( K ) FACS - isolated SCs from YY1iKO mice were infected with YY1 expressing or control viruses , followed by immunostaining for Pax7 ( green ) and MyoD ( red ) 36 hrs post - transfection . Scale bar = 100 µm . Quantifications of the numbers of each population , Pax7 + MyoD - , Pax7 - MyoD + , or Pax7 + MyoD + , are shown on the right ( n = 3 mice , each ) .

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(K) FACS-isolated SCs from YY1iKO mice were infected with YY1 expressing or control viruses, followed by immunostaining for Pax7 (green) and MyoD (red) 36 hrs post-transfection. Scale bar = 100 µm. Quantifications of the numbers of each population, Pax7+MyoD-, Pax7-MyoD+, or Pax7+MyoD+, are shown on the right (n=3 mice, each).


( L ) Single EDL myofibers were cultured for 3 days , followed by immunostaining for Myogenin ( red ) and MyoD ( green ) . Scale bar = 50 µm . Quantifications of the numbers of each population , MyoD + Myog + , MyoD - Myog + , or MyoD + Myog - , are shown on the right ( n = 3 mice , each ) .

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(L) Single EDL myofibers were cultured for 3 days, followed by immunostaining for Myogenin (red) and MyoD (green). Scale bar = 50 µm. Quantifications of the numbers of each population, MyoD+Myog+, MyoD-Myog+, or MyoD+Myog-, are shown on the right (n=3 mice, each).


( M ) FACS - isolated SCs were cultured for 2 days in proliferation medium followed by 2 days in differentiation medium ; the degree of differentiation was assessed by IF staining of MF20 + . Scale bar = 100 µm . Quantifications of the numbers of MF20 + area per nucleus are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(M) FACS-isolated SCs were cultured for 2 days in proliferation medium followed by 2 days in differentiation medium; the degree of differentiation was assessed by IF staining of MF20+. Scale bar = 100 µm. Quantifications of the numbers of MF20+ area per nucleus are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.


( A ) RNA - seq was performed with RNAs extracted from ASCs ( FACS purified and cultured for 36hrs ) of YY1iKO or Ctrl mice ; scatter plot shows differentially expressed genes with a fold change > = 2 ( red dots ) in YY1iKO vs Ctrl .

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(A) RNA-seq was performed with RNAs extracted from ASCs (FACS purified and cultured for 36hrs) of YY1iKO or Ctrl mice; scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl.


( C ) Genomic snapshots showing the examples of mitochondrial genes ( Uqcr11 , Mrp11 , Apo5o and Nme4 ) up - regulated in YY1iKO vs Ctrl cells .

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(C) Genomic snapshots showing the examples of mitochondrial genes (Uqcr11, Mrp11, Apo5o and Nme4) up-regulated in YY1iKO vs Ctrl cells.


( D ) GSEA analysis shows oxidative phosphorylation gene set is enriched in YY1iKO vs Ctrl cells ( p - value < 0 . 0001 ) .

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(D) GSEA analysis shows oxidative phosphorylation gene set is enriched in YY1iKO vs Ctrl cells (p-value < 0.0001).


( E ) RT - qPCR validation of the expression of the selected up - regulated mitochondrial genes in YY1iKO vs Ctrl .

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(E) RT-qPCR validation of the expression of the selected up-regulated mitochondrial genes in YY1iKO vs Ctrl.


( F ) Protein levels of the respiratory chain complexes I - V components in YY1iKO vs Ctrl ASCs were detected using a mitoprofile antibody cocktail . β - actin was used as the loading control .

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(F) Protein levels of the respiratory chain complexes I-V components in YY1iKO vs Ctrl ASCs were detected using a mitoprofile antibody cocktail. β-actin was used as the loading control.


( G ) RNA - seq was performed using RNAs from freshly isolated SCs ( FISCs ) ; scatter plot shows differentially expressed genes with a fold change > = 2 ( red dots ) in YY1iKO vs Ctrl .

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(G) RNA-seq was performed using RNAs from freshly isolated SCs (FISCs); scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl.


( I ) Genomic snapshots show the selected mitochondrial genes ( Cox8b , Nme4 , Slc25a29 , Nnt ) up - regulated in YY1iKO vs Ctrl FISCs .

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(I) Genomic snapshots show the selected mitochondrial genes (Cox8b, Nme4, Slc25a29, Nnt) up-regulated in YY1iKO vs Ctrl FISCs.


( J ) ChIPseq was performed using chromatins from WT ASCs and the genomic distribution of 2031 YY1 binding peaks is shown .

(J) ChIPseq was performed using chromatins from WT ASCs and the genomic distribution of 2031 YY1 binding peaks is shown.


( N ) Genomic snapshots of two of the above identified mitochondrial genes ( Tomm40I and Ndufa13 ) that are bound by YY1 in their TSS ( ChIPseq tracks ) and up - regulated by YY1 deletion ( RNAseq tracks ) .

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(N) Genomic snapshots of two of the above identified mitochondrial genes (Tomm40I and Ndufa13) that are bound by YY1 in their TSS (ChIPseq tracks) and up-regulated by YY1 deletion (RNAseq tracks).


( O ) ChIP - qPCR validation of YY1 binding on some mitochondrial genes . Negative control ( NC ) represents a genomic region on Chromosome 11 with no YY1 binding peak identified . Enrichment fold was calculated as the amount of amplified DNA from YY1 binding sites normalized to values obtained from IgG control .

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(O) ChIP-qPCR validation of YY1 binding on some mitochondrial genes. Negative control (NC) represents a genomic region on Chromosome 11 with no YY1 binding peak identified. Enrichment fold was calculated as the amount of amplified DNA from YY1 binding sites normalized to values obtained from IgG control.


ChIP - qPCR was performed to show the enrichment of Ezh2 ( P ) binding on selected mitochondrial genes .

ChIP-qPCR was performed to show the enrichment of Ezh2 (P) binding on selected mitochondrial genes.


ChIP - qPCR was performed to show the enrichment of Suz12 ( Q ) binding on selected mitochondrial genes .

ChIP-qPCR was performed to show the enrichment of Suz12 (Q) binding on selected mitochondrial genes.


ChIP - qPCR was performed to show the enrichment of H3K27 me3 ( R ) binding on selected mitochondrial genes .

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ChIP-qPCR was performed to show the enrichment of H3K27 me3 (R) binding on selected mitochondrial genes.


( S ) A construct to express wild type YY1 ( WT ) , or two mutants lacking DNA binding activity , YY1 ( 1 - 394 ) and YY1 ( S365D ) were transfected into YY1iKO cells ; the expression of mitochondrial gene was measured . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(S) A construct to express wild type YY1(WT), or two mutants lacking DNA binding activity, YY1(1-394) and YY1(S365D) were transfected into YY1iKO cells; the expression of mitochondrial gene was measured. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


( A ) An equal number of FACS - isolated SCs from C57 mice were cultured for 24 hrs and treated with 0 , 1 , 10 , or 20mM 2 - DG ( glycolytic inhibitor ) or NaN3 ( respiration inhibitor ) for 3 hrs before measurement of ATP production . The relative ATP levels normalized to the 0 mM values are plotted . N = 3 mice .

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(A) An equal number of FACS-isolated SCs from C57 mice were cultured for 24 hrs and treated with 0, 1, 10, or 20mM 2-DG (glycolytic inhibitor) or NaN3 (respiration inhibitor) for 3 hrs before measurement of ATP production. The relative ATP levels normalized to the 0 mM values are plotted. N=3 mice.


( B ) SCs from C57 mice were treated with 10mM 2 - DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS assay ( n = 3 mice , each ) .

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(B) SCs from C57 mice were treated with 10mM 2-DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS assay (n=3 mice, each).


( C ) SCs from C57 mice were treated with 10mM 2 - DG for 24 hrs and subject to EdU labeling for 6 hrs . The percentage of Pax7 + / EdU + cells over the total number of Pax7 + cells was quantified ( n = 3 mice , each ) .

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(C) SCs from C57 mice were treated with 10mM 2-DG for 24 hrs and subject to EdU labeling for 6 hrs. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified (n=3 mice, each).


( D ) Ctrl and YY1iKO ASCs were stained with 60 ug / mL 2 - NBDG for 45 mins and the fluorescence intensity ( MFI ) of 2 - NBDG was measured by flow cytometry ( n = 3 mice , each ) .

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(D) Ctrl and YY1iKO ASCs were stained with 60 ug/mL 2-NBDG for 45 mins and the fluorescence intensity (MFI) of 2-NBDG was measured by flow cytometry (n=3 mice, each).


An equal number of Ctrl and YY1iKO cells were cultured for 36h ; basal extracellular acidification rate ( ECAR ) level were measured and normalized with cell numbers . N = 3 mice .

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An equal number of Ctrl and YY1iKO cells were cultured for 36h; basal extracellular acidification rate (ECAR) level were measured and normalized with cell numbers. N=3 mice.


An equal number of Ctrl and YY1iKO cells were cultured for 36h Lactate concentration ( F ) were measured and normalized with cell numbers . N = 3 mice .

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An equal number of Ctrl and YY1iKO cells were cultured for 36h Lactate concentration (F) were measured and normalized with cell numbers. N=3 mice. by


An equal number of Ctrl and YY1iKO cells were cultured for 36h ATP production ( G ) were measured and normalized with cell numbers . N = 3 mice .

An equal number of Ctrl and YY1iKO cells were cultured for 36h ATP production (G) were measured and normalized with cell numbers. N=3 mice.


( H ) Genomic snapshots depict RNAseq profiles of the selected glycolytic genes ( Hk2 , Glut1 , Ldha , Pdk1 , Eno1 ) down - regulated in YY1iKO vs Ctrl ASCs .

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(H) Genomic snapshots depict RNAseq profiles of the selected glycolytic genes (Hk2, Glut1, Ldha, Pdk1, Eno1) down-regulated in YY1iKO vs Ctrl ASCs.


( I ) Expression of the selected glycolytic genes was quantified by RT - qPCR . Hsp90ab1 was used as the normalization control .

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(I) Expression of the selected glycolytic genes was quantified by RT-qPCR. Hsp90ab1 was used as the normalization control.


( J ) Re - expression of YY1 by transfecting YY1iKO ASCs with a YY1 WT expressing plasmid led to up - regulation of glycolytic genes including Glut1 , Pfkl , Gapdh , Eno1 , Pgam1 , Pkm . Hsp90ab1 was used as the normalization control .

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(J) Re-expression of YY1 by transfecting YY1iKO ASCs with a YY1 WT expressing plasmid led to up-regulation of glycolytic genes including Glut1, Pfkl, Gapdh, Eno1, Pgam1, Pkm. Hsp90ab1 was used as the normalization control.


( K ) FACS - isolated SCs from Pax7 - nGFP mice were transfected with Hif1α or control siRNAs and EdU labeled for 5 hrs . The percentage of MyoD + EdU + cells over the total number of MyoD + cells was quantified . Scale bar = 100 µm . N = 3 mice .

(K) FACS-isolated SCs from Pax7-nGFP mice were transfected with Hif1α or control siRNAs and EdU labeled for 5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.


( L ) The expression of glycolytic genes and cell cycle related genes was detected by RT - qPCR in the above transfected cells . β - actin was used as the normalization control .

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(L) The expression of glycolytic genes and cell cycle related genes was detected by RT-qPCR in the above transfected cells. β-actin was used as the normalization control.


( M ) Hif1α protein level was detected by Western blotting in YY1iKO vs Ctrl ASCs . β - actin was used as the loading control .

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(M) Hif1α protein level was detected by Western blotting in YY1iKO vs Ctrl ASCs. β-actin was used as the loading control.


( N ) Left : Image of flow cytometric analysis of Hif1α protein or IgG control in YY1iKO vs Ctrl ASCs . Right : Relative mean fluorescence intensity ( MFI ) of Hif1α protein is shown . N = 3 mice .

(N) Left: Image of flow cytometric analysis of Hif1α protein or IgG control in YY1iKO vs Ctrl ASCs. Right: Relative mean fluorescence intensity (MFI) of Hif1α protein is shown. N=3 mice.


( O ) YY1iKO ASCs were transfected with empty YY1 WT viruses ; the relative expression level of Hif1a protein was detected by the above described flow cytometric analysis N = 3 mice . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(O) YY1iKO ASCs were transfected with empty YY1 WT viruses; the relative expression level of Hif1a protein was detected by the above described flow cytometric analysis N=3 mice. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


( A - B ) Ctrl or YY1iKO ASCs were treated with CHX ( Cycloheximide ) for 90 mins and Hif1α protein was measured by flow cytometry . The numbers indicate the degradation rate after CHX treatment . N = 3 mice . ( C ) The relative degradation rate was calculated by the ratio of degradation rate in YY1iKO ( 11 . 14 % ± 1 . 71 % ) vs Ctrl ( 5 . 08 % ± 0 . 796 % ) . N = 3 mice .

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(A-B) Ctrl or YY1iKO ASCs were treated with CHX (Cycloheximide) for 90 mins and Hif1α protein was measured by flow cytometry. The numbers indicate the degradation rate after CHX treatment. N=3 mice. (C) The relative degradation rate was calculated by the ratio of degradation rate in YY1iKO (11.14% ± 1.71%) vs Ctrl (5.08% ± 0.796%). N=3 mice.


( D ) Lysates from C2C12 myoblasts ( GM ) were subjected to co - immunoprecipitation ( Co - IP ) assays with anti - YY1 or anti - IgG antibodies and blotted with anti - Hif1α or anti - YY1 antibodies .

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(D) Lysates from C2C12 myoblasts (GM) were subjected to co-immunoprecipitation (Co-IP) assays with anti-YY1 or anti-IgG antibodies and blotted with anti-Hif1α or anti-YY1 antibodies.


( E ) The above lysates were also IPed with anti - Hif1α or anti - IgG antibodies and blotted with anti - YY1 or anti - Hif1α antibodies .

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(E) The above lysates were also IPed with anti-Hif1α or anti-IgG antibodies and blotted with anti-YY1 or anti-Hif1α antibodies.


( F ) ASCs from YY1iKO mice were infected with pRlenti - Hif1α ( P402A / P564A ) ( Hif1α - DM ) viruses to overexpress a non - degradable Hif1α protein or GFP - expressing viruses . ASCs from Ctrl mice were also infected with the GFP viruses . 36 hrs after infection , the expression of target glycolytic genes was detected by RT - qPCR . Hsp90ab1 was used as the normalization control .

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(F) ASCs from YY1iKO mice were infected with pRlenti-Hif1α (P402A/P564A) (Hif1α-DM) viruses to overexpress a non-degradable Hif1α protein or GFP-expressing viruses. ASCs from Ctrl mice were also infected with the GFP viruses. 36 hrs after infection, the expression of target glycolytic genes was detected by RT-qPCR. Hsp90ab1 was used as the normalization control.


( G ) SCs were isolated from Ctrl or YY1iKO mice and infected with the above viruses . EdU labeling was then performed for 5 . 5 hrs . The percentage of MyoD + EdU + cells over the total number of MyoD + cells was quantified . Scale bar = 100 µm . N = 3 mice .

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(G) SCs were isolated from Ctrl or YY1iKO mice and infected with the above viruses. EdU labeling was then performed for 5.5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.


Heat maps indicating gene expression levels ( Log2 [ FPKM ] ) of pentose cycle enzymes ( H ) in YY1iKO vs Ctrl .

Heat maps indicating gene expression levels (Log2[FPKM]) of pentose cycle enzymes (H) in YY1iKO vs Ctrl.


Heat maps indicating gene expression levels ( Log2 [ FPKM ] ) of amino acid transporter ( I ) in YY1iKO vs Ctrl .

Heat maps indicating gene expression levels (Log2[FPKM]) of amino acid transporter (I) in YY1iKO vs Ctrl.


( J ) YY1 ( WT ) or YY1 ( S365D ) mutant was expressed in YY1iKO ASCs and EdU labeled for 6 hrs . ( K ) Similarly , YY1 ( WT ) or YY1 ( 1 - 394 ) mutant was expressed and EdU labeled for 6hrs . A GFP expressing plasmid was used the negative control . The percentage of Pax7 + / EdU + cells over the total number of Pax7 + cells was quantified . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(J) YY1(WT) or YY1(S365D) mutant was expressed in YY1iKO ASCs and EdU labeled for 6 hrs. (K) Similarly, YY1 (WT) or YY1 (1-394) mutant was expressed and EdU labeled for 6hrs. A GFP expressing plasmid was used the negative control. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


A . Results of CRISPR screening analyzed by the MAGeCK algorithm .

A. Results of CRISPR screening analyzed by the MAGeCK algorithm.


B . Wild type ( WT ) and NCOA4 KO MEFs were pretreated with 25 μg / ml ferric ammonium citrate ( FAC ) for 16 h and then chased with 20 μg / ml cycloheximide ( CHX ) for the indicated times . Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies .

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B. Wild type (WT) and NCOA4 KO MEFs were pretreated with 25 μg/ml ferric ammonium citrate (FAC) for 16 h and then chased with 20 μg/ml cycloheximide (CHX) for the indicated times. Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies.


C . WT MEFs untreated or treated with 10 μg / ml FAC for 12 h were lysed with SDS - containing sample buffer or Triton buffer , and analyzed by immunoblotting with the indicated antibodies .

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C. WT MEFs untreated or treated with 10 μg/ml FAC for 12 h were lysed with SDS-containing sample buffer or Triton buffer, and analyzed by immunoblotting with the indicated antibodies.


E . Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h , 10 μg / ml FAC for 12 h , or 100 μM Deferoxamine ( Dfo ) for 6 h after treatment with 10 μg / ml FAC for 12 h were prepared by differential centrifugation as described in ( D ) . Each fraction was subjected to SDS - PAGE and analyzed by immunoblotting with the indicated antibodies .

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E. Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12 h, or 100 μM Deferoxamine (Dfo) for 6 h after treatment with 10 μg/ml FAC for 12 h were prepared by differential centrifugation as described in (D). Each fraction was subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


F . WT MEFs were untreated , treated with 10 μg / ml FAC for 12h or treated with 10 μg / ml FAC , 10 μM E64d and 10 μg / ml pepstatin A ( pep A ) for 12h . The cells were immunostained with anti - NCOA4 and anti - LAMP1 . Scale bar , 10 μm . Quantitative data are shown as the means ± SD of three biological replicates . At least 30 cells were quantified in each replicate . P = 0 . 033 ( Welch Two Sample t - test ) .

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F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test).


G . NCOA4 puncta were photobleached in MEFs stably expressing NCOA4 - GFP after treatment with 10 μg / ml FAC for 12 h , and then fluorescent recovery was monitored . p62 puncta were photobleached in MEFs stably expressing GFP - human p62 , and then fluorescent recovery was monitored . Representative images are shown . Time 0 indicates the start of recovery after photobleaching . Scale bars , 2 μm . H . Quantitative data of fluorescence recovery in ( G ) shown as means ± SD . 26 dots ( NCOA4 ) or 25 dots ( p62 ) were quantified from three biological replicates .

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G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm. H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates.


I . Time - lapse imaging of MEFs stably expressing NCOA4 - GFP . Representative fusion and fission images are shown . Red arrowheads indicate the fission event . Scale bars , 5 μm .

I. Time-lapse imaging of MEFs stably expressing NCOA4-GFP. Representative fusion and fission images are shown. Red arrowheads indicate the fission event. Scale bars, 5 μm.


B . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h and then fixed for imaging . Cells were immunostained with anti - myc antibody . Representative images are shown . Scale bar , 10 μm .

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B. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with anti-myc antibody. Representative images are shown. Scale bar, 10 μm.


C . Number of puncta per cell in ( B ) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates .

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C. Number of puncta per cell in (B) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates.


D . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h , fractionated , and analyzed by immunoblotting with the indicated antibodies .

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D. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.


E . DISOPRED3 disorder score of human NCOA4 .

E. DISOPRED3 disorder score of human NCOA4.


F . WT MEFs stably expressing NCOA4 IDR ( aa 167 - 334 ) fused with tandem FKBP were treated with 10 μg / ml FAC or 20 μM Dfo for 12 h and then cultured with 0 . 1 nM FKBP ligand ( AP20187 ) for 6h . The cells were fixed for imaging and immunostained with anti - myc to analyze puncta of the fusion protein . Scale bar , 10μm . G . Number of puncta in ( F ) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates . P < 0 . 0001 ( Dfo + ligand vs FAC + ligand ) and P = 0 . 048 ( FAC vs FAC + ligand ) ( Kruskal - Wallis ANOVA with Dunn ' s multiple comparison test ) . P values were adjusted using the Bonferroni method .

O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O 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. WT MEFs stably expressing NCOA4 IDR (aa 167-334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6h. The cells were fixed for imaging and immunostained with anti-myc to analyze puncta of the fusion protein. Scale bar, 10μm. G. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P<0.0001 (Dfo+ligand vs FAC+ligand) and P=0.048 (FAC vs FAC+ligand) (Kruskal-Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.


H . Coomassie - stained SDS - PAGE gel of purified proteins . I . The amount of co - purifying iron in purified proteins in ( H ) was measured by inductively coupled plasma mass spectrometry ( ICP - MS ) . Data are shown as means ± SD of three biological replicates . P = 0 . 032 ( Welch Two Sample t - test ) .

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H. Coomassie-stained SDS-PAGE gel of purified proteins. I. The amount of co-purifying iron in purified proteins in (H) was measured by inductively coupled plasma mass spectrometry (ICP-MS). Data are shown as means ± SD of three biological replicates. P=0.032 (Welch Two Sample t-test).


A . Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated . Samples were subjected to SDS - PAGE and stained with Coomassie Brilliant Blue . B . Quantitative results in ( A ) are shown as means ± SEM of three biological replicates .

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A. Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated. Samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. B. Quantitative results in (A) are shown as means ± SEM of three biological replicates.


F . Electron micrograph of NCOA4 KO MEFs reconstituted with myc - hNCOA4 . Cryosections were labeled with anti - myc antibody . Scale bar , 200nm . G . Quantification of the diameter of NCOA4 condensates . Red line shows the mean . The dot plot represents the diameter of 36 condensates from two biological replicates .

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F. Electron micrograph of NCOA4 KO MEFs reconstituted with myc-hNCOA4. Cryosections were labeled with anti-myc antibody. Scale bar, 200nm. G. Quantification of the diameter of NCOA4 condensates. Red line shows the mean. The dot plot represents the diameter of 36 condensates from two biological replicates.


A . MEFs were treated with 10 μg / ml FAC for the indicated times and harvested . Cell lysates were fractionated and analyzed by SDS - PAGE with the indicated antibodies . Asterisks indicate a non - specific band . TfR1 ; Transferrin receptor

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A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1


B . MEFs were cultured for 12 h in medium containing 10 μg / ml FAC supplemented with DMSO or 10 μM E64d and 10 μg / ml pepstatin A ( pepA ) . Samples were analyzed by immunoblotting with the indicated antibodies . The black arrowhead indicates an intact ferritin band , and the white arrowhead indicates a partial degradation band of ferritin .

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B. MEFs were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin band, and the white arrowhead indicates a partial degradation band of ferritin.


C . NCOA4 KO MEFs reconstituted with hNCOA4 - myc were cultured with 10 μg / ml FAC for the indicated times and fixed for imaging . Cells were immunostained with ferritin and myc antibodies . Scale bar , 10μm .

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C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm.


D . Myc ( NCOA4 ) puncta were extracted , and average signal intensities of myc and ferritin in the puncta in ( C ) were plotted .

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D. Myc (NCOA4) puncta were extracted, and average signal intensities of myc and ferritin in the puncta in (C) were plotted.


E . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min . Cells were lysed with Triton buffer , and lysates were pulled down by streptavidin beads . Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies .

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E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies.


F . WT MEFs pretreated with 10 μg / ml FAC for 6 h or 24 h were chased with 20 μg / ml CHX and analyzed by immunoblotting with the indicated antibodies .

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F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies.


G . NCOA4 KO MEFs reconstituted with myc - hNCOA4 were pretreated with 10 μg / ml FAC and then cultured with 20 μg / ml CHX or 200 nM bafilomycin A1 ( BafA ) . Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies . Scale bar , 10μm .

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G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.


A . WT , ATG7 KO , TAX1BP1 KO , and FIP200KO MEFs were treated or not treated with 10 μg / ml FAC for 12 h . Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies .

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A. WT, ATG7 KO, TAX1BP1 KO, and FIP200KO MEFs were treated or not treated with 10 μg/ml FAC for 12 h. Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.


C . HEK293T cells were transfected with GFP - NCOA4 and the indicated FLAG - His - TAX1BP1 variants . Soluble lysates extracted from transfected cells were immunoprecipitated with anti - FLAG antibody . Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies .

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C. HEK293T cells were transfected with GFP-NCOA4 and the indicated FLAG-His-TAX1BP1 variants. Soluble lysates extracted from transfected cells were immunoprecipitated with anti-FLAG antibody. Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.


WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg / ml FAC for 12 h . ( D , F ) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies .

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WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg/ml FAC for 12 h. (D, F) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.


WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were treated with 10 μg / ml FAC for 12 h . ( E ) Cells were fixed for imaging and immunostained to analyze ferritin localization . Scale bar , 10μm .

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WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were treated with 10 μg/ml FAC for 12 h. (E) Cells were fixed for imaging and immunostained to analyze ferritin localization. Scale bar, 10μm.


A . MEFs were pretreated with 10 μg / ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time . Cells were lysed with SDS - containing sample buffer or Triton buffer . Lysates were analyzed by immunoblotting with the indicated antibodies .

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A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates were analyzed by immunoblotting with the indicated antibodies.


B . MEFs were treated with 20 μM Dfo for 12 h , 10 μg / ml FAC for 12h , or 100 μM Dfo for 6 h after treatment with 10 μg / ml FAC for 12 h . Triton - soluble lysates were subjected to glycerol gradient ultracentrifugation analysis .

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B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis.


C . WT and TAX1BP1 KO MEFs were pretreated with 25 μg / ml FAC for 16 h and then cultured with 100 μM Dfo . Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies . D . Soluble FTL bands in ( C ) were quantified by densitometry . Data are shown as the means ± SD of three biological replicates .

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C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies. D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates.


E . TAX1BP1 KO MEFs ( sg1 clone ) stably expressing FLAG - OsTIR1 ( F74G ) and FLAG - mAID - mTAX1BP1 were pretreated with 10 μg / ml FAC for 12 h , cultured with DMSO or 1 μM 5 - Ph - IAA for 2 h , and then treated with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with the indicated antibodies . 5 - Ph - IAA , a derivative of Auxin ; mAID , mini auxin - inducible

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E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1 were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM 5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degron


F . TAX1BP1 KO MEFs ( clone sg1 ) in which ATG7 was knocked down were pretreated with 10 μg / ml FAC for 12 h and then cultured with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with the indicated antibodies .

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F. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.


G . TAX1BP1 KO MEFs ( clone sg1 ) stably expressing myc - hNCOA4 pretreated with 25 μg / ml FAC for 16 h were cultured with 100 μM Dfo , and soluble lysates were analyzed by immunoblotting with the indicated antibodies .

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G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies.


H , I . NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg / ml FAC for 12 h and then chased with ( H ) 20 μg / ml CHX or ( I ) 100 μM Dfo for the indicated times . Soluble lysates were analyzed by immunoblotting with the indicated antibodies .

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H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.


J . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 3 h ( indicated as F ) or 100 μM DFO for 3 h after treatment with 10 μg / ml FAC for 12 h ( indicated as D ) , and then cultured with DMSO or 50 μM biotin for 30 min . Triton - soluble cell lysates were pulled down by streptavidin beads . Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies . The black and white arrowheads indicate hNCOA4 - TurboID and endogenous NCOA4 , respectively .

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J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.


A IMR90 ER : RasV12 primary fibroblasts were cultured in the presence of EtOH ( proliferating ) or 100nM 4OHT ( for 6 - 8days to induce RasV12 expression and oncogene - induced senescence , OIS ) . mRNA levels of 4OHT cells , normalised to PUM1 and presented relative to EtOH control . ( n = 4 independent experimental repeats ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All data are analysed by 2 - tailed , nonpaired Student ' s t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 )

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A IMR90 ER:RasV12 primary fibroblasts were cultured in the presence of EtOH (proliferating) or 100nM 4OHT (for 6-8days to induce RasV12 expression and oncogene-induced senescence, OIS). mRNA levels of 4OHT cells, normalised to PUM1 and presented relative to EtOH control. (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001)


B , C Cells as in A were analysed by Western blot for Lamp1 , p62 and Cathepsin B and quantified , normalised to GAPDH and expressed relative to EtOH . ( n = 3 independent experimental repeats ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All data are analysed by 2 - tailed , nonpaired Student ' s t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 .

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B, C Cells as in A were analysed by Western blot for Lamp1, p62 and Cathepsin B and quantified, normalised to GAPDH and expressed relative to EtOH. (n=3 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001


D , E Cells as in A were fixed and immunostained for Lamp1 and signal intensity was quantified . Scale bars : 10µm ( n = 3 independent experimental repeats ( at least 40 cells analysed per condition , from at least 4 fields of view , per repeat ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All data are analysed by 2 - tailed , nonpaired Student ' s t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 )

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D, E Cells as in A were fixed and immunostained for Lamp1 and signal intensity was quantified. Scale bars: 10µm (n=3 independent experimental repeats (at least 40 cells analysed per condition, from at least 4 fields of view, per repeat). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001)


K , Cells as in A were incubated with DQ - BSA and TR - BSA for 2 hours , fixed and imaged by confocal microscopy . Scale bar : 20µm ( n = 3 independent experimental repeats ( at least 240 cells analysed from 8 fields of view per condition ) .

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K, Cells as in A were incubated with DQ-BSA and TR-BSA for 2 hours, fixed and imaged by confocal microscopy. Scale bar: 20µm (n=3 independent experimental repeats (at least 240 cells analysed from 8 fields of view per condition).


O , Cells as in A were incubated with 50µg / ml cycloheximide ( CHX ) for the timepoints indicated . Degradation of autophagy receptors , p62 and NDP52 was assessed by Western blot ( n = 3 independent experimental repeats ) .

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O, Cells as in A were incubated with 50µg/ml cycloheximide (CHX) for the timepoints indicated. Degradation of autophagy receptors, p62 and NDP52 was assessed by Western blot (n=3 independent experimental repeats).


A Representative images of GFP - TFEB localisation in EtOH and 4OHT - treated fibroblasts , in full nutrient conditions . Scale bar : 20µm .

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A Representative images of GFP-TFEB localisation in EtOH and 4OHT-treated fibroblasts, in full nutrient conditions. Scale bar: 20µm.


B , EtOH and 4OHT - treated fibroblasts immunostained for endogenous TFE3 in the conditions indicated ; starvation indicates serum starvation overnight and one - hour amino acid starvation , refeeding was with full nutrient medium ( including FCS ) for 2 hours . Cells were incubated with leptomycin B ( LMB ) for 3 hours and where indicated , wash out involved replacement of the media for a further 2 hours . Scale bar : 20µm ; ( n = 3 independent experimental repeats ( at least 150 cells analysed from at least 5 fields of view per repeat ) .

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B, EtOH and 4OHT-treated fibroblasts immunostained for endogenous TFE3 in the conditions indicated; starvation indicates serum starvation overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. Scale bar: 20µm; (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat).


C EtOH and 4OHT - treated fibroblasts in the conditions indicated ; starvation indicates serum starvation overnight and one - hour amino acid starvation , refeeding was with full nutrient medium ( including FCS ) for 2 hours . Cells were incubated with leptomycin B ( LMB ) for 3 hours and where indicated , wash out involved replacement of the media for a further 2 hours . % cells with nuclear TFE3 was quantified . ( n = 3 independent experimental repeats ( at least 150 cells analysed from at least 5 fields of view per repeat ) . Data information : Violin blots include all datapoints from n = 3 , lines represent median and upper and lower quartiles ; Analysis : one - way ANOVA with Tukey ' s multiple comparison test * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

C EtOH and 4OHT-treated fibroblasts in the conditions indicated; starvation indicates serum starvation overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. % cells with nuclear TFE3 was quantified. (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat). Data information: Violin blots include all datapoints from n=3, lines represent median and upper and lower quartiles; Analysis: one-way ANOVA with Tukey's multiple comparison test * p<0.05; ** p<0.01; *** p<0.001.


D Cells treated as in C were analysed by Western blotting .

D Cells treated as in C were analysed by Western blotting.


H Representative images of cells treated as in C , D before being fixed and immunostained with antibodies against Lamp2 and p62 . Scale bar :

H Representative images of cells treated as in C,D before being fixed and immunostained with antibodies against Lamp2 and p62. Scale bar: 20µm


A Fibroblasts stably expressing GFP - TFEB were treated 4OHT to induce senescence ( or EtOH control ) . Cells were lysed and subject to Western blotting for antibodies as indicated . B Quantification of A . ( n = 4 independent experimental repeats ) C Quantification of A . ( n = 3 independent experimental repeats ) Data information : All graphs show individual data points , mean and error bars represent standard deviation . B and C : analysed by 2 - tailed , unpaired T - Test * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

A Fibroblasts stably expressing GFP-TFEB were treated 4OHT to induce senescence (or EtOH control). Cells were lysed and subject to Western blotting for antibodies as indicated. B Quantification of A. (n=4 independent experimental repeats) C Quantification of A. (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. B and C: analysed by 2-tailed, unpaired T-Test * p<0.05; ** p<0.01; *** p<0.001.


D Western blot of GFP immunoprecipitation of control ( EtOH ) and senescent ( 4OHT ) cells expressing GFP or GFP - CDK4 and FLAG - TFEB .

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

D Western blot of GFP immunoprecipitation of control (EtOH) and senescent (4OHT) cells expressing GFP or GFP-CDK4 and FLAG-TFEB.


H Fibroblasts stably expressing GFP - TFEB were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were lysed and subject to Western blotting . I Quantification of H ( n = 4 independent experimental repeats ) . Data information : All graphs show individual data points , mean and error bars represent standard deviation . All other panels analysed by one - way ANOVA with Tukey ' s multiple comparison test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 )

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

H Fibroblasts stably expressing GFP-TFEB were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. I Quantification of H (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)


J Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were lysed and subject to Western blotting . K , L Quantification of J ( n = 3 independent experimental repeats ) Data information : All graphs show individual data points , mean and error bars represent standard deviation . All other panels analysed by one - way ANOVA with Tukey ' s multiple comparison test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 )

J Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. K, L Quantification of J (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)


M Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were fixed and immunostained with antibodies against p62 and Lamp2 , Scale bar : 20µm .

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

M Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were fixed and immunostained with antibodies against p62 and Lamp2, Scale bar: 20µm.


O Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT . Cells were incubated with serum - free media overnight which was collected and subject to ELISA analysis for IL - 6 ( n = 3 independent experimental repeats ) Data information : All graphs show individual data points , mean and error bars represent standard deviation . All other panels analysed by one - way ANOVA with Tukey ' s multiple comparison test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 )

O Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were incubated with serum-free media overnight which was collected and subject to ELISA analysis for IL-6 (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)


B . Representative 2D projection of confocal images showing caspase activation at the dissection time point ( red channel ) and in the recent past ( green channel , arrows ) within putative escort somatic cells via DBS - S - QF sensor ; TUNEL staining indicates apoptosis ( grey , arrowhead ) ; DAPI labels the nuclei in the entire Figure . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection . Genotype : Actin DBS - S - QF , UAS - mCD8 - GFP , QUAS - tomato - HA / + ; ; QUAS - Gal4 / + ( BL83123 ) . C . Representative 2D projection of confocal images showing escort and follicular somatic cells permanently labelled with DBS - S - QF sensor ( green channel , arrows ) ; the arrowhead indicates the presence of germline cells positive for TUNEL staining ( red , arrowhead ) . Notice the lack of TUNEL signal and therefore apoptosis in somatic cells permanently labelled with DBS - S - QF sensor ( green ) . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection . Genotype : Actin DBS - S - QF , UAS - mCD8 - GFP , QUAS - tomato - HA / + ; QUAS - flippase ( BL30126 ) / + ; Actin5C FRT - stop - FRT lacZ - nls / + ( BL6355 ) Data information : Scale bars represents 10 µm in the entire Figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative 2D projection of confocal images showing caspase activation at the dissection time point (red channel) and in the recent past (green channel, arrows) within putative escort somatic cells via DBS-S-QF sensor; TUNEL staining indicates apoptosis (grey, arrowhead); DAPI labels the nuclei in the entire Figure. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; ;QUAS-Gal4 /+ (BL83123). C. Representative 2D projection of confocal images showing escort and follicular somatic cells permanently labelled with DBS-S-QF sensor (green channel, arrows); the arrowhead indicates the presence of germline cells positive for TUNEL staining (red, arrowhead). Notice the lack of TUNEL signal and therefore apoptosis in somatic cells permanently labelled with DBS-S-QF sensor (green). Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; QUAS-flippase (BL30126)/+; Actin5C FRT-stop-FRT lacZ-nls/+ (BL6355) Data information: Scale bars represents 10 µm in the entire Figure.


D . Graph showing the percentage of germaria permanently labelled with DBS - S - QF overtime at 25 ° C ( N = 4 biological replicates ; n = 520 and n = 536 germaria were inspected at 7 and 14 days post adult eclosion , respectively ) and 29 ° C ( N = 7 , n = 799 at 7 days and n = 588 at 14 days ) . A two - way ANOVA and Tukey ' s multiple comparison tests were used to determine statistical significance ( n . s = not significant ; * * * * p < 0 . 0001 ) . Genotype : Actin DBS - S - QF , UAS - mCD8 - GFP , QUAS - tomato - HA / + ; QUAS - flippase ( BL30126 ) / + ; Actin5C FRT - stop - FRT lacZ - nls / + ( BL6355 ) . Data information : The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Graph showing the percentage of germaria permanently labelled with DBS-S-QF overtime at 25 °C (N=4 biological replicates; n=520 and n=536 germaria were inspected at 7 and 14 days post adult eclosion, respectively) and 29 °C (N=7, n=799 at 7 days and n=588 at 14 days). A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; QUAS-flippase (BL30126)/+; Actin5C FRT-stop-FRT lacZ-nls/+ (BL6355). Data information: The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets.


E . Percentage of germaria showing permanently labelled with DBS - S - QF the ECD over time at 25 ° C and 29 ° C . FasIII immunostaining was used to locate the DBS - S - QF labelling in the inspected germaria . The N and n numbers of these experiments are indicated in D . A two - way ANOVA and Tukey ' s multiple comparison tests were used to determine statistical significance ( n . s = not significant ; * * * * p < 0 . 0001 ) . Genotypes are shown in D . F . Percentage of germaria showing permanently labelled with DBS - S - QF the ECD over time at 25 ° C and 29 ° C . FasIII immunostaining was used to locate the DBS - S - QF labelling in the inspected germaria . The N and n numbers of these experiments are indicated in D . A two - way ANOVA and Tukey ' s multiple comparison tests were used to determine statistical significance ( n . s = not significant ; * * * * p < 0 . 0001 ) . Genotypes are shown in D . Data information : The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percentage of germaria showing permanently labelled with DBS-S-QF the ECD over time at 25 °C and 29 °C. FasIII immunostaining was used to locate the DBS-S-QF labelling in the inspected germaria. The N and n numbers of these experiments are indicated in D. A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotypes are shown in D. F. Percentage of germaria showing permanently labelled with DBS-S-QF the ECD over time at 25 °C and 29 °C. FasIII immunostaining was used to locate the DBS-S-QF labelling in the inspected germaria. The N and n numbers of these experiments are indicated in D. A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotypes are shown in D. Data information: The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets.


A - B . Representative 2D projection of confocal images showing the expression of the cell identity Castor in follicular cells control with Dronc expression ( A : 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + ) and without Dronc ( B : 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ) . Notice the reduction in the number of Castor - expressing cells in the FCD and the reduction in size of this region in B . Nuclei are labelled with DAPI ( blue ) ; Castor ( green ) and FasIII ( grey ) label the follicular cells ; UAS - Histone - RFP labels the nuclei of Gal4 - expressing cells ( red ) . Data information : Scale bars represents 10 µm . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B. Representative 2D projection of confocal images showing the expression of the cell identity Castor in follicular cells control with Dronc expression (A: 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+) and without Dronc (B: 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF). Notice the reduction in the number of Castor-expressing cells in the FCD and the reduction in size of this region in B. Nuclei are labelled with DAPI (blue); Castor (green) and FasIII (grey) label the follicular cells; UAS-Histone-RFP labels the nuclei of Gal4-expressing cells (red). Data information: Scale bars represents 10 µm. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).


C . Quantification of total number of follicular cells ( left ) and Castor - expressing cells ( right ) within the FCD ( FasIII positive region ) in either heterozygous or homozygous Dronc mutant conditions : Genotypes : CTRL = 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + ; Dronc - / - = 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF . CTRL = ptc - Gal4 ( BL2017 ) / + ; UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + . Dronc - / - = ptc - Gal4 ( BL2017 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF . N = 2 ; the n number for each column in order of appearance n = 16 , n = 14 , n = 20 , n = 17 , n = 19 , n = 18 . Statistical significance was determined by using an unpaired parametric Welch ' s t - test ( * * * * p ≤ 0 . 001 ) . The median and quartiles are indicated in the violin plots . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of total number of follicular cells (left) and Castor-expressing cells (right) within the FCD (FasIII positive region) in either heterozygous or homozygous Dronc mutant conditions: Genotypes: CTRL= 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+; Dronc -/- = 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF.CTRL=ptc-Gal4 (BL2017)/+; UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+. Dronc -/- = ptc-Gal4 (BL2017)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF. N=2; the n number for each column in order of appearance n=16, n=14, n=20, n=17, n=19, n=18. Statistical significance was determined by using an unpaired parametric Welch's t-test (****p≤0.001). The median and quartiles are indicated in the violin plots. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).


E . Percentage of germaria showing TUNEL staining in different cell types of the germarium ( somatic cells and germline cells ) upon altering Dronc expression in all of the escort cells and follicular stem cells . N = 3 biological replicates ; the n number of inspected germaria per condition is indicated close to the genotype . The genotypes in the graph are : White triangle ( n = 378 germaria ) = ptc - Gal4 ( BL2017 ) / + ; + / + . Yellow circle ( n = 381 ) = ptc - Gal4 ( BL2017 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + . Red square ( n = 196 ) = ptc - Gal4 ( BL2017 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - suntag - HA . Blue circle ( n = 184 ) = ptc - Gal4 ( BL2017 ) / UAS - Dronc ( BL56198 ) ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + . An ordinary one - way ANOVA and Holm - Sidak ' s multiple comparison test was used to determine statistical significance ( n . s = not significant ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . The graph shows the mean and standard deviations . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percentage of germaria showing TUNEL staining in different cell types of the germarium (somatic cells and germline cells) upon altering Dronc expression in all of the escort cells and follicular stem cells. N=3 biological replicates; the n number of inspected germaria per condition is indicated close to the genotype. The genotypes in the graph are: White triangle (n=378 germaria) = ptc-Gal4 (BL2017)/+; +/+. Yellow circle (n=381) = ptc-Gal4 (BL2017)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ +. Red square (n=196) = ptc-Gal4 (BL2017)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-suntag-HA. Blue circle (n=184)= ptc-Gal4 (BL2017)/UAS-Dronc (BL56198); DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+. An ordinary one-way ANOVA and Holm-Sidak's multiple comparison test was used to determine statistical significance (n.s = not significant; ** p≤0.01; *** p≤0.001; ****p≤0.0001). The graph shows the mean and standard deviations. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).


J - K . Representative 2D projection of confocal images of the genotypes quantified in I . Nuclei are labelled with DAPI ( blue ) . Red and green nuclei show the expression of different Fucci markers . FasIII ( grey ) was used to locate the FCD . Notice the reduction of follicular cells and limited overlap between the green and red fluorescent signals ( white arrowheads ) in K . Data information : Scale bars represents 10 µm . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) .

J-K. Representative 2D projection of confocal images of the genotypes quantified in I. Nuclei are labelled with DAPI (blue). Red and green nuclei show the expression of different Fucci markers. FasIII (grey) was used to locate the FCD. Notice the reduction of follicular cells and limited overlap between the green and red fluorescent signals (white arrowheads) in K. Data information: Scale bars represents 10 µm. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).


A . Quantification of total number of follicular cells in germaria with follicular stem cells expressing normal levels of Dronc ( DroncKO / + ) , without Dronc expression ( DroncKO / DroncKO - Suntag - HA - Cherry ) , and expressing a catalytically inactive form of Dronc ( DroncKO / DroncKO - FL - CAEA - Suntag - HA - Cherry ) . The genotypes are as follows : 109 - 30 - Gal4 ( BL7023 ) / + ( n = 13 ) . 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT Dronc - GFP - Apex FRT - Suntag - HA - Cherry ( n = 15 ) . 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT Dronc - GFP - Apex FRT - Dronc FL - CAEA - Suntag - HA - Cherry ( n = 14 ) . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of total number of follicular cells in germaria with follicular stem cells expressing normal levels of Dronc (DroncKO/+), without Dronc expression (DroncKO/DroncKO- Suntag-HA-Cherry), and expressing a catalytically inactive form of Dronc (DroncKO/DroncKO- FL-CAEA-Suntag-HA-Cherry ). The genotypes are as follows: 109-30-Gal4 (BL7023)/+ (n=13). 109-30-Gal4 (BL7023)/+; DroncKO Tub-G80ts (BL7019) / UAS-flippase (BL8209) DroncKO-FRT Dronc-GFP-Apex FRT-Suntag-HA-Cherry (n=15). 109-30-Gal4 (BL7023)/+; DroncKO Tub-G80ts (BL7019) / UAS-flippase (BL8209) DroncKO-FRT Dronc-GFP-Apex FRT-Dronc FL-CAEA-Suntag-HA-Cherry (n=14). A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (***p≤0.001; ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


B . Quantification of total Castor - expressing cells within the FasIII cellular domain in the genotypes shown in A . n numbers are shown in A . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * * p ≤ 0 . 0001 ) . C . Percentage of Castor - expressing cells versus the total number of Follicular cells in germaria of the genotypes indicated in A . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of total Castor-expressing cells within the FasIII cellular domain in the genotypes shown in A. n numbers are shown in A. A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (****p≤0.0001). C. Percentage of Castor-expressing cells versus the total number of Follicular cells in germaria of the genotypes indicated in A. A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


D . Quantification of total number of follicular cells within the FasIII cellular domain in the following genotypes : 109 - 30 - Gal4 / + ; Tub - G80ts ( BL7019 ) / + ( n = 16 ) . 109 - 30 - Gal4 ( BL7023 ) / UAS - DriceRNAi UAS - DecayRNAi ( a gift from Pascal Meier ) ; UAS - DammRNAi , UAS - Dcp1RNAi ( a gift from Pascal Meier ) ( n = 15 ) . 109 - 30 - Gal4 ( BL7023 ) / + ; Tub - G80ts ( BL7019 ) / UAS - Dcp1RNAi ( BL28909 ) ( n = 25 ) 109 - 30 - Gal4 ( BL7023 ) / + ; Tub - G80ts ( BL7019 ) / UAS - DriceRNAi ( BL32403 ) ( n = 16 ) 109 - 30 - Gal4 ( BL7023 ) / UAS - Dark - sh ; Tub - G80ts ( BL7019 ) / + ( a gift from M . Miura ) ( n = 18 ) . 109 - 30 - Gal4 ( BL7023 ) / UAS - microRNA - RHG ; Tub - G80ts ( BL7019 ) / + ( a gift from Iswar Hariharan ) ( n = 10 ) . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of total number of follicular cells within the FasIII cellular domain in the following genotypes: 109-30-Gal4/+; Tub-G80ts (BL7019)/+ (n=16). 109-30-Gal4 (BL7023)/UAS-DriceRNAi UAS-DecayRNAi (a gift from Pascal Meier); UAS-DammRNAi, UAS-Dcp1RNAi (a gift from Pascal Meier) (n=15). 109-30-Gal4 (BL7023)/+; Tub-G80ts (BL7019)/ UAS-Dcp1RNAi (BL28909) (n=25) 109-30-Gal4 (BL7023)/+; Tub-G80ts (BL7019)/ UAS-DriceRNAi (BL32403) (n=16) 109-30-Gal4 (BL7023)/UAS-Dark-sh; Tub-G80ts (BL7019)/+ (a gift from M. Miura) (n=18). 109-30-Gal4 (BL7023)/UAS-microRNA-RHG; Tub-G80ts (BL7019)/+ (a gift from Iswar Hariharan) (n=10). A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (***p≤0.001; ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


E . Quantification of total number of Castor - expressing cells in germaria of the genotypes indicated in D . n numbers are shown in D . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . F . Percentage of Castor - expressing cells versus the total number of Follicular cells ( FasIII + cells ) in germaria of the genotypes indicated in A . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( n . s . = not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of total number of Castor-expressing cells in germaria of the genotypes indicated in D. n numbers are shown in D. A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (***p≤0.001; ****p≤0.0001). F. Percentage of Castor-expressing cells versus the total number of Follicular cells (FasIII+ cells) in germaria of the genotypes indicated in A. A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (n.s.= not significant, *p≤0.05, **p≤0.01, ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


A - B . Representative 2D projections of confocal images showing the expression of Ci - 155 ( blue and grey channels ) , ptc - GFP ( green and grey ; ptc - GFP is a bona - fide transcriptional read out of Hh - pathway and weak hypomorph allele and Castor ( red and grey ) in germaria with either normal ( A ) or deficient Dronc expression ( B ) . Genotypes : 109 - 30 - Gal4 ( BL7023 ) / ptc - GFPCB02030 ( a gift from Isabel Guerrero ) ( A ) and 109 - 30 - Gal4 ( BL7023 ) / ptc - GFPCB02030 ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase DroncKO - FRT - Dronc - GFP - APEX - FRT - QF . Data information : Scale bars represents 10 µm in the entire figure . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B. Representative 2D projections of confocal images showing the expression of Ci-155 (blue and grey channels), ptc-GFP (green and grey; ptc-GFP is a bona-fide transcriptional read out of Hh-pathway and weak hypomorph allele and Castor (red and grey) in germaria with either normal (A) or deficient Dronc expression (B). Genotypes: 109-30-Gal4 (BL7023)/ptc-GFPCB02030 (a gift from Isabel Guerrero) (A) and 109-30-Gal4 (BL7023)/ptc-GFPCB02030; DroncKOTub-G80ts (BL7019)/ UAS-flippase DroncKO-FRT-Dronc-GFP-APEX-FRT-QF. Data information: Scale bars represents 10 µm in the entire figure. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).


D . Western blot showing Caspase - 9 expression ( upper lane ) and actin ( bottom lane , loading control ) in either control or Caspase - 9 deficient OVCAR - 3 cells ( 24h and 72h post - transfection of an shRNA against Caspase - 9 ) . Notice the strong downregulation of Caspase - 9 72h after siRNA treatment .

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

D. Western blot showing Caspase-9 expression (upper lane) and actin (bottom lane, loading control) in either control or Caspase-9 deficient OVCAR-3 cells (24h and 72h post-transfection of an shRNA against Caspase-9). Notice the strong downregulation of Caspase-9 72h after siRNA treatment.


H . Quantification of total number of follicular cells ( left ) or Castor - expressing cells ( right ) within the FasIII cellular domain in the following genotypes from left to right : Dronc - / - = CTRL = 109 - 30 - Gal4 ( BL7023 ) / ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ( n = 16 ) . CTRL = 109 - 30 - Gal4 ( BL7023 ) / UAS - smoAct ( BL44621 ) ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 11 ) . Dronc - / - = 109 - 30 - Gal4 ( BL7023 ) / UAS - smoAct ( BL44621 ) ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ( n = 8 ) . CTRL = 109 - 30 - Gal4 ( BL7023 ) / UAS - Ci ( BL28984 ) ; DroncKOTub - G80ts ( BL7019 ) / + . ( n = 7 ) . Dronc - / - = 109 - 30 - Gal4 ( BL7023 ) / UAS - UAS - Ci ( BL28984 ) ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ( n = 21 ) . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( n . s . = not significant ; * * * * p ≤ 0 . 0001 ; ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . Unless otherwise indicated , all experimental data presented were obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of total number of follicular cells (left) or Castor-expressing cells (right) within the FasIII cellular domain in the following genotypes from left to right: Dronc -/- = CTRL= 109-30-Gal4 (BL7023)/; DroncKOTub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF (n=16). CTRL= 109-30-Gal4 (BL7023)/UAS-smoAct (BL44621); DroncKOTub-G80ts (BL7019)/+ (n=11). Dronc -/- = 109-30-Gal4 (BL7023)/UAS-smoAct (BL44621); DroncKOTub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF (n=8). CTRL= 109-30-Gal4 (BL7023)/UAS-Ci (BL28984); DroncKOTub-G80ts (BL7019)/+. (n=7). Dronc -/- = 109-30-Gal4 (BL7023)/UAS- UAS-Ci (BL28984); DroncKOTub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF (n=21). A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (n.s.= not significant; ****p≤0.0001;). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). Unless otherwise indicated, all experimental data presented were obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


J . Quantification of total number of follicular cells ( left ) or Castor - expressing cells ( right ) within the FasIII cellular domain in the following genotypes from left to right : Dronc + / + = ptc - Gal4 ( BL2017 ) / + ; Tub - G80ts ( BL7019 ) ( n = 19 ) . Dronc + / - = ptc - Gal4 ( BL2017 ) / + ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 23 ) . Dronc + / - UAS - Dronc = ptc - Gal4 ( BL2017 ) / + ; DroncKOTub - G80ts ( BL7019 ) / UAS - Dronc ( BL56198 ) ( n = 14 ) . A one - way ordinary ANOVA and Welch ' s correction were used to establish statistical significance ( n . s . = not significant ; * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . Unless otherwise indicated , all experimental data presented were obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of total number of follicular cells (left) or Castor-expressing cells (right) within the FasIII cellular domain in the following genotypes from left to right: Dronc +/+ = ptc-Gal4 (BL2017)/+; Tub-G80ts (BL7019) (n=19). Dronc +/- = ptc-Gal4 (BL2017)/+; DroncKOTub-G80ts (BL7019)/+ (n=23). Dronc +/- UAS-Dronc = ptc-Gal4 (BL2017)/+; DroncKOTub-G80ts (BL7019)/UAS-Dronc (BL56198) (n=14). A one-way ordinary ANOVA and Welch's correction were used to establish statistical significance (n.s.= not significant; ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). Unless otherwise indicated, all experimental data presented were obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


B . Relative number of Ptc - positive puncta per germaria of the following genotypes , from left to right : DroncKO Tub - G80ts / + ( n = 10 ) . ptc - Gal4 / + ; Tub - G80ts / + ( n = 9 ) . ptc - Gal4 / + ; DroncKO Tub - G80ts / + ( n = 10 ) . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( * * * p ≤ 0 . 001 Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative number of Ptc-positive puncta per germaria of the following genotypes, from left to right: DroncKO Tub-G80ts /+ (n=10). ptc-Gal4 /+; Tub-G80ts/+ (n=9). ptc-Gal4 /+; DroncKO Tub-G80ts /+ (n=10). A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (***p≤0.001 Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


C . Western blot showing Ptc ( upper lane ) and actin ( bottom lane , loading control ) expression in ovaries of the genotypes shown in A . Notice the Ptc accumulation in double heterozygous germaria ( ptc - Gal4 / + ; DroncKO Tub - G80ts / + ) .

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

C. Western blot showing Ptc (upper lane) and actin (bottom lane, loading control) expression in ovaries of the genotypes shown in A. Notice the Ptc accumulation in double heterozygous germaria (ptc-Gal4 /+; DroncKO Tub-G80ts /+).


E . Pearson colocalization index between Ptc and Atg8 in germaria of the following genotypes : ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ( n = 12 ) . ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 18 ) . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * * p ≤ 0 . 01 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

E. Pearson colocalization index between Ptc and Atg8 in germaria of the following genotypes: ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749) (n=12). ptc-Gal4 (BL2017)/UAS-GFP-mCherry-Atg8 (BL37749); DroncKOTub-G80ts (BL7019)/+ (n=18). An unpaired parametric Welch's t-test was used to establish the statistical significance (** p≤0.01). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.