O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O


c , VX680 ( 0 . 5 μM , a concentration that will specifically allow neuronal differentiation ) was used to inhibit Aurora A activity . Bar diagrams below show quantifications . Compared to DMSO - treated control NPCs , a higher proportion of VX680 treated NPCs are ciliated ( * * * p < 0 . 001 ) , exhibiting longer cilia ( * * * p < 0 . 001 ) and show Tuj1 positive neurons ( * * * p < 0 . 001 ) . Error bars are s . e . m . For ciliated cells and cilium length : n = 3 independent experiments . 164 cells for DMSO - treated and 77 cells for VX680 - treated . For NPC differentiation : n = 4 independent experiments . 286 cells for DMSO - treated and 118 cells for VX680 - treated . Scale bar 1 μm .

O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE 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-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O

c, VX680 (0.5 μM, a concentration that will specifically allow neuronal differentiation) was used to inhibit Aurora A activity. Bar diagrams below show quantifications. Compared to DMSO-treated control NPCs, a higher proportion of VX680 treated NPCs are ciliated (***p<0.001), exhibiting longer cilia (***p<0.001) and show Tuj1 positive neurons (***p<0.001). Error bars are s. e. m. For ciliated cells and cilium length: n = 3 independent experiments. 164 cells for DMSO-treated and 77 cells for VX680-treated. For NPC differentiation: n = 4 independent experiments. 286 cells for DMSO-treated and 118 cells for VX680-treated. Scale bar 1 μm.


d , Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours . Inhibition of Ofd1 ( * p < 0 . 05 ) , Nde1 ( * * p < 0 . 01 ) and Aurora A ( * * * p < 0 . 001 ) caused significantly reduced incorporation of EdU .

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d, Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours. Inhibition of Ofd1 (*p<0.05), Nde1 (**p<0.01) and Aurora A (***p<0.001) caused significantly reduced incorporation of EdU.


e - g , Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP . Both low - and high - exposures are given ( e ) . Increased cilia length and NPC differentiation upon expression of RNAi - resistant CPAP E1235V compared to WT CPAP ( f - g ) . Error bars are s . e . m . * * * p < 0 . 001 ( cilia length ) , * p < 0 . 05 ( differentiation ) , t - test . For cilia length : n = 4 independent experiments . 25 cells for CPAP , 47 cells for CPAP E1235V . For differentiation : n = 4 independent experiments . 145 for CPAP and 81 for CPAP E1235V . Scale bar 1 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O B-GENEPROD 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O

e-g, Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP. Both low -and high -exposures are given (e). Increased cilia length and NPC differentiation upon expression of RNAi-resistant CPAP E1235V compared to WT CPAP (f-g). Error bars are s. e. m. *** p<0.001 (cilia length), * p<0.05 (differentiation), t-test. For cilia length: n = 4 independent experiments. 25 cells for CPAP, 47 cells for CPAP E1235V. For differentiation: n = 4 independent experiments. 145 for CPAP and 81 for CPAP E1235V. Scale bar 1 μm.


a , iPS - derived 14 - day old organoid displaying complex morphological structures of brain - like tissues , parenthesis . Scale bar 500 μm . The ventricular zone ( VZ ) with radial glial cells stained with nestin ( magenta ) and neurons stained with TUJ1 is shown at higher magnification . Scale bar 20 μm .

O O B-CELL_TYPE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O

a, iPS-derived 14-day old organoid displaying complex morphological structures of brain-like tissues, parenthesis. Scale bar 500 μm. The ventricular zone (VZ) with radial glial cells stained with nestin (magenta) and neurons stained with TUJ1 is shown at higher magnification. Scale bar 20 μm.


b , Reduced Nestin / Pax - 6 - positive progenitors and increased TUJ1 - positive neurons in Seckel organoids . In ventricular zones of WT organoids , there are significantly more Pax6 positive cells than TUJ1 positive cells , which is not the case in Seckel organoids ( * p < 0 . 05 ) . TUJ1 / Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs . Pax6 cells in Seckel organoid ventricular zones ( * p < 0 . 05 ) . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 10 WT organoids ( 2520 cells ) . Seckeln = 10 organoids ( 2024 cells ) . Scale bar 20 μm .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Reduced Nestin/Pax-6-positive progenitors and increased TUJ1-positive neurons in Seckel organoids. In ventricular zones of WT organoids, there are significantly more Pax6 positive cells than TUJ1 positive cells, which is not the case in Seckel organoids (*p<0.05). TUJ1/Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs. Pax6 cells in Seckel organoid ventricular zones (* p<0.05). Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 10 WT organoids (2520 cells). Seckeln = 10 organoids (2024 cells). Scale bar 20 μm.


c , ( i ) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen ( * p < 0 . 05 ) . Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids . ( * p < 0 . 05 ) . Error bars are s . e . m . t - test . n = 6 for WT organoid and 6 for Seckel organoid . Scale bar 20 μm . ( ii ) Seckel organoid cells display longer cilia . Error bars are s . e . m . * * * p < 0 . 001 , t - test . n = 3 for WT organoids and 4 for Seckel organoids . Scale bar 1 μm .

O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O 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, (i) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen (* p<0.05). Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids. (*p<0.05). Error bars are s. e. m. t-test. n=6 for WT organoid and 6 for Seckel organoid. Scale bar 20 μm. (ii) Seckel organoid cells display longer cilia. Error bars are s. e. m. *** p<0.001, t-test. n=3 for WT organoids and 4 for Seckel organoids. Scale bar 1 μm.


d , Seckel organoids electroporated with constructs expressing GFP alone ( i ) , GFP with CC5 - GFP and ( ii ) GFP with CPAP - GFP ( iii ) . Middle panel shows staining with anti - GFP antibodies ( red ) to show the transfected area . Note the presence of increased Pax6 - positive ( magenta ) NPCs in CC5 and CPAP electroporated tissues compared to GFP control . Likewise , there is a larger neural epithelium with multiple RGs ( arrowheads ) . Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone ( iv ) lower TUJ1 / Pax6 ratio ( * * p < 0 . 01 and * * * p < 0 . 001 respectively ) ( v ) smaller lumen diameter ( * * p < 0 . 01 ) and ( vi ) shorter cilia length ( * * p < 0 . 01 and * * * p < 0 . 001 respectively ) . Error bars are s . e . m . One - way ANOVA followed by Dunnett ' s multiple comparisons test . n = 3 organoids with 225 cells for GFP , 537 cells for CPAP and 569 for CC5 . At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter . t test . Error bars are s . e . m . Scale bar 20 μm . e , Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho - vimentin - positive RGs in Seckel organoids . Compared to GFP control , electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs ( for CC5 * * * p < 0 . 001 ) and decreases the portion of obliquely ( for CC5 * p < 0 . 05 ) or vertically oriented SeckelGFP ( for CPAP and CC5 * * p < 0 . 01 ) . Error bars are s . e . m . Two - way ANOVA followed by Tukey ' s multiple comparisons test . n = 4 organoids . 100 cells for GFP control , 112 cells for CPAP and 99 cells for CC5 .

O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD 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-TISSUE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O

d, Seckel organoids electroporated with constructs expressing GFP alone (i), GFP with CC5-GFP and (ii) GFP with CPAP-GFP (iii). Middle panel shows staining with anti-GFP antibodies (red) to show the transfected area. Note the presence of increased Pax6-positive (magenta) NPCs in CC5 and CPAP electroporated tissues compared to GFP control. Likewise, there is a larger neural epithelium with multiple RGs (arrowheads). Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone (iv) lower TUJ1/Pax6 ratio (**p<0.01 and ***p<0.001 respectively) (v) smaller lumen diameter (**p<0.01) and (vi) shorter cilia length (**p<0.01 and ***p<0.001 respectively). Error bars are s. e. m. One-way ANOVA followed by Dunnett's multiple comparisons test. n = 3 organoids with 225 cells for GFP, 537 cells for CPAP and 569 for CC5. At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter. t test. Error bars are s.e.m. Scale bar 20 μm.e, Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho-vimentin-positive RGs in Seckel organoids. Compared to GFP control, electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs (for CC5 ***p<0.001) and decreases the portion of obliquely (for CC5 *p<0.05) or vertically oriented SeckelGFP (for CPAP and CC5 **p<0.01). Error bars are s.e.m. Two-way ANOVA followed by Tukey's multiple comparisons test. n = 4 organoids. 100 cells for GFP control, 112 cells for CPAP and 99 cells for CC5.


B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths . Yeast cells expressing clogger proteins ( cytochrome b2 - DHFR and cytochrome b2 ∆ 19 - DHFR ) or cytosolic DHFR for comparison under control of the galactose - inducible promoter were grown to mid - log phase on lactate medium . Ten - fold serial dilutions were dropped on lactate ( no induction ) or lactate with 0 . 5 % galactose medium ( induction ) . MTS , matrix - targeting signal .

O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths. Yeast cells expressing clogger proteins (cytochrome b2-DHFR and cytochrome b2∆19-DHFR) or cytosolic DHFR for comparison under control of the galactose-inducible promoter were grown to mid-log phase on lactate medium. Ten-fold serial dilutions were dropped on lactate (no induction) or lactate with 0.5% galactose medium (induction). MTS, matrix-targeting signal.


E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates . Cells were grown in lactate medium and clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before samples were harvested and lysed . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

O O O O B-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with 0.5% galactose before samples were harvested and lysed. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


A The clogger was expressed in wild type ( WT ) and ∆ rpn4 ( ∆ ) cells for 4 . 5 h with medium containing 0 . 5 % galactose . Medium was exchanged for a non - inducing lactate medium . Precursor ( pre ) and mature ( m ) forms of the b2 ∆ - DHFR clogger were visualized by Western blotting using a DHFR - specific antibody .

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A The clogger was expressed in wild type (WT) and ∆rpn4 (∆) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a non-inducing lactate medium. Precursor (pre) and mature (m) forms of the b2∆-DHFR clogger were visualized by Western blotting using a DHFR-specific antibody.


G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆ rpn4 cells . The cytosolic soluble DHFR protein was expressed for control . The strains were grown in lactate medium and clogger expression was induced with 0 . 5 % galactose for 6 h .

O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O

G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆rpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.


A Cytosolic DHFR ( control ) or the clogger protein b2 - DHFR were expressed in wild type and ∆ rpn4 cells for 4 . 5 h . The distribution of constitutively expressed Hsp104 - GFP was visualized by fluorescence microscopy . Diameter of Hsp104 - GFP - bound aggregates were quantified . . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates . Significance was assessed using two - sided , paired Student ' s t - test . P values are indicated as asterisks * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . See also Fig . EV4A for quantification of the number of aggregates per cell .

O B-SUBCELLULAR B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cytosolic DHFR (control) or the clogger protein b2-DHFR were expressed in wild type and ∆rpn4 cells for 4.5 h. The distribution of constitutively expressed Hsp104-GFP was visualized by fluorescence microscopy. Diameter of Hsp104-GFP-bound aggregates were quantified. . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates. Significance was assessed using two-sided, paired Student's t-test. P values are indicated as asterisks ** p ≤ 0.01, **** p ≤ 0.0001. See also Fig. EV4A for quantification of the number of aggregates per cell.


B Hsp104 - GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures . SN , supernatant representing the non - bound fraction . The signals for Ssa1 and Sod1 are shown for control .

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B Hsp104-GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures. SN, supernatant representing the non-bound fraction. The signals for Ssa1 and Sod1 are shown for control.


A The mitochondrial proteins Pdb1 , Mam33 and Aim17 were co - expressed for 4 . 5 h with clogger as fusions with the red fluorescent protein RFP ( mCherry ) . Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4 . 5 h after induction of the clogger or cytosolic DHFR for control . For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17 - expressing plasmids and grown to mid - log phase in lactate medium . Clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before microscopy was perfomed . Please note that in the absence of Hsp42 and Hsp104 , no Aim17 - RFP - containing granules are formed . C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N - terminally truncated version of Mam33 - RFP ( both under control of GAL promoter ) together with constitutively expressed Hsp104 - GFP ( TPI promoter ) . The cells were grown on lactate plus 0 . 5 % galactose - containing medium for 4 . 5 h . The distribution of the fluorescent proteins was visualized by microscopy .

O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

A The mitochondrial proteins Pdb1, Mam33 and Aim17 were co-expressed for 4.5 h with clogger as fusions with the red fluorescent protein RFP (mCherry). Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4.5 h after induction of the clogger or cytosolic DHFR for control. For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17-expressing plasmids and grown to mid-log phase in lactate medium. Clogger expression was induced for 4.5 h with 0.5 % galactose before microscopy was perfomed. Please note that in the absence of Hsp42 and Hsp104, no Aim17-RFP-containing granules are formed. C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N-terminally truncated version of Mam33-RFP (both under control of GAL promoter) together with constitutively expressed Hsp104-GFP (TPI promoter) . The cells were grown on lactate plus 0.5% galactose-containing medium for 4.5 h. The distribution of the fluorescent proteins was visualized by microscopy.


D Wild type cells expressing Hsp104 - GFP were grown on glucose or lactate medium to log phase . Cells were diluted and visualized by life cell imaging . Please note , that in glucose media , Hsp104 - GFP is initially dispersed in cells and only aggregates when cells become stationary . In contrast , Hsp104 - GFP - containing granules are frequent on lactate medium even at log phase . See movie EV1 for life cell imaging of glucose - grown cells .

O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O

D Wild type cells expressing Hsp104-GFP were grown on glucose or lactate medium to log phase. Cells were diluted and visualized by life cell imaging. Please note, that in glucose media, Hsp104-GFP is initially dispersed in cells and only aggregates when cells become stationary. In contrast, Hsp104-GFP-containing granules are frequent on lactate medium even at log phase. See movie EV1 for life cell imaging of glucose-grown cells.


E Clogger and Mam33 - HA were co - expressed for 4 . 5 h in wild type and ∆ hsp42 cells . Mitochondria were isolated and subjected to Western blotting to detect mature ( m ) and precursor ( pre ) forms of Mam33 - HA . The matrix protein Tim16 served as loading control .

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E Clogger and Mam33-HA were co-expressed for 4.5 h in wild type and ∆hsp42 cells. Mitochondria were isolated and subjected to Western blotting to detect mature (m) and precursor (pre) forms of Mam33-HA. The matrix protein Tim16 served as loading control.


F The mitochondria used for E were treated with proteinase K ( PK ) to remove surface - exposed proteins . Mitochondrial membranes were lysed with NP - 40 and soluble ( S ) and aggregated ( P ) proteins were separated by centrifugation . T , total .

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F The mitochondria used for E were treated with proteinase K (PK) to remove surface-exposed proteins. Mitochondrial membranes were lysed with NP-40 and soluble (S) and aggregated (P) proteins were separated by centrifugation. T, total.


G To measure the toxicity of clogger expression , clogger proteins were induced for 24 h with lactate medium containing 0 . 5 % galactose in the mutants indicated . Aliquots were removed and the number of living cells were assessed by a plating assay on glucose - containing plates . The resistance of ∆ rpn4 to clogger expression depends on Hsp104 and Hsp42 . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

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G To measure the toxicity of clogger expression, clogger proteins were induced for 24 h with lactate medium containing 0.5 % galactose in the mutants indicated. Aliquots were removed and the number of living cells were assessed by a plating assay on glucose-containing plates. The resistance of ∆rpn4 to clogger expression depends on Hsp104 and Hsp42. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


A . 3D image of an EGFP - CaaX - expressing Ramos B cell embedded in Matrigel ( left ) and segmentation of the inner dome ( center ) using our own script based on Watershed segmentation and segmentation of the cell surface ( right ) using an nnU - Net model that we trained based on our own - generated ground truth .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

A. 3D image of an EGFP-CaaX-expressing Ramos B cell embedded in Matrigel (left) and segmentation of the inner dome (center) using our own script based on Watershed segmentation and segmentation of the cell surface (right) using an nnU-Net model that we trained based on our own-generated ground truth.


C . Cluster density at surface features . IgM - BCR cluster density within different topographical features quantified as cluster count per surface feature volume .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

C. Cluster density at surface features. IgM-BCR cluster density within different topographical features quantified as cluster count per surface feature volume.


F , G , H . Continuous analysis of IgM - BCR cluster density using geodesic distance maps initiated from ( F ) microvilli tips towards microvilli roots , ( G ) centerline of ridges towards cell body surface , and ( H ) nodes towards cell body surface .

O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O

F, G, H. Continuous analysis of IgM-BCR cluster density using geodesic distance maps initiated from (F) microvilli tips towards microvilli roots, (G) centerline of ridges towards cell body surface, and (H) nodes towards cell body surface.


A , B . Sum projections of the skeletonized ridge network and microvilli ( A ) and segmented IgM - BCR clusters ( B ) in an untreated and 100 µM CK - 666 - treated cell for 15 consecutive time frames amounting to 37 . 5 s .

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A, B. Sum projections of the skeletonized ridge network and microvilli (A) and segmented IgM-BCR clusters (B) in an untreated and 100 µM CK-666-treated cell for 15 consecutive time frames amounting to 37.5 s.


G . Continuous analysis of IgM - BCR cluster density in 15 - 30 min 100 µM CK - 666 - treated cells using geodesic distance maps initiating from distinct surface features ( centerline of ridges , nodes and microvilli tips ) ( to compare to untreated in Fig 3F ) . Data points and the dotted lines represent mean ± SD . H . Time - course images of EGFP - CaaX and IgM - BCR intensity in an elongating microvillus ( purple , MV mask ) .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O 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-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O

G. Continuous analysis of IgM-BCR cluster density in 15-30 min 100 µM CK-666-treated cells using geodesic distance maps initiating from distinct surface features (centerline of ridges, nodes and microvilli tips) (to compare to untreated in Fig 3F). Data points and the dotted lines represent mean ± SD. H. Time-course images of EGFP-CaaX and IgM-BCR intensity in an elongating microvillus (purple, MV mask).


A . IgM - BCR cluster distribution upon Ag stimulation . Ramos B cells in the Matrigel were stimulated with 50 ng / ml NIP - 15 - BSA Ag for at least 15 minutes . Ag - MC on the B cell surface were segmented and overlaid with the ridge network ( including microvilli ) with a 1 . 25x and 3 . 0x local intensity cut - off for cluster detection .

O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

A. IgM-BCR cluster distribution upon Ag stimulation. Ramos B cells in the Matrigel were stimulated with 50 ng/ml NIP-15-BSA Ag for at least 15 minutes. Ag-MC on the B cell surface were segmented and overlaid with the ridge network (including microvilli) with a 1.25x and 3.0x local intensity cut-off for cluster detection.


G , H . Staticity scores for different surface features ( R + N , MV ) ( G ) and for segmented IgM - BCR clusters assigned to different surface features ( H ) in unstimulated and Ag - stimulated cells . Violin plot solid lines represent the median and dotted lines represent the quartiles .

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G, H. Staticity scores for different surface features (R+N, MV) (G) and for segmented IgM-BCR clusters assigned to different surface features (H) in unstimulated and Ag-stimulated cells. Violin plot solid lines represent the median and dotted lines represent the quartiles.


A . Phalloidin - Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP - CaaX and IgM - BCR with or without 30 min 2 µM LatA treatment .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O

A. Phalloidin-Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP-CaaX and IgM-BCR with or without 30 min 2 µM LatA treatment.


D . Surface - masked EGFP - CaaX and IgM - BCR signal and overlay of segmented elevations ( yellow ) with the surface - masked IgM - BCR signal in cells treated with LatA and LatA + Ag ( 50 ng / ml NIP - 15 - BSA ) . E . Time frames from a LatA + Ag cell showing directed movement of Ag - MC towards the elevations ( yellow ) . Each Ag - MC shown with a different colored arrow .

O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O 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. Surface-masked EGFP-CaaX and IgM-BCR signal and overlay of segmented elevations (yellow) with the surface-masked IgM-BCR signal in cells treated with LatA and LatA+Ag (50 ng/ml NIP-15-BSA). E. Time frames from a LatA+Ag cell showing directed movement of Ag-MC towards the elevations (yellow). Each Ag-MC shown with a different colored arrow.


F . IgM - BCR intensity enrichment within all the segmented clusters in LatA and Lat + Ag cells . G . Cluster density at surface features . Quantification of the IgM - BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM - BCR clusters ( left panel , local contrast cutoff = 1x ) and for Ag - MC ( right panel , local contrast cutoff = 3x ) .

O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O 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. IgM-BCR intensity enrichment within all the segmented clusters in LatA and Lat+Ag cells. G. Cluster density at surface features. Quantification of the IgM-BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM-BCR clusters (left panel, local contrast cutoff = 1x) and for Ag-MC (right panel, local contrast cutoff = 3x). H


I . Surface - masked EGFP - CaaX and IgM - BCR signal for a LatA and LatA + Ag cell and the sum projections of their segmented IgM - BCR clusters ( duration : 37 . 5 s ) . IgM - BCR clusters are color - coded ( green and yellow ) according to their assigned surface region .

O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

I. Surface-masked EGFP-CaaX and IgM-BCR signal for a LatA and LatA+Ag cell and the sum projections of their segmented IgM-BCR clusters (duration: 37.5 s). IgM-BCR clusters are color-coded (green and yellow) according to their assigned surface region.


D ) Cells expressing empty vector ( EV ) , or PRKAA1 - myc , PRKAB2 and PRKAG2 - FLAG ( AMPK ) alone or with myc - Ulk1 WT ( ULK ) were cultured in full medium ( F ) , EBSS ( St ) , or serum - free medium ( SFM ) alone or in the presence of 991 ( 1µM ) or MRT68921 ( 1μM ) for 90 minutes as indicated . 2 % Loading control and pull down samples were analysed by Western blot .

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D) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.


A ) VPS15 - ZZ was co - expressed with Ulk1 and Ulk2 wild type ( WT ) or kinase inactive ( KI ) before analysis by Western blot . An electrophoretic mobility shift in VPS15 - ZZ was noted with WT only .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

A) VPS15-ZZ was co-expressed with Ulk1 and Ulk2 wild type (WT) or kinase inactive (KI) before analysis by Western blot. An electrophoretic mobility shift in VPS15-ZZ was noted with WT only.


A ) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot . LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA - derived clones .

O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O

A) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot. LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA-derived clones.


B ) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr , fixed and imaged for WIPI2 ( green ) . Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles ( red asterisks ) . Quantification of WIPI2 spots , mean + / - SEM , n = 3 .

O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O

B) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr, fixed and imaged for WIPI2 (green). Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles (red asterisks). Quantification of WIPI2 spots, mean +/- SEM, n=3.


D ) Wild type ( WT ) , 6SA or 6SE VPS15 - HA , or empty vector ( EV ) , were expressed in VPS15 KO or HEK293A cells . Cells were starved for 1 hr and the distributions of WIPI2 ( green ) , HA ( red ) and LC3 ( blue ) were assessed , mean + / - SEM , n = 4 .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O

D) Wild type (WT), 6SA or 6SE VPS15-HA, or empty vector (EV), were expressed in VPS15 KO or HEK293A cells. Cells were starved for 1 hr and the distributions of WIPI2 (green), HA (red) and LC3 (blue) were assessed, mean +/- SEM, n=4.


D ) VPS34 CI components expressed with Ulk1 1 - 427 WT or KI as indicated . After starvation for 1 hr in the presence or absence of MRT68921 ( ULK inhibitor ) , cells were lysed and VPS34 CI coimmunoprecipitated via ATG14 - ZZ . VPS15 S861 phosphorylation was increased in the presence of active Ulk1 . VPS15 pS861 / total VPS15 was quantified , mean + / - SEM , n = 3 .

O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

D) VPS34 CI components expressed with Ulk1 1-427 WT or KI as indicated. After starvation for 1 hr in the presence or absence of MRT68921 (ULK inhibitor), cells were lysed and VPS34 CI coimmunoprecipitated via ATG14-ZZ. VPS15 S861 phosphorylation was increased in the presence of active Ulk1. VPS15 pS861/total VPS15 was quantified, mean +/- SEM, n=3.


E ) HEK293A were transfected with VPS34 CII components and Ulk1 1 - 427 WT or KI and treated as indicated before VPS34 CII co - immunopurification via UVRAG - ZZ . Samples were analysed by Western blot as in D , revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII . Quantifications show mean of 4 independent experiments + / - SEM , * * * p < 0 . 001 [ one - tailed ANOVA ] .

O O B-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O 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) HEK293A were transfected with VPS34 CII components and Ulk1 1-427 WT or KI and treated as indicated before VPS34 CII co-immunopurification via UVRAG-ZZ. Samples were analysed by Western blot as in D, revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII. Quantifications show mean of 4 independent experiments +/-SEM, ***p<0.001 [one-tailed ANOVA].


A ) HEK293A , VPS15 KO control and stably rescued VPS15 KOs ( sgA - B3 ) were starved for 1 hr before WIPI2 ( green ) , LC3 ( red ) and DNA ( Hoechst , blue ) were visualised . Quantification of WIPI2 puncta number per cell , mean + / - SEM , n = 5 .

O O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

A) HEK293A, VPS15 KO control and stably rescued VPS15 KOs (sgA-B3) were starved for 1 hr before WIPI2 (green), LC3 (red) and DNA (Hoechst, blue) were visualised. Quantification of WIPI2 puncta number per cell, mean +/-SEM, n=5.


B ) Stably rescued VPS15 KO ( sgA - B3 ) , HEK293A and VPS15 KO control cells were starved for 1 hr with ( SB ) or without ( St ) 100nM Bafilomycin A1 , or cultured in full media ( F ) . Quantification shows LC3II / Actin , mean + / - SEM , n = 5 .

O O O O B-GENEPROD O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

B) Stably rescued VPS15 KO (sgA-B3), HEK293A and VPS15 KO control cells were starved for 1 hr with (SB) or without (St) 100nM Bafilomycin A1, or cultured in full media (F). Quantification shows LC3II/Actin, mean +/-SEM, n=5.


A ) HEK293A were treated with VPS34 - IN1 ( 1μM ) alone or in the presence of MRT68921 ( 1μM ) for the indicated time . Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot . Identical samples were loaded in parallel for VPS34 , ATG13 , PRKAB2 analysis , indicated by dashed line .

O O B-CELL_LINE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O

A) HEK293A were treated with VPS34-IN1 (1μM) alone or in the presence of MRT68921 (1μM) for the indicated time. Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot. Identical samples were loaded in parallel for VPS34, ATG13, PRKAB2 analysis, indicated by dashed line.


E ) HEK293A and VPS15 KO cells were fed , or starved for 2 hrs before fixation , and labelling with ULK1 ( green ) , ATG9A ( red ) and GM130 ( blue ) . Dashed boxes show magnified regions of interest .

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E) HEK293A and VPS15 KO cells were fed, or starved for 2 hrs before fixation, and labelling with ULK1 (green), ATG9A (red) and GM130 (blue). Dashed boxes show magnified regions of interest.


B ) HEK293A were transfected with siRNAs targeting ATG14 , UVRAG or p62 , or with non - targeting siRNA ( RISC - free , RF ) and cells were treated with VPS34 - IN1 ( IN1 ; 1μM ) for 0 , 4 or 18 hrs as indicated . Quantification of ATG13 pS318 / Total ATG13 , mean + / - SEM , n = 3 . Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis , indicated by dashed line .

O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

B) HEK293A were transfected with siRNAs targeting ATG14, UVRAG or p62, or with non-targeting siRNA (RISC-free, RF) and cells were treated with VPS34-IN1 (IN1; 1μM) for 0, 4 or 18 hrs as indicated. Quantification of ATG13 pS318/Total ATG13, mean +/- SEM, n=3. Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis, indicated by dashed line.


C ) Cells transfected as in B were treated with IN1 for 18 hours as indicated , fixed and labelled for ubiquitin ( green ) , FIP200 ( red ) and p62 ( blue ) . FIP200 - positive bodies were quantified from 4 independent experiments with mean + / - SEM plotted . Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested .

O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O

C) Cells transfected as in B were treated with IN1 for 18 hours as indicated, fixed and labelled for ubiquitin (green), FIP200 (red) and p62 (blue). FIP200-positive bodies were quantified from 4 independent experiments with mean +/- SEM plotted. Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested.


Plasma corticosterone levels at baseline , 30 minutes after induction of stress by placing mice in a clean cage , and after 90 minutes upon return in the home cage . DEX mice received an injection of dexamethasone at 0 . 1 mg / kg body weight 150 minutes before collecting the first blood sample ( n = 5 - 6 animals per group ) . Flox and GRBATKO mice were injected with 0 . 02 mg / kg dexamethasone and the procedure described in ( A ) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in ( A ) ( n = 6 - 7 animals per group ) .

B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O

Plasma corticosterone levels at baseline, 30 minutes after induction of stress by placing mice in a clean cage, and after 90 minutes upon return in the home cage. DEX mice received an injection of dexamethasone at 0.1 mg/kg body weight 150 minutes before collecting the first blood sample (n = 5-6 animals per group). Flox and GRBATKO mice were injected with 0.02 mg/kg dexamethasone and the procedure described in (A) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in (A) (n = 6-7 animals per group).


GR mRNA levels in BAT of Flox and GRBATKO mice . Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage . Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure , controls received an injection with saline ( n = 5 - 6 animals per group ) .

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GR mRNA levels in BAT of Flox and GRBATKO mice. Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage. Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure, controls received an injection with saline (n = 5-6 animals per group).


Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice ( n = 5 - 6 animals per group ) . Blood glucose levels ( n = 5 - 6 animals per group ) .

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Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice (n = 5-6 animals per group). Blood glucose levels (n = 5-6 animals per group).


Knockdown of GR in BAT assessed by immunoblotting . Β - Tubulin was used as loading control . Quantification of immunoblot in ( A ) using Image Lab ( n = 6 animals per group ) .

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Knockdown of GR in BAT assessed by immunoblotting. Β-Tubulin was used as loading control. Quantification of immunoblot in (A) using Image Lab (n = 6 animals per group).


Body temperature over the course of the cold exposure , determined daily ( n = 6 animals per group ) .

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Body temperature over the course of the cold exposure, determined daily (n = 6 animals per group).


Body weight at the end of the cold exposure ( n = 6 animals per group ) .

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Body weight at the end of the cold exposure (n = 6 animals per group).


BAT weight at the end of the cold exposure ( n = 6 animals per group ) .

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BAT weight at the end of the cold exposure (n = 6 animals per group).


Representative Hematoxilin and eosin staining image of BAT of cold - exposed Flox and GRBATKO mice . Scale bar 50 µm .

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Representative Hematoxilin and eosin staining image of BAT of cold-exposed Flox and GRBATKO mice. Scale bar 50 µm.


Triglyceride content in BAT from Flox and GRBATKO animals ( n = 5 - 6 animals per group ) .

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Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group).


Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals , indicating unchanged thermogenic marker gene expression . Color gradient ranges from 2 - fold higher expression in GRBATKO over Flox controls ( red ) to 0 . 1 - fold ( lower ) expression ( blue ) ( n = 4 animals per group ) .

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Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals, indicating unchanged thermogenic marker gene expression. Color gradient ranges from 2-fold higher expression in GRBATKO over Flox controls (red) to 0.1-fold (lower) expression (blue) (n = 4 animals per group).


Volcano plot from microarray gene expression data as in ( I ) , with cut - off values for the adjusted p - value < 0 . 1 and fold change > 1 . 5 .

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Volcano plot from microarray gene expression data as in (I), with cut-off values for the adjusted p-value <0.1 and fold change >1.5.


Serum corticosterone levels ( n = 6 animals per group ) .

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Serum corticosterone levels (n = 6 animals per group).


Body weight after 16 h fasting and 6 h chow refeeding at 22 ° C ( n = 6 animals per group )

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Body weight after 16 h fasting and 6 h chow refeeding at 22°C (n = 6 animals per group)


Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h ( n = 6 animals per group ) .

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Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h (n = 6 animals per group).


Serum insulin and corticosterone levels determined by ELISA ( n = 6 animals per group ) .

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Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group).


Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding ( n = 6 animals per group ) .

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Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).


Body weight gain over a 24 - week period of high - fat feeding ( n = 13 - 14 animals per group ) . Panel shows experimental design for Figures 4 - 6 .

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Body weight gain over a 24-week period of high-fat feeding (n = 13-14 animals per group). Panel shows experimental design for Figures 4-6.


Oxygen consumption over a 90 - hour period . Grays columns indicate dark phase ( n = 6 - 8 mice per group ) . Average oxygen consumption specified for light phase , dark phase , and total ( n = 6 - 8 animals per genotype ) .

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Oxygen consumption over a 90-hour period. Grays columns indicate dark phase (n = 6-8 mice per group). Average oxygen consumption specified for light phase, dark phase, and total (n = 6-8 animals per genotype).


Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight ( n = 6 - 8 animals per group ) .

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Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight (n = 6-8 animals per group).


Locomotor activity over a 90 - hour period ( n = 6 - 8 animals per group ) .

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Locomotor activity over a 90-hour period (n = 6-8 animals per group).


Intraperitoneal GTT after 19 weeks of HFD - feeding , glucose injections at 1 . 5 mg per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve calculated from ( A ) ( n = 11 - 13 animals per group ) .

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Intraperitoneal GTT after 19 weeks of HFD-feeding, glucose injections at 1.5 mg per g of body weight (n = 11-13 animals per group). Area under the curve calculated from (A) (n = 11-13 animals per group).


Plasma insulin levels during GTT ( n = 11 - 13 animals per group ) .

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Plasma insulin levels during GTT (n = 11-13 animals per group).


Intraperitoneal ITT after 20 weeks of high - fat diet intervention . Insulin was adjusted to 1 mU per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve determined from ( D ) ( n = 11 - 13 animals per group ) .

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Intraperitoneal ITT after 20 weeks of high-fat diet intervention. Insulin was adjusted to 1 mU per g of body weight (n = 11-13 animals per group). Area under the curve determined from (D) (n = 11-13 animals per group).


Plasma insulin levels after a 5 - hour fast , before injecting insulin ( n = 11 - 13 animals per group ) .

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Plasma insulin levels after a 5-hour fast, before injecting insulin (n = 11-13 animals per group).


Body weight in Flox and GRBATKO after 24 weeks of high - fat feeding ( n = 10 - 13 animals per group ) .

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Body weight in Flox and GRBATKO after 24 weeks of high-fat feeding (n = 10-13 animals per group).


BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks ( n = 10 - 13 animals per group ) .

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BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks (n = 10-13 animals per group).


GR levels in BAT after 24 weeks high - fat feeding ( n = 10 - 13 mice per group ) .

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GR levels in BAT after 24 weeks high-fat feeding (n=10-13 mice per group).


Representative H & E stained sections of BAT from Flox and GRBATKO animals . Scale bar 100 µm .

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Representative H&E stained sections of BAT from Flox and GRBATKO animals. Scale bar 100 µm.


Triglyceride content in BAT per gram of wet tissue ( n = 10 - 13 animals per group ) .

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Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).


qRT - PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high - fat feeding ( n = 9 - 13 animals per group ) .

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qRT-PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high-fat feeding (n = 9-13 animals per group).


Representative immunohistochemistry for UCP1 in BAT . Scale bar 100 µm .

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Representative immunohistochemistry for UCP1 in BAT. Scale bar 100 µm.


Serum insulin levels determined by ELISA ( n = 10 - 13 animals per group ) .

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Serum insulin levels determined by ELISA (n = 10-13 animals per group).


Blood glucose in Flox and GRBATKO animals after 24 weeks HFD ( n = 10 - 13 animals per group ) .

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Blood glucose in Flox and GRBATKO animals after 24 weeks HFD (n = 10-13 animals per group).


B - Congo red plates with colonies of E . coli strain MC4100 relA + and its target mutation csgD : M4 - ompR : mut2 derivative , harboring sRNA - producing plasmids pOmrA , pOmrB , or an empty vector ( pControl ) . Incubation at 28 ° C for 48 h .

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B - Congo red plates with colonies of E. coli strain MC4100 relA+ and its target mutation csgD:M4-ompR:mut2 derivative, harboring sRNA-producing plasmids pOmrA, pOmrB, or an empty vector (pControl). Incubation at 28°C for 48 h.


B - Relative fluorescence / OD600 of cells of E . coli strain MC4100 relA + ∆ omrAB or MC4100 relA + ∆ omrAB ∆ hfq harboring the translational fusion plasmid pDgcM : : GFP in combination with plasmids expressing OmrA , OmrB , or an empty control vector , after 16 h of growth at 37 ° C . Error bars SD , N = 3 . Average fluorescence intensities ( a . u . ) / OD600 used to calculate ratios Note that absolute dgcM : : gfp expression values are ≈ 3 - 4 - fold lower in the ∆ hfq

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B - Relative fluorescence/OD600 of cells of E. coli strain MC4100 relA+ ∆omrAB or MC4100 relA+ ∆omrAB ∆hfq harboring the translational fusion plasmid pDgcM::GFP in combination with plasmids expressing OmrA, OmrB, or an empty control vector, after 16 h of growth at 37°C. Error bars SD, N=3. Average fluorescence intensities (a.u.)/ OD600 used to calculate ratios Note that absolute dgcM::gfp expression values are ≈3-4-fold lower in the ∆hfq strain


C - Western blot analysis of cell lysates from strains MC4100 relA + ∆ omrAB harboring plasmids pOmrA , pOmrB , or mutant variants as indicated , in combination with a plasmid expressing C - terminal FLAG - tagged dgcM or dgcM _ M2 . As a loading control , part of a protein - stained gel is shown below .

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C - Western blot analysis of cell lysates from strains MC4100 relA+ ∆omrAB harboring plasmids pOmrA, pOmrB, or mutant variants as indicated, in combination with a plasmid expressing C-terminal FLAG-tagged dgcM or dgcM_M2. As a loading control, part of a protein-stained gel is shown below.


A - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA ( 210 nt from + 1 ) by OmrA and OmrB . Inclusion of 30S ribosomes , tRNAfmet , OmrA , and OmrB is indicated . The unrelated IstR - 1 sRNA is a negative control . The 30S / tRNAfmet - dependent reverse transcription block ( toeprint ) , is indicated by an arrow . Sequencing reactions were loaded next to the primer extension products ( UCGA ) . The AUG start codon is indicated by asterisks .

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A - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA (210 nt from +1) by OmrA and OmrB. Inclusion of 30S ribosomes, tRNAfmet, OmrA, and OmrB is indicated. The unrelated IstR-1 sRNA is a negative control. The 30S/ tRNAfmet-dependent reverse transcription block (toeprint), is indicated by an arrow. Sequencing reactions were loaded next to the primer extension products (UCGA). The AUG start codon is indicated by asterisks.


B - In vitro translation of dgcM : : 3xflag mRNA in the presence or absence of OmrA / OmrB , with or without purified Hfq protein , was carried out As an internal control , the non - target ompA : : 3xflag mRNA was included . The Western blot shows DgcM - 3xFLAG and OmpA - 3xFLAG protein levels .

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B - In vitro translation of dgcM::3xflag mRNA in the presence or absence of OmrA/ OmrB, with or without purified Hfq protein, was carried out As an internal control, the non-target ompA::3xflag mRNA was included. The Western blot shows DgcM-3xFLAG and OmpA-3xFLAG protein levels.


A - 5 ' end - labeled dgcM mRNA ( 10 nM ) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram . When added , Hfq hexamers were at 50 nM , the sRNAs OmrA and OmrB at 250 nM . The accessibility of each nucleotide was probed with water ( lanes 1 - 4 , negative control for RNA integrity ) , RNase T1 , or lead ( II ) acetate . Lane T1 : RNase T1 under denaturing conditions . Lane OH : alkaline ladders . Binding sites of Hfq ( green arrows and boxes ) , and site 1 ( red arrows and boxes ) and 2 ( blue arrows and boxes ) of the sRNAs , are highlighted . Note that site 2 is bound differently by OmrA and OmrB ( blue boxes on gel ) . Black boxes indicate Hfq - dependent T1 cleavage enhancements .

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A - 5' end-labeled dgcM mRNA (10 nM) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram. When added, Hfq hexamers were at 50 nM, the sRNAs OmrA and OmrB at 250 nM. The accessibility of each nucleotide was probed with water (lanes 1-4, negative control for RNA integrity), RNase T1, or lead(II) acetate. Lane T1: RNase T1 under denaturing conditions. Lane OH: alkaline ladders. Binding sites of Hfq (green arrows and boxes), and site 1 (red arrows and boxes) and 2 (blue arrows and boxes) of the sRNAs, are highlighted. Note that site 2 is bound differently by OmrA and OmrB (blue boxes on gel). Black boxes indicate Hfq-dependent T1 cleavage enhancements.


Mobility shift assay with end - labeled dgcM RNA ( 210 nt species from + 1 ) in the presence or absence of unlabeled OmrA or OmrB . When indicated , single - stranded DNA oligos were pre - annealed to site 1 ( dgcM _ site1 ) , site 2 ( dgcM _ site2 ) , or both , before addition of sRNAs . The additions for each sample are indicated above the Figure , and the identities of the nucleic acid complexes are indicated on the left for guidance .

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Mobility shift assay with end-labeled dgcM RNA (210 nt species from +1) in the presence or absence of unlabeled OmrA or OmrB. When indicated, single-stranded DNA oligos were pre-annealed to site 1 (dgcM_site1), site 2 (dgcM_site2), or both, before addition of sRNAs. The additions for each sample are indicated above the Figure, and the identities of the nucleic acid complexes are indicated on the left for guidance.


Western blots showing DgcM - 3xFLAG translated in vitro in the presence or absence of OmrA or OmrB . A - The inclusion of purified wild - type or mutant Hfq proteins is indicated , and the translation assay was conducted The different Hfq interaction site mutant proteins are indicated . Data information : Quantification of DgcM translation rates , relative to that of the unaffected OmpA control , is shown below the gels . The ratio of DgcM / OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations ( - sRNA , + OmrA , + OmrB ) , indicated by " 1 . 00 * " . Effects due to OmrA / OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set .

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Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. A - The inclusion of purified wild-type or mutant Hfq proteins is indicated, and the translation assay was conducted The different Hfq interaction site mutant proteins are indicated. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set.


Western blots showing DgcM - 3xFLAG translated in vitro in the presence or absence of OmrA or OmrB . B - An ssDNA - oligo ( Hfq _ BS that base - pairs specifically to the Hfq binding site on dgcM mRNA ( nt 42 - 66 ) was preannealed to the mRNA prior to OmrA / B addition and translation . OmpA - 3xFLAG was included as internal control . Data information : Quantification of DgcM translation rates , relative to that of the unaffected OmpA control , is shown below the gels . The ratio of DgcM / OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations ( - sRNA , + OmrA , + OmrB ) , indicated by " 1 . 00 * " . Effects due to OmrA / OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set .

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Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. B - An ssDNA-oligo (Hfq_BS that base-pairs specifically to the Hfq binding site on dgcM mRNA (nt 42-66) was preannealed to the mRNA prior to OmrA/B addition and translation. OmpA-3xFLAG was included as internal control. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set.


A - Histograms show flow cytometry analysis of exponentially growing cells of E . coli strain MC4100 relA - , and its mutant derivatives hfq - F42A ( hfq - prox ) , hfq - Y25D ( hfq - dist ) , hfq - R16S _ R17A _ R19A _ K47E ( hfq - rim ) , ∆ hfq , harboring the translational fusion plasmid pDgcM : : GFP in combination with pOmrA , pOmrB , or a control vector . Data was obtained on a MACSQuant VYB instrument .

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A - Histograms show flow cytometry analysis of exponentially growing cells of E. coli strain MC4100 relA-, and its mutant derivatives hfq-F42A (hfq-prox), hfq-Y25D (hfq-dist), hfq-R16S_R17A_R19A_K47E (hfq-rim), ∆hfq, harboring the translational fusion plasmid pDgcM::GFP in combination with pOmrA, pOmrB, or a control vector. Data was obtained on a MACSQuant VYB instrument.


B - Northern blot analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry , probed for OmrA ( strains containing pOmrA ) or OmrB ( strains containing pOmrB ) . 5S rRNA was used as loading control .

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B - Northern blot analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry, probed for OmrA (strains containing pOmrA) or OmrB (strains containing pOmrB). 5S rRNA was used as loading control.


LGG - 1 : : GFP - positive puncta labeling autophagic membranes [ 22 ] were counted in wild - type or in food - limited animals . ( A ) Micrographs of eat - 2 ( ad1116 ) L3 larvae expressing GFP - tagged lgg - 1 / LC3 . Arrow indicates autophagic focus . Magnification is indicated . ( B ) Average number of LGG - 1 : : GFP - containing puncta in eat - 2 ( ad1116 ) mutants and N2 wild - type animals ( WT ) , p < 0 . 0001 . ( C ) Average number of LGG - 1 : : GFP - containing puncta in N2 wild - type , food - restricted animals grown in liquid media ( WT , food limited ) and N2 wild - type animals grown in liquid with a higher concentration of bacteria ( WT , fully fed ) , p < 0 . 0001 ; see Methods . Between three and ten seam cells were counted in each of 20 - 40 animals using high - power microscopy and averaged . n , total number of seam cells observed . Error bars : ± SEM . p - Values were calculated as unpaired , two - tailed t - test . Animals were raised at 20 ° C . Please see Table S1 for quantification of all data .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LGG-1::GFP-positive puncta labeling autophagic membranes [22] were counted in wild-type or in food-limited animals.(A) Micrographs of eat-2(ad1116) L3 larvae expressing GFP-tagged lgg-1/LC3. Arrow indicates autophagic focus. Magnification is indicated.(B) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) mutants and N2 wild-type animals (WT), p < 0.0001.(C) Average number of LGG-1::GFP-containing puncta in N2 wild-type, food-restricted animals grown in liquid media (WT, food limited) and N2 wild-type animals grown in liquid with a higher concentration of bacteria (WT, fully fed), p < 0.0001; see Methods.Between three and ten seam cells were counted in each of 20-40 animals using high-power microscopy and averaged. n, total number of seam cells observed. Error bars: ±SEM. p-Values were calculated as unpaired, two-tailed t-test. Animals were raised at 20 °C. Please see Table S1 for quantification of all data.


( A ) Survival curves of eat - 2 ( ad1116 ) animals fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . Mean lifespan was 23 . 7 d for control and 19 . 6 d for bec - 1 RNAi , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . This experiment was performed a total of six times , and bec - 1 RNAi shortened the lifespan of eat - 2 animals ∼ 15 % - 30 % . Please see Table 1 for additional data . ( B ) Survival curves of N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . These assays were performed concurrently with the eat - 2 mutant lifespan analysis in Figure 2A . Mean lifespan was 17 . 3 d for control and 18 . 9 d for bec - 1 RNAi . p = 0 . 045 , Log - rank ( Mantel - Cox ) test . Depletion of bec - 1 did not significantly change the lifespan of N2 or sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals in any of six experiments . Please see Table 1 for additional data . ( C ) Survival curves of eat - 2 ( ad1116 ) animals fed either control bacteria or bacteria expressing vps - 34 dsRNA during adulthood at 20 ° C . Mean lifespan was 27 . 6 d for control and 22 . 8 d for vps - 34 RNAi , p = 0 . 0003 , Log - rank ( Mantel - Cox ) test . This experiment was performed a total of four times . Please see Table 1 for additional data . ( D ) Survival curves of sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals ( WT ) fed either control bacteria or bacteria expressing vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at the same time as the eat - 2 lifespan analysis shown in Figure 2C . Mean lifespan was 21 . 5 d for control and 23 . 3 d for bec - 1 RNAi . p = 0 . 14 , Log - rank ( Mantel - Cox ) test . Depletion of vps - 34 did not significantly change the lifespan of N2 or sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals in each of six different experiments . Please see Table 1 for additional data .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O

(A) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan was 23.7 d for control and 19.6 d for bec-1 RNAi, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed a total of six times, and bec-1 RNAi shortened the lifespan of eat-2 animals ∼15%-30%. Please see Table 1 for additional data.(B) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. These assays were performed concurrently with the eat-2 mutant lifespan analysis in Figure 2A. Mean lifespan was 17.3 d for control and 18.9 d for bec-1 RNAi. p = 0.045, Log-rank (Mantel-Cox) test. Depletion of bec-1 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in any of six experiments. Please see Table 1 for additional data.(C) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.6 d for control and 22.8 d for vps-34 RNAi, p = 0.0003, Log-rank (Mantel-Cox) test. This experiment was performed a total of four times. Please see Table 1 for additional data.(D) Survival curves of sterile fer-15(b26); fem-1(hc17) animals (WT) fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the eat-2 lifespan analysis shown in Figure 2C. Mean lifespan was 21.5 d for control and 23.3 d for bec-1 RNAi. p = 0.14, Log-rank (Mantel-Cox) test. Depletion of vps-34 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in each of six different experiments. Please see Table 1 for additional data.


( A ) Average number of LGG - 1 : : GFP - containing puncta in eat - 2 ( ad1116 ) progeny of animals fed either control bacteria or bacteria expressing bec - 1 , vps - 34 or pha - 4 dsRNA their entire lives . p ≤ 0 . 0001 for bec - 1 , vps - 34 and pha - 4 RNAi treatments compared to control , respectively , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

(A) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34 or pha-4 dsRNA their entire lives. p ≤ 0.0001 for bec-1, vps-34 and pha-4 RNAi treatments compared to control, respectively, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details.


( B ) Average number of LGG - 1 : : GFP - containing puncta in rab - 10 ( ok1494 ) progeny of animals fed either control bacteria or bacteria expressing pha - 4 dsRNA their entire lives ; p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . Please see Figure 1 for details and Table S1 for quantification of data . Feeding mutants for several generations with pha - 4 dsRNA , inhibited development and sharply decreased the number of eggs laid ( [ 46 ] , data not shown ) . However , the loss of many embryos was unlikely to bias our findings , since many progeny of daf - 2 mutants treated with pha - 4 RNAi also died but those that did not exhibited a robust autophagy phenotype ( Figure 6 ) .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O 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-CELL_TYPE I-CELL_TYPE O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) progeny of animals fed either control bacteria or bacteria expressing pha-4 dsRNA their entire lives; p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details and Table S1 for quantification of data. Feeding mutants for several generations with pha-4 dsRNA, inhibited development and sharply decreased the number of eggs laid ([46], data not shown). However, the loss of many embryos was unlikely to bias our findings, since many progeny of daf-2 mutants treated with pha-4 RNAi also died but those that did not exhibited a robust autophagy phenotype (Figure 6).


( A ) Average number of LGG - 1 : : GFP - containing puncta in let - 363 / TOR RNAi - arrested animals compared to N2 wild - type animals ( WT ) grown on control ( vector - only ) bacteria , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . We were not able to detect increased LGG - 1 : : GFP puncta in long - lived let - 363 ( RNAi ) adults ; however , one generation of daf - 2 RNAi , our positive control , did not significantly increase the number of foci in adults either ( data not shown ) . Please see Table S1 for quantification of all data . ( B ) Average number of LGG - 1 : : GFP - containing puncta in daf - 15 ( m81 ) / unc - 24 ( e138 ) heterozygotes ( daf - 15 / + ) compared to N2 wild - type animals ( WT ) , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Please see Table S1 for quantification of all data . daf - 15 encodes the TOR - binding partner Raptor .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O 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-CELL_TYPE I-CELL_TYPE 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

(A) Average number of LGG-1::GFP-containing puncta in let-363/TOR RNAi-arrested animals compared to N2 wild-type animals (WT) grown on control (vector-only) bacteria, p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. We were not able to detect increased LGG-1::GFP puncta in long-lived let-363(RNAi) adults; however, one generation of daf-2 RNAi, our positive control, did not significantly increase the number of foci in adults either (data not shown). Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+) compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data. daf-15 encodes the TOR-binding partner Raptor.


( C ) Survival curves of daf - 15 ( m81 ) / unc - 24 ( e138 ) heterozygotes ( daf - 15 / + , strain DR412 ) fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . Mean lifespan : daf - 15 / + animals grown on control RNAi - bacteria : 25 . 1 d , daf - 15 / + animals on bec - 1 RNAi : 20 . 8 d , p = 0 . 0008 , Log - rank ( Mantel - Cox ) test . The lifespan of daf - 15 / + animals grown on bec - 1 RNAi - bacteria during adulthood was measured again , yielding similar results ; the lifespan of daf - 15 / + animals was also measured three times following whole - life RNAi exposure . In these experiments , bec - 1 RNAi generally shortened the mean lifespan of daf - 15 / + animals to a greater extent than it shortened the lifespan of wild type . ( We also attempted to perform double - RNAi experiments , in which animals were cultured on a 50 : 50 mixture of let - 363 / TOR and bec - 1 [ or control ] RNAi bacteria . Although the trends we saw were in the expected direction , the effects produced by half - strength RNAi were small and not statistically significant [ data not shown ] . ) Please see Table 2 for additional data . ( D ) Survival curves of wild - type animals derived from strain DR412 ( WT ) fed either control bacteria or bacteria expressing bec - 1 dsRNA throughout their whole life at 20 ° C . These assays were performed concurrently with the daf - 15 / + lifespan analysis shown in Figure 4C . WT grown on control RNAi - bacteria : 21 . 4 d , WT on bec - 1 RNAi - bacteria : 21 . 1 d ( p = 0 . 34 ) , p between daf - 15 / + and WT grown on control RNAi - bacteria , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . Please see Table 2 for additional data .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O 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) Survival curves of daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+, strain DR412) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan: daf-15/+ animals grown on control RNAi-bacteria: 25.1 d, daf-15/+animals on bec-1 RNAi: 20.8 d, p = 0.0008, Log-rank (Mantel-Cox) test. The lifespan of daf-15/+ animals grown on bec-1 RNAi-bacteria during adulthood was measured again, yielding similar results; the lifespan of daf-15/+ animals was also measured three times following whole-life RNAi exposure. In these experiments, bec-1 RNAi generally shortened the mean lifespan of daf-15/+ animals to a greater extent than it shortened the lifespan of wild type. (We also attempted to perform double-RNAi experiments, in which animals were cultured on a 50:50 mixture of let-363/TOR and bec-1 [or control] RNAi bacteria. Although the trends we saw were in the expected direction, the effects produced by half-strength RNAi were small and not statistically significant [data not shown].) Please see Table 2 for additional data.(D) Survival curves of wild-type animals derived from strain DR412 (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA throughout their whole life at 20 °C. These assays were performed concurrently with the daf-15/+ lifespan analysis shown in Figure 4C. WT grown on control RNAi-bacteria: 21.4 d, WT on bec-1 RNAi-bacteria: 21.1 d (p = 0.34), p between daf-15/+ and WT grown on control RNAi-bacteria, p < 0.0001, Log-rank (Mantel-Cox) test. Please see Table 2 for additional data.


( A ) Average number of LGG - 1 : : GFP - containing puncta in N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing daf - 2 ( as a control ) or rab - 10 dsRNA for two generations . p < 0 . 0001 for either daf - 2 or rab - 10 RNAi treatment compared to control RNAi treatment , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Please see Table S1 for quantification of all data . ( B ) Average number of LGG - 1 : : GFP - containing puncta in rab - 10 ( ok1494 ) mutants compared to N2 wild - type animals ( WT ) , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . Please see Figure 1 for details . Please see Table S1 for quantification of all data .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O 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-CELL_TYPE I-CELL_TYPE O 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) Average number of LGG-1::GFP-containing puncta in N2 wild-type animals (WT) fed either control bacteria or bacteria expressing daf-2 (as a control) or rab-10 dsRNA for two generations. p < 0.0001 for either daf-2 or rab-10 RNAi treatment compared to control RNAi treatment, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) mutants compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details. Please see Table S1 for quantification of all data.


( C ) Survival curves of rab - 10 ( ok1494 ) animals fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . Mean lifespan was 27 . 9 d for control , 19 . 9 d for bec - 1 RNAi , and 22 . 1 d for vps - 34 RNAi , all pair - wise comparisons to control , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . This experiment was performed two times . Please see Table 3 for additional data . ( D ) Survival curves of N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at the same time as the rab - 10 lifespan analysis shown in Figure 5C . Mean lifespan was 22 . 9 d for control , 23 . 6 d for bec - 1 RNAi , and 24 . 5 d for vps - 34 RNAi , pair - wise comparison to control , p = 0 . 057 and p = 0 . 16 , respectively , Log - rank ( Mantel - Cox ) test . Please see Table 1 for additional data .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O 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) Survival curves of rab-10(ok1494) animals fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.9 d for control, 19.9 d for bec-1 RNAi, and 22.1 d for vps-34 RNAi, all pair-wise comparisons to control, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed two times. Please see Table 3 for additional data.(D) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the rab-10 lifespan analysis shown in Figure 5C. Mean lifespan was 22.9 d for control, 23.6 d for bec-1 RNAi, and 24.5 d for vps-34 RNAi, pair-wise comparison to control, p = 0.057 and p = 0.16, respectively, Log-rank (Mantel-Cox) test. Please see Table 1 for additional data.


( A ) Average number of LGG - 1 : : GFP - containing puncta in daf - 2 ( e1370 ) progeny of animals fed either control bacteria or bacteria expressing bec - 1 , vps - 34 , or pha - 4 dsRNA for their entire lives . p < 0 . 0001 for bec - 1 and vps - 34 RNAi - bacteria compared to control RNAi - bacteria , respectively , p = 0 . 17 for pha - 4 RNAi - bacteria compared to control RNAi - bacteria , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Feeding daf - 2 mutants for several generations with pha - 4 dsRNA sharply decreased the number of eggs laid ( data not shown ) . The mean lifespan of daf - 2 ( e1370 ) animals was shortened 12 . 5 % by pha - 4 RNAi ( [ 28 ] and data not shown ) , and we measured an 11 % decrease in puncta in daf - 2 ( e1370 ) animals fed pha - 4 RNAi . Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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) Average number of LGG-1::GFP-containing puncta in daf-2(e1370) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34, or pha-4 dsRNA for their entire lives. p < 0.0001 for bec-1 and vps-34 RNAi-bacteria compared to control RNAi-bacteria, respectively, p = 0.17 for pha-4 RNAi-bacteria compared to control RNAi-bacteria, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Feeding daf-2 mutants for several generations with pha-4 dsRNA sharply decreased the number of eggs laid (data not shown). The mean lifespan of daf-2(e1370) animals was shortened 12.5% by pha-4 RNAi ([28] and data not shown), and we measured an 11% decrease in puncta in daf-2(e1370) animals fed pha-4 RNAi. Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan.