocalizes with Atg8a - GFP in punctuate structures during stages 12 and 13 ( insets in B and insets and arrows in C ) and not during stage 10 ( insets in A ) .

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

dBruce colocalizes with the autophagic marker Atg8a-GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants. Confocal micrographs of late stage egg chambers expressing Atg8a-GFP and stained for dBruce. (A-C) Stage 10 (A), early stage 12 (B), and late stage 12 (C). dBruce colocalizes with Atg8a-GFP in punctuate structures during stages 12 and 13 (insets in B and insets and arrows in C) and not during stage 10 (insets in A).


( D - G ) Confocal micrographs of stage 14 wild - type ( D ) and autophagy mutant egg chambers ( E - G ) stained for dBruce . dBruce is accumulated in atg1 , atg13 , and vps34 mutant nurse cells ( arrows ) . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . Nurse cells are outlined with a white line . OC , oocyte ; PhC , phase contrast . Bars , 10 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

(D-G) Confocal micrographs of stage 14 wild-type (D) and autophagy mutant egg chambers (E-G) stained for dBruce. dBruce is accumulated in atg1, atg13, and vps34 mutant nurse cells (arrows). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. OC, oocyte; PhC, phase contrast. Bars, 10 µm.


dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis . ( A ) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA . Arrows point to degenerating stage 8 / 9 egg chambers that are TUNEL positive .

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

dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis. (A) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA. Arrows point to degenerating stage 8/9 egg chambers that are TUNEL positive.


( B and C ) Confocal micrographs of vps34 / dBruceE81 and atg , dBruceE81 double mutant egg chambers stained for TUNEL ( green and red , respectively ) and DNA . ( B ) vps34 − / − GLCs ; dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse cell nuclei ( outlined ; arrow ) . ( C ) atg1 − / − GLCs , dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse cell nuclei . atg1 mutant nurse cells are identified by the lack of GFP staining ( outlined ; arrow ) . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . Bars , 50 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 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 O O O

(B and C) Confocal micrographs of vps34/dBruceE81 and atg, dBruceE81 double mutant egg chambers stained for TUNEL (green and red, respectively) and DNA. (B) vps34−/−GLCs; dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei (outlined; arrow). (C) atg1−/−GLCs, dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei. atg1 mutant nurse cells are identified by the lack of GFP staining (outlined; arrow). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


B - E ) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation ( pH3 , red ) in the adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm .

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

B-E) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.


withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation ( pH3 , red ) in the adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm .

O O O B-CONTROLLED_VAR 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

withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.


wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , and significantly increased by dietary histidine depletion ( 25 % his ) , quantified in ( F ) ( n = 28 , 22 , 7 ) , scale bar = 50

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

wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his), quantified in (F) (n=28,22,7), scale bar = 50 μm


F - H ) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , and significantly increased by dietary histidine depletion ( 25 % his ) ,

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

F-H) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his),


I - K ) Representaitve pictures showing that adult nerfin - 1 clonal growth was significantly reduced on - his diet compared to CDD ( measured at day 0 , and day 9 ) quantified in ( K ) ( n = 27 , 110 , 136 ) . Scale bar = 75μm . * * * p < 0 . 001 L - N ) Representaitve pictures showing that larval nerfin - 1 clonal growth was significantly reduced on 25 % his diet compared to CDD ( after 6 days ) , quantified in ( N ) ( n = 53 , 36 ) . Scale bar = 50 μm .

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 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

I-K) Representaitve pictures showing that adult nerfin-1 clonal growth was significantly reduced on -his diet compared to CDD (measured at day 0, and day 9) quantified in (K) (n= 27, 110, 136). Scale bar = 75μm. ***p<0.001 L-N) Representaitve pictures showing that larval nerfin-1 clonal growth was significantly reduced on 25% his diet compared to CDD (after 6 days), quantified in (N) (n=53,36). Scale bar = 50 μm.


O - Q ) Representaitve pictures showing that adult pros clonal growth was not significantly altered on - his diet compared to CDD ( after 7 days ) , quantified in ( Q ) ( n = 12 , 23 , 16 ) . Scale bar = 75μm . R - T ) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction ( 25 % his ) compared to CDD ( after 6 days ) , quantified in ( T ) ( n = 19 , 13 ) . Scale bar = 50 μm .

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 B-CONTROLLED_VAR 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-Q) Representaitve pictures showing that adult pros clonal growth was not significantly altered on -his diet compared to CDD (after 7 days), quantified in (Q) (n = 12, 23,16). Scale bar = 75μm. R-T) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction (25% his) compared to CDD (after 6 days), quantified in (T) (n=19,13). Scale bar = 50 μm.


U - W ) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction ( 25 % his ) compared to CDD , quantified in ( W ) ( n = 27 , 19 ) . Scale bar = 100 μm .

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 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

U-W) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction (25% his) compared to CDD, quantified in (W) (n=27,19). Scale bar = 100 μm.


B ) Concentration of histidine and 3 - methyl - L - histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or - his diet ( n = 3 , 3 ) . Both compounds are reduced to below detection levels ( < 0 . 3mM ) upon dietary his depletion .

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

B) Concentration of histidine and 3-methyl-L-histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or -his diet (n=3,3). Both compounds are reduced to below detection levels (<0.3mM) upon dietary his depletion.


C ) Dietary supplementation of 3 - methyl - L - his for 4 days significantly increased adult nerfin - 1 clonal growth , and rescued nerfin - 1 growth inhibition due to his dietary withdrawal ( n = 18 , 14 , 7 , 9 ) .

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

C) Dietary supplementation of 3-methyl-L-his for 4 days significantly increased adult nerfin-1 clonal growth, and rescued nerfin-1 growth inhibition due to his dietary withdrawal (n =18,14, 7,9).


D ) Dietary supplementation of histamine does not significantly increase adult nerfin - 1 clone size , but can rescue nerfin - 1 clonal growth inhibition due to his dietary withdrawal ( n = 42 , 14 , 23 , 25 ) .

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

D) Dietary supplementation of histamine does not significantly increase adult nerfin-1 clone size, but can rescue nerfin-1 clonal growth inhibition due to his dietary withdrawal (n =42,14,23,25).


E ) Larval nerfin - 1 clonal growth is significantly decreased upon overexpression of HDC RNAi ( n = 23 , 36 ) .

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) Larval nerfin-1 clonal growth is significantly decreased upon overexpression of HDC RNAi (n=23,36).


G ) Hdc inhibition significantly reduced the percentage of Dpn + NBs ( n = 16 , 17 ) , and significantly increased the percentage of Elav + neurons per clone ( n = 16 , 17 ) .

O O O O O O O O O O O O O O O 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) Hdc inhibition significantly reduced the percentage of Dpn+ NBs (n=16,17), and significantly increased the percentage of Elav+ neurons per clone (n=16,17).


H ) HdcRi overexpression reduced cellular growth of nerfin - 1 NBs ( n = 17 , 19 ) and neurons ( n = 36 , 19 ) measured as the ratio of nucleolus / nuclear volume .

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

H) HdcRi overexpression reduced cellular growth of nerfin-1 NBs (n=17,19) and neurons (n=36,19) measured as the ratio of nucleolus/nuclear volume.


A - E ) Representative images of nerfin - 1 clones in the adult VNC after 6 days of feeding on CDD or - his diet , scale bar = 100μm . Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in ( C ) ( n = 6 , 7 ) .

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 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

A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm. Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7).


Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav + neurons ( red ) quantified in ( D ) ( n = 16 , 17 ) and significantly fewer Mira + NBs ( blue ) , quantified in ( E ) ( n = 12 , 22 ) ,

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

Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav+ neurons (red) quantified in (D) (n = 16,17) and significantly fewer Mira+ NBs (blue), quantified in (E) (n = 12,22),


F - G ) Larval nerfin - 1 clones consisted of significantly greater proportion of differentiated neurons ( n = 10 , 7 ) and significantly reduced proportion of NBs per clone upon his dietary depletion ( 25 % his , n = 9 , 11 ) .

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

F-G) Larval nerfin-1 clones consisted of significantly greater proportion of differentiated neurons (n=10,7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n=9,11).


Representative images of adult nerfin - 1 ( I - J ' , green ) and pros clones ( K - L ' , green ) , scale bar = 5μm . In nerfin - 1 clones , the size of the NBs ( Ase + , blue ) was significantly reduced upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 33 , 20 ) . In pros clones , the size of the NBs ( Ase + blue ) , and cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 9 , 8 )

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

Representative images of adult nerfin-1 (I-J', green ) and pros clones (K-L', green), scale bar = 5μm. In nerfin-1 clones, the size of the NBs (Ase+, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n=33,20). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n=9,8)


Cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was also significantly reduced , quantified in ( M ) ( n = 33 , 20 ) .

Cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was also significantly reduced, quantified in (M) (n=33,20).


Cellular growth measured as the ratio of nucleolus / nuclear volume was significantly reduced in larval nerfin - 1 NBs after 6 days his dietary reduction ( 25 % his compared to CDD , n = 14 , 6 ) quantified in ( N ) .

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

Cellular growth measured as the ratio of nucleolus /nuclear volume was significantly reduced in larval nerfin-1 NBs after 6 days his dietary reduction (25% his compared to CDD, n=14,6) quantified in (N).


In pros clones , the size of the NBs ( Ase + blue ) , and cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( O ) ( n = 9 , 7 ) .

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

In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (O) (n=9,7).


Panel ( P ) shows that the larval nerfin - 1 neuron to NB reversion requires 15 - fold increase in cellular volume ( n = 10 ) and pros GMC to NB reversion requires approximately 3 . 5 - fold increase in cellular volume ( n = 31 ) .

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

Panel (P) shows that the larval nerfin-1 neuron to NB reversion requires 15-fold increase in cellular volume (n=10) and pros GMC to NB reversion requires approximately 3.5-fold increase in cellular volume (n=31).


A - D ) Representative pictures showing that pros NBs and GMCs are smaller than wildtype ( NBs and GMCs are distinguished at telophase , as a doublet of unequal size ) , quantified in C ( n = 25 , 31 , 21 , 16 ) . pros NBs exhibit similar nucleolus / nuclear ratio ( white arrows , nucleolus marked by Fib ) compared to wild type and pros ; TorDN NBs , quantified in D ( n = 10 , 18 , 15 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in C (n=25,31,21,16). pros NBs exhibit similar nucleolus /nuclear ratio (white arrows, nucleolus marked by Fib) compared to wild type and pros;TorDN NBs, quantified in D (n=10,18,15).


E - F ) Representative pictures showing pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / nuclear ratio ( E - 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 O O O O

E-F) Representative pictures showing pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F)


pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / nuclear ratio ( E - F ) , nucleolus labelled with Fib ( red ) [ quantified in G ( n = 44 , 43 ) ] , clone size [ quantified in H ( n = 41 , 34 ) ] , or the proportion of Dpn + NBs and Elav + neurons per clone [ quantified in I ( n = 15 , 15 , 15 , 15 ) ] .

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

pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F), nucleolus labelled with Fib (red) [quantified in G (n=44,43)], clone size [quantified in H (n=41,34)], or the proportion of Dpn+ NBs and Elav+ neurons per clone [quantified in I (n=15,15,15,15)].


J - K ) Representative pictures showing that nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K )

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

J-K) Representative pictures showing that nerfin-1 clones (green) generated in myc hypomorphic background (J-K)


nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K ) exhibited significantly reduced nucleolus / nuclear ratio , [ nucleolus labelled with Fib ( red ) ] and reduced clone size , quantified in L ( n = 34 , 27 ) and M ( n = 106 , 75 ) , and displayed an increased proportion of differentiated Elav + neurons , and reduced proportion of Dpn + NBs per clone , quantified in N ( n = 20 , 20 , 20 , 20 ) . scale bar =

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

nerfin-1 clones (green) generated in myc hypomorphic background (J-K) exhibited significantly reduced nucleolus/nuclear ratio, [nucleolus labelled with Fib (red)] and reduced clone size, quantified in L (n=34,27) and M (n= 106,75), and displayed an increased proportion of differentiated Elav+ neurons, and reduced proportion of Dpn+ NBs per clone, quantified in N (n=20,20,20,20). scale bar =10μm


O ) Reducing Myc dosage with the hypomorphic Myc allele , mycP0 , abolished the increase in nerfin - 1 clone size mediated by 2x histidine dietary intake , 2x his ( n = 41 , 18 , 10 , 57 ) .

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

O) Reducing Myc dosage with the hypomorphic Myc allele, mycP0, abolished the increase in nerfin-1 clone size mediated by 2x histidine dietary intake, 2x his (n=41,18,10,57).


P ) Overexpression of myc restored the growth of nerfin - 1 clones inhibited by histidine dietary reduction , 25 % his ( n = 51 , 36 , 39 , 29 ) .

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

P) Overexpression of myc restored the growth of nerfin-1 clones inhibited by histidine dietary reduction, 25% his (n=51,36,39,29).


Q , R ) pros ( Q ) and nerfin - 1 ( R ) clones are reduced upon Tor inhibition ( n = 22 , 63 ) compared to control ( n = 51 , 65 ) , but their growth differentially depend on amino acid transporter Slif ( n = 12 , 32 ) and downstream growth regulators eIF4E ( n = 21 , 47 ) and S6K ( n = 39 , 37 ) .

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

Q, R) pros (Q) and nerfin-1 (R) clones are reduced upon Tor inhibition (n=22,63) compared to control (n=51,65), but their growth differentially depend on amino acid transporter Slif ( n=12,32) and downstream growth regulators eIF4E (n= 21,47) and S6K (n=39,37).


The graphs depict the recovery of CAR1 - mcherry ( left ) and tPH - CynA - KikGR ( right ) . Mean ± SEM is shown for n = 2 cells .

O O O O O O O O O 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 graphs depict the recovery of CAR1-mcherry (left) and tPH-CynA-KikGR (right). Mean ± SEM is shown for n = 2 cells.


Time - lapse confocal images of tPH - CynA - KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell . Front and back of the cell 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

Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell. Front and back of the cell are shown.


Time - lapse confocal images of AX3 cell co - expressing PH - Crac - GFP ( green ) and another established PI ( 3 , 4 ) P2 biosensor tPH - TAPP1 - RFP ( red ) . Box shows the vesicle .

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

Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4)P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle.


Time - lapse confocal images of tPH - CynA - KikGR ( green ) expressing wild - type AX3 cells . FM4 - 64 ( red ) was added at time 0 during live cell imaging . Front and back of the cell are shown . Box shows the vesicle .

Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.


Time - lapse confocal images of wild - type AX3 cells expressing tPH - CynA - KikGR ( green ) and PH - crac ( red ) before and after PI3K inhibition by 5 µM Wortmannin .

O O O O O O O O O O O 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

Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin.


Confocal images of tPH - CynA - KikGR ( green ) and PH - Crac ( red ) expressing wild - type Dictyostelium AX3 cells ( left ) and PI3K1 - 2 - Dictyostelium cells ( right ) treated with 5 µM Latrunculin A .

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

Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A.


Time - lapse confocal images showing dynamic localization of PI ( 3 , 4 ) P2 . Nocodazole was added at time 0 during the live cell imaging . Quantification of vesicle number , vesicle number / min , and vesicle lifetime ( s ) from experiment in E . T - test was carried out by GraphPad Prism , * * P ≤ 0 . 01 versus After Nocodazole group ; mean ± SEM ( n = 10 ) .

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

Time-lapse confocal images showing dynamic localization of PI(3,4)P2. Nocodazole was added at time 0 during the live cell imaging.   Quantification of vesicle number, vesicle number/min, and vesicle lifetime (s) from experiment in E. T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus After Nocodazole group; mean±SEM (n = 10).


( L ) Representative confocal images of tPH - CynA - KikGR in wild - type Dictyostelium AX2 or RacH − cells treated with 5 μM latrunculin A Scale bar : 5 μm . ( M ) Ratio of membrane to cytosol intensity of tPH - CynA - KikGR in wild - type Dictyostelium AX2 and RacH − cells . T - test was carried out by GraphPad Prism , * * * * P ≤ 0 . 0001 versus AX2 group ; mean ± SEM ( n = 33 ) .

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

(L) Representative confocal images of tPH-CynA-KikGR in wild-type Dictyostelium AX2 or RacH− cells treated with 5 μM latrunculin A Scale bar: 5 μm. (M) Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wild-type Dictyostelium AX2 and RacH− cells. T-test was carried out by GraphPad Prism, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 33).


Time - lapse confocal images of tPH - CynA - KikGR expressing Dictyostelium AX3 cells . The macropinosome at the front of the cell was photoconverted at time 0 . White arrow points to PI ( 3 , 4 ) P2 localization at the back of the membrane . Cell front and back are shown , box showing the photoconverted region .

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

Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. The macropinosome at the front of the cell was photoconverted at time 0. White arrow points to PI(3,4)P2 localization at the back of the membrane. Cell front and back are shown, box showing the photoconverted region.


Senescence - associated β - galactosidase ( SA - β - gal ) staining in A549 , HAFF , HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses ( shCtrl ) or those targeting GUARDIN ( shGUARDIN ) . The graph shows percentage of SA - β - gal - positive cells .

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

Senescence-associated β-galactosidase (SA-β-gal) staining in A549, HAFF, HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses (shCtrl) or those targeting GUARDIN (shGUARDIN). The graph shows percentage of SA-β-gal-positive cells.


Senescence - associated heterochromatin foci formation ( SAHF ) in A549 cells with either shCtrl or shGUARDIN . Representative IF staining with H3K9me3 antibodies ( green ) and nuclear Hoechst counterstaining ( blue ) .

Senescence-associated heterochromatin foci formation (SAHF) in A549 cells with either shCtrl or shGUARDIN. Representative IF staining with H3K9me3 antibodies (green) and nuclear Hoechst counterstaining (blue).


Secretion of SASP factors IL - 6 and IL - 8 in A549 cells with either shCtrl or shGUARDIN .

Secretion of SASP factors IL-6 and IL-8 in A549 cells with either shCtrl or shGUARDIN.


Western blotting for p21 , p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN - 1 , - 2 , respectively ( left ) . Knockdown efficiencies analyzed by qPCR ( right ) .

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

Western blotting for p21, p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN-1, -2, respectively (left). Knockdown efficiencies analyzed by qPCR (right).


SA - β - gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA ( shp21 ) . The graph shows percentage of SA - β - gal - positive cells .

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

SA-β-gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA (shp21). The graph shows percentage of SA-β-gal-positive cells.


qPCR measurement of p21 mRNA in A549 , HepG2 , HAFF and H1299 cells with either GUARDIN shRNA knockdown ( top ) or GUARDIN overexpression ( bottom ) .

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

qPCR measurement of p21 mRNA in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).


Western blotting for p53 and p21 in A549 , HepG2 , HAFF and H1299 cells with either GUARDIN shRNA knockdown ( top ) or GUARDIN overexpression ( bottom ) .

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

Western blotting for p53 and p21 in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).


qPCR assays of NOXA , TIGAR , BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN .

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

qPCR assays of NOXA, TIGAR, BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN.


SDS - PAGE of RNA pulldown assays using biotin - labelled sense / antisense probes against GUARDIN from whole - cell lysates of A549 cells indicating putative GUARDIN - binding proteins ( left ) ; protein identities with high probabilities were determined by mass spectrometry ( right ) .

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

SDS-PAGE of RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells indicating putative GUARDIN-binding proteins (left); protein identities with high probabilities were determined by mass spectrometry (right).


RNA pulldown assays interrogating putative GUARDIN - associated proteins identified in ( A ) from whole - cell lysates of A549 and HAFF cells . BRCA1 , BARD1 served as positive controls and β - actin served as negative controls .

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

RNA pulldown assays interrogating putative GUARDIN-associated proteins identified in (A) from whole-cell lysates of A549 and HAFF cells. BRCA1, BARD1 served as positive controls and β-actin served as negative controls.


RNA immunoprecipitation ( RIP ) assays against IgG / LRP130 antibodies in whole - cell lysates of A549 cells .

RNA immunoprecipitation (RIP) assays against IgG/LRP130 antibodies in whole-cell lysates of A549 cells.


Subcellular localization of GUARDIN and its co - localization with LRP130 . RNA FISH for GUARDIN ( red ) and IF for LRP130 ( green ) in A549 cells with either shCtrl or shGUARDIN . Nucleus were counterstained with Hoechst ( blue ) .

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

Subcellular localization of GUARDIN and its co-localization with LRP130. RNA FISH for GUARDIN (red) and IF for LRP130 (green) in A549 cells with either shCtrl or shGUARDIN. Nucleus were counterstained with Hoechst (blue).


RNA pulldown assays using biotin - labelled sense / antisense probes against GUARDIN from whole - cell lysates of A549 cells . GUARDIN levels were measured by RT - PCR and co - precipitated LRP130 and PGC1α detected by Western blotting . BRCA1 and β - actin served as positive and negative controls , respectively .

RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells. GUARDIN levels were measured by RT-PCR and co-precipitated LRP130 and PGC1α detected by Western blotting. BRCA1 and β-actin served as positive and negative controls, respectively.


RIP assay using IgG / PGC1α antibodies from whole - cell lysates of A549 cells . GUARDIN , LRP130 and PGC1α levels were measured as per ( E ) .

RIP assay using IgG/PGC1α antibodies from whole-cell lysates of A549 cells. GUARDIN, LRP130 and PGC1α levels were measured as per (E).


Two - step IP assays in whole - cell lysates of A549 cells transfected with FLAG - tagged PGC1α . First phase IPs were conducted with FLAG antibodies ( left ) and following elution with FLAG peptides , eluates were further subjected to second phase IPs with LRP130 antibodies ( right ) . Samples were subjected to Western blotting and qPCR analysis for LRP130 , PGC1α and GUARDIN , respectively .

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

Two-step IP assays in whole-cell lysates of A549 cells transfected with FLAG-tagged PGC1α. First phase IPs were conducted with FLAG antibodies (left) and following elution with FLAG peptides, eluates were further subjected to second phase IPs with LRP130 antibodies (right). Samples were subjected to Western blotting and qPCR analysis for LRP130, PGC1α and GUARDIN, respectively.


Co - immunoprecipitation ( co - IP ) between LRP130 and PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN . LRP130 was precipitated and samples subjected to Western blotting analysis for LRP130 , PGC1α and β - actin as loading control .

Co-immunoprecipitation (co-IP) between LRP130 and PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. LRP130 was precipitated and samples subjected to Western blotting analysis for LRP130, PGC1α and β-actin as loading control.


Mammalian two - hybrid assays between pACT - LRP130 and pBIND - PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN . Samples were subjected to the luciferase activity assays .

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

Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. Samples were subjected to the luciferase activity assays.


LRP130 / PGC1α and p21 protein expression was measured by Western blot in A549 and HAFF cells after 48h transduction with shCtrl or shLRP130 ( top left ) or shPGC1α ( bottom left ) as indicated . qPCR assays for p21 mRNA levels were performed in parallel ( right panels ) .

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

LRP130/PGC1α and p21 protein expression was measured by Western blot in A549 and HAFF cells after 48h transduction with shCtrl or shLRP130 (top left) or shPGC1α (bottom left) as indicated. qPCR assays for p21 mRNA levels were performed in parallel (right panels).


Western blotting analysis of LRP130 , PGC1α and p21 protein levels in HAFF and A549 cells after 48h transduction with shCtrl or shGUARDIN .

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

Western blotting analysis of LRP130, PGC1α and p21 protein levels in HAFF and A549 cells after 48h transduction with shCtrl or shGUARDIN.


ChIP assays detecting binding of LRP130 / PGC1α to the p21 promoter using qPCR and RT - PCR ( top left and right , respectively ) . IgG and p53 served as a negative and positive controls , respectively . Schematic illustrations of the putative LRP130 / PGC1α binding sites within DNase I hypersensitive regions of the p21 promoter ( bottom ) .

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

ChIP assays detecting binding of LRP130/PGC1α to the p21 promoter using qPCR and RT-PCR (top left and right, respectively). IgG and p53 served as a negative and positive controls, respectively. Schematic illustrations of the putative LRP130/PGC1α binding sites within DNase I hypersensitive regions of the p21 promoter (bottom).


qPCR assays for GUARDIN , FOXO1 , FOXO3a and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN .

qPCR assays for GUARDIN, FOXO1, FOXO3a and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN.


Western blotting assays for FOXO4 and p21 protein expression in A549 cells after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4 . β - actin served as loading 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

Western blotting assays for FOXO4 and p21 protein expression in A549 cells after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4. β-actin served as loading control.


Half - life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg / ml of actinomycin ( ActD ) for the indicated times .

O O 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 B-CONTROLLED_VAR O O O O O O

Half-life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg/ml of actinomycin (ActD) for the indicated times.


ChIP assays detecting binding of LRP130 / PGC1α to putative binding sites in the FOXO4 promoter using qPCR ( top ) . Data were normalized to the IgG negative control . Schematic illustration of the LRP130 / PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter ( bottom ) .

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

ChIP assays detecting binding of LRP130/PGC1α to putative binding sites in the FOXO4 promoter using qPCR (top). Data were normalized to the IgG negative control. Schematic illustration of the LRP130/PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter (bottom).


Luciferase assays conducted in A549 cells transfected with LRP130 or PGC1α using the pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids .

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

Luciferase assays conducted in A549 cells transfected with LRP130 or PGC1α using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.


qPCR assays for LRP130 and FOXO4 mRNA levels ( upper ) and Western blotting analysis for LRP130 protein level ( lower ) in A549 cells after 48h transduction with LRP130 overexpression or knockdown .

qPCR assays for LRP130 and FOXO4 mRNA levels (upper) and Western blotting analysis for LRP130 protein level (lower) in A549 cells after 48h transduction with LRP130 overexpression or knockdown.


qPCR assays for PGC1α and FOXO4 mRNA level ( upper ) and Western blotting analysis for PGC1α protein level ( lower ) in A549 cells after 48h transduction with PGC1α overexpression or knockdown .

qPCR assays for PGC1α and FOXO4 mRNA level (upper) and Western blotting analysis for PGC1α protein level (lower) in A549 cells after 48h transduction with PGC1α overexpression or knockdown.


Luciferase activity assays in A549 cells transduced with shCtrl or shGUARDIN using the pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids .

Luciferase activity assays in A549 cells transduced with shCtrl or shGUARDIN using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.


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qPCR assays for GUARDIN expression in A549 cells treated with increasing doses rapamycin for 24h


qPCR assays for GUARDIN expression in A549 cells treated with 100nM of rapamycin for the indicated times ( B ) .

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qPCR assays for GUARDIN expression in A549 cells treated with 100nM of rapamycin for the indicated times (B).


Western blotting assays for p53 , LRP130 , FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle ( DMSO ) for 48h .

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Western blotting assays for p53, LRP130, FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle (DMSO) for 48h.


A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h . Western blotting was used to measure p21 levels ( left ) while conducting SA - β - gal staining in parallel ( middle ) with the percentage of senescent cells calculated from the SA - β - gal staining ( right ) .

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A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h. Western blotting was used to measure p21 levels (left) while conducting SA-β-gal staining in parallel (middle) with the percentage of senescent cells calculated from the SA-β-gal staining (right).


Mammalian two - hybrid assays between pACT - LRP130 and pBIND - PGC1α in A549 cells treated with DMSO or 100nM rapamycin . Samples were subjected to the luciferase activity assays .

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Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells treated with DMSO or 100nM rapamycin. Samples were subjected to the luciferase activity assays.


SIX4 and FOSL2 mRNA ( left ) and protein levels ( right ) measured by qPCR and Western blotting , respectively in A549 cells treated with DMSO or 100nM rapamycin . TBP served as a control .

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SIX4 and FOSL2 mRNA (left) and protein levels (right) measured by qPCR and Western blotting, respectively in A549 cells treated with DMSO or 100nM rapamycin. TBP served as a control.


qPCR ( upper ) and Western blotting ( lower ) assays for GUARDIN , SIX4 and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 ( left ) , shFOSL2 ( middle ) or FOSL2 ( right ) after transduction for 48h .

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qPCR (upper) and Western blotting (lower) assays for GUARDIN, SIX4 and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 (left), shFOSL2 (middle) or FOSL2 (right) after transduction for 48h.


Luciferase assays in A549 cells transduced with shCtrl , shSIX4 or shFOSL2 using pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids .

Luciferase assays in A549 cells transduced with shCtrl, shSIX4 or shFOSL2 using pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.


ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT - PCR assay . UPe and UPk served as a positive and negative controls , respectively [ 54 ] .

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ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT-PCR assay. UPe and UPk served as a positive and negative controls, respectively [54].


( D ) Heatmap showing the expression change of representative NF - κB target genes from the genes with significantly changed expression ( fold change > 2 ) . The data represent the log2 ( gene FPKM ) normalized for each row . RNA - seq data is from 2 independent experiments .

(D) Heatmap showing the expression change of representative NF-κB target genes from the genes with significantly changed expression (fold change >2). The data represent the log2 (gene FPKM) normalized for each row. RNA-seq data is from 2 independent experiments.


( E ) Intracellular cytokine staining assay of human TNF - α and IL - 2 in sh - NC and sh - p38IP Jurkat E6 . 1 cells stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for 6 hours ( average frequency of positive staining YFP + cells in sh - NC cells without stimulation = 1 ) .

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(E) Intracellular cytokine staining assay of human TNF-α and IL-2 in sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for 6 hours (average frequency of positive staining YFP+ cells in sh-NC cells without stimulation = 1).


( F ) ELISA analysis of IL - 2 and IL - 6 production in the culture supernatants of human PBMCs transfected with si - NC or si - p38IP , and then stimulated with 10 μg / ml anti - CD3 plus 10 μg / ml anti - CD28 ( αCD3 / CD28 ) for 24 hours .

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(F) ELISA analysis of IL-2 and IL-6 production in the culture supernatants of human PBMCs transfected with si-NC or si-p38IP, and then stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 (αCD3/CD28) for 24 hours.


( G and H ) Real - time PCR analysis of TNF - α , IL - 6 and IL - 1β ( G ) and of IFN - β , ISG54 and ISG56 ( H ) mRNA production in RAW264 . 7 cells transfected with si - NC and si - mp38IP - 1 and then stimulated with 500 ng / ml LPS for the indicated times ( average mRNA production in si - NC cells at 0 hour = 1 ) .

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(G and H) Real-time PCR analysis of TNF-α, IL-6 and IL-1β (G) and of IFN-β, ISG54 and ISG56 (H) mRNA production in RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times (average mRNA production in si-NC cells at 0 hour = 1).


( I ) ELISA analysis of TNF - α , IL - 6 , IL - 1β and IFN - β production in the culture supernatants of human PBMCs transfected with si - NC and si - p38IP and then stimulated with 1μg / mL LPS for 24 hours .

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(I) ELISA analysis of TNF-α, IL-6, IL-1β and IFN-β production in the culture supernatants of human PBMCs transfected with si-NC and si-p38IP and then stimulated with 1μg/mL LPS for 24 hours.


( A ) Immunoblot analysis with the indicated antibodies of total cell lysates of sh - NC and sh - p38IP Jurkat E6 . 1 cells stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times . Actin served as a loading control ( throughout ) .

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(A) Immunoblot analysis with the indicated antibodies of total cell lysates of sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times. Actin served as a loading control (throughout).


( B ) Statistical analysis of p65 nuclear translocation of sh - NC and sh - p38IP Jurkat E6 . 1 T cells stimulated with 50 ng / ml PMA plus 1µM ionomycin for the indicated times . p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell . * * * P < 0 . 001 ( two - tailed unpaired t - test , mean and s . e . m . ) .

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(B) Statistical analysis of p65 nuclear translocation of sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated with 50 ng/ml PMA plus 1µM ionomycin for the indicated times. p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).


( C ) Luciferase reporter assay of sh - NC and sh - p38IP Jurkat E6 . 1 cells transfected with NF - κB luc plus an internal control Renilla luciferase reporter for 36 hours , followed by stimulation with ( + ) or without ( - ) 50ng / ml PMA plus 1 µM ionomycin for 8 hours . * * * P < 0 . 001 ; n . s . , not significant ( two - tailed unpaired t - test , mean and s . e . m . ) .

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(C) Luciferase reporter assay of sh-NC and sh-p38IP Jurkat E6.1 cells transfected with NF-κB luc plus an internal control Renilla luciferase reporter for 36 hours, followed by stimulation with ( + ) or without ( - ) 50ng/ml PMA plus 1 µM ionomycin for 8 hours. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).


( D , E ) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264 . 7 cells transfected with si - NC and si - mp38IP - 1 and then stimulated with 500 ng / ml LPS for the indicated times .

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(D,E) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times.


( A ) Fragmentation spectrum of the p38IP peptide identified in TAK1 complex immunoprecipitated from Jurkat TAg cells by liquid chromatography - tandem mass spectrometry . m / z , mass / charge ratio .

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(A) Fragmentation spectrum of the p38IP peptide identified in TAK1 complex immunoprecipitated from Jurkat TAg cells by liquid chromatography-tandem mass spectrometry. m/z, mass/charge ratio.


( B ) HEK293T cells were transfected with YFP - p38IP , and the cell lysates aliquots were immunoprecipitated with IgG , anti - TAB1 , anti - TAB2 and anti - TAK1 respectively , and then immunoblotted with the indicated antibodies .

(B) HEK293T cells were transfected with YFP-p38IP, and the cell lysates aliquots were immunoprecipitated with IgG, anti-TAB1, anti-TAB2 and anti-TAK1 respectively, and then immunoblotted with the indicated antibodies.


( C ) sh - NC or sh - p38IP Jurkat E6 . 1 T cells were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times and lysed , followed byTAK1 immunoprecipitation . TAK1 beads were washed three times with lysis buffer and two times with kinase buffer , then resuspended in kinase buffer containing 100 μM ATP and 1μg recombinant GST - IKKβ ( 111 - 250aa ) protein as substrate to perform in vitro kinase assay , and then immunoblot analysis with p - IKKβ antibody .

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(C) sh-NC or sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times and lysed, followed byTAK1 immunoprecipitation. TAK1 beads were washed three times with lysis buffer and two times with kinase buffer, then resuspended in kinase buffer containing 100 μM ATP and 1μg recombinant GST-IKKβ(111-250aa) protein as substrate to perform in vitro kinase assay, and then immunoblot analysis with p-IKKβ antibody.


( D ) HEK293T cells were transfected with cMyc vector ( - ) or increasing amounts of cMyc - p38IP ( wedge ) , and 24 hours post transfection , the cell lysates were immunoprecipitated with IgG or anti - TAB2 and then immunoblotted with anti - TAK1 and anti - TAB2 . Expression of cMyc - p38IP was determined in the immunoblotted lysates with anti - cMyc .

(D) HEK293T cells were transfected with cMyc vector ( - ) or increasing amounts of cMyc-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAB2 and then immunoblotted with anti-TAK1 and anti-TAB2. Expression of cMyc-p38IP was determined in the immunoblotted lysates with anti-cMyc.


( E ) HEK293T cells were transfected with HA vector ( - ) or increasing amount of HA - p38IP ( wedge ) , and 24 hours post transfection , the cell lysates were immunoprecipitated with IgG or anti - TAK1 and then immunoblotted with anti - TAB2 , anti - TAB1 , anti - HA and anti - TAK1 . Expression of HA - p38IP was determined in the immunoblotted lysates with anti - HA .

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(E) HEK293T cells were transfected with HA vector ( - ) or increasing amount of HA-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAK1 and then immunoblotted with anti-TAB2, anti-TAB1, anti-HA and anti-TAK1. Expression of HA-p38IP was determined in the immunoblotted lysates with anti-HA.


( F ) sh - NC and sh - p38IP Jurkat E6 . 1 T cells were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times , and the cell lysates were immunoprecipitated with anti - TAB2 and immunoblotted with anti - TAK1 and anti - TAB2 .

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(F) sh-NC and sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and the cell lysates were immunoprecipitated with anti-TAB2 and immunoblotted with anti-TAK1 and anti-TAB2.


( H ) Jurkat TAg T cells transfected with cMyc vector or cMyc - p38IP were stimulated with 10μg / ml anti - CD3 for the indicated times , and then were lysed and immunoprecipitated with anti - TAK1 antibody and immunoblotted with anti - K63Ub , anti - cMyc and anti - TAK1 antibodies . Lysates were blotted with the indicated antibodies , and actin served as the loading control .

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(H) Jurkat TAg T cells transfected with cMyc vector or cMyc-p38IP were stimulated with 10μg/ml anti-CD3 for the indicated times, and then were lysed and immunoprecipitated with anti-TAK1 antibody and immunoblotted with anti-K63Ub, anti-cMyc and anti-TAK1 antibodies. Lysates were blotted with the indicated antibodies, and actin served as the loading control.


( I ) sh - NC and sh - p38IP Jurkat E6 . 1 T cells stimulated as in ( F ) were lysed and immunoprecipitated with anti - TAK1 and immunoblotted with anti - K63Ub and anti - TAK1 . Lysates were immunoblotted with the indicated antibodies .

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(I) sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-K63Ub and anti-TAK1. Lysates were immunoblotted with the indicated antibodies.


( J ) Jurkat TAg T cells transfected and stimulated as in ( H ) were lysed and immunoprecipitated with anti - TAB2 and immunoblotted with anti - K63Ub and anti - TAB2 .

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(J) Jurkat TAg T cells transfected and stimulated as in (H) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2.


( K ) sh - NC and sh - p38IP Jurkat E6 . 1 cells stimulated as in ( F ) were lysed and immunoprecipitated with anti - TAB2 and immunoblotted with anti - K63Ub and anti - TAB2 . Data ( B - F , H - K ) are representatives of at least three independent experiments .

(K) sh-NC and sh-p38IP Jurkat E6.1 cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2. Data (B-F, H-K) are representatives of at least three independent experiments.


( A ) Jurkat E6 . 1 T cells were stimulated with 10 μg / ml anti - CD3 and 2 μg / ml anti - CD28 for the indicated times , the cell lysates were immunoprecipitated and immunoblotted as indicated . The relative intensity of the IP bands was quantified by densitometry and is shown below the graph .

(A) Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for the indicated times, the cell lysates were immunoprecipitated and immunoblotted as indicated. The relative intensity of the IP bands was quantified by densitometry and is shown below the graph.


( B ) HEK293T cells transfected with Flag - tagged TAB2 ( open arrow ) or TAB2 - ub ( solid arrow ) were lysed and immunoprecipitated with anti - Flag , and then immunoblotted with the indicated antibodies . The asterisk indicates a non - specific band . The relative intensity of the IP bands was quantified by densitometry and is presented in the right .

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(B) HEK293T cells transfected with Flag-tagged TAB2 (open arrow) or TAB2-ub (solid arrow) were lysed and immunoprecipitated with anti-Flag, and then immunoblotted with the indicated antibodies. The asterisk indicates a non-specific band. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.


( C ) HEK293T cells co - transfected with HA - tagged p38IP together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) were lysed and immunoprecipitated with anti - cMyc , and then immunoblotted with the indicated antibodies . Asterisk indicates non - specific bands . The relative intensity of the IP bands was quantified by densitometry and is presented in the right .

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(C) HEK293T cells co-transfected with HA-tagged p38IP together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow) were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. Asterisk indicates non-specific bands. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.


( D ) HEK293T cells co - transfected with HA - tagged p38IP , Flag - tagged TAB2 - ub , together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) , were lysed and immunoprecipitated with anti - cMyc , and then immunoblotted with the indicated antibodies . The relative intensity of the IP bands was quantified by densitometry and is presented in the right .

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(D) HEK293T cells co-transfected with HA-tagged p38IP, Flag-tagged TAB2-ub, together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow), were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.


( A ) GSH - Sepharose beads - immobilized full length or truncated GST - TAK1 proteins were incubated with K63 - linked polyUb chains in 300 μl binding buffer , followed by washing and resolving in SDS - PAGE , and then immunoblotted with anti - ub . GST - fused NEMO serve as positive control and NEMO L329P mutant as negative control . The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST - fused proteins ( lower panel ) .

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(A) GSH-Sepharose beads-immobilized full length or truncated GST-TAK1 proteins were incubated with K63-linked polyUb chains in 300 μl binding buffer, followed by washing and resolving in SDS-PAGE, and then immunoblotted with anti-ub. GST-fused NEMO serve as positive control and NEMO L329P mutant as negative control. The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST-fused proteins (lower panel).