Figure 2 ( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs ( top panels ) or siRNAs ( bottom panels ) . GFP level was measured in Cy5 + cells by flow cytometry 72 hours ( hrs ) post - transfection . ( B ) Mavs + / - and Mavs - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs . The level of d2GFP in Cy5 + cells was measured at various timepoints post - transfection as indicated . Histograms plots show d2GFP level 48 hrs post - transfection . Bar graphs show 3 timepoints as indicated . ( C ) WT , Ifnar1 + / - or Ifnar1 - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry at 48 hrs post - transfection .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs (top panels) or siRNAs (bottom panels). GFP level was measured in Cy5+ cells by flow cytometry 72 hours (hrs) post-transfection.(B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated.(C) WT, Ifnar1+/- or Ifnar1-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry at 48 hrs post-transfection.


Figure 3 ( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR ( α - IFNAR Ab ) , an isotype control antibody ( IgG1 ) or no antibody ( no Ab ) . Cells were then were transfected or not ( mock ) with Cy5 - labeled dsRNA as indicated and GFP level monitored in Cy5 + cells 72 hrs post - transfection . ( B ) Mavs - / - MEFs stably expressing d2GFP were treated with recombinant IFN ( 200 U / ml ) for 24 hrs prior to transfection or not ( mock ) with Cy5 - labeled dsRNA , as indicated . IFN treatment was maintained for 2 days post - transfection and then GFP level monitored in Cy5 + cells .

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Figure 3(A) Mavs+/- and Mavs-/-MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+cells 72 hrs post-transfection.(B) Mavs-/-MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+cells.


Figure 4 ( A ) Northern blot analysis of Ifnar1 - / - MEFs at 24 h . p . t with the indicated dsRNA or transfection reagent alone ( mock ) using a probe specific for dsRNA - GFP ( top ) or dsRNA - RL ( bottom ) . Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG - human Dicer were loaded in parallel . Two lanes ( delimited by dashed lines ) were left empty between the Ifnar1 - / - MEFs transfected samples and the in vitro dicing assay reactions . A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17 , 21 and 25 - nt in length was run in parallel . Endogenous U6 was used as a loading control . Signals corresponding to the dsRNA - derived siRNAs are indicated with an arrow . The membrane was first probed for dsRNA - GFP ( top ) , then probed for the miRNA marker ( top left ) and subsequently stripped and reprobed for dsRNA - RL ( middle ) and finally stripped and reprobed for both U6 ( bottom ) and for the miRNA marker again ( middle left ) . ( B ) Western blot of Dicer in d2GFP - expressing Mavs - / - MEFs either not transduced ( NT ) or transduced to express a non - targeting shRNA control ( CTL shRNA ) or two different Dicer - specific targeting shRNAs ( DICER shRNA 1 and 2 ) . P97 served as loading control . ( C ) Cells described in ( B ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later . Please note that the biphasic pattern of d2GFP level observed in dsRNA - GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP ( D ) Western blots of Ago2 in parental Mavs - / - MEFs stably expressing d2GFP and three Mavs - / - Ago2 - / - clones generated by CRISPR / CAS9 - mediated genome engineering . P97 served as loading control . ( E ) Mavs - / - ( parental line ) and Mavs - / - Ago2 - / - MEFs ( clone 5 . 1 ) stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

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Figure 4(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left).(B) Western blot of Dicer in d2GFP-expressing Mavs-/- MEFs either not transduced (NT) or transduced to express a non-targeting shRNA control (CTL shRNA) or two different Dicer-specific targeting shRNAs (DICER shRNA 1 and 2). P97 served as loading control.(C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP(D) Western blots of Ago2 in parental Mavs-/- MEFs stably expressing d2GFP and three Mavs-/- Ago2-/- clones generated by CRISPR/CAS9-mediated genome engineering. P97 served as loading control.(E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


Figure 5 ( A ) Western blots for Ago2 ( top ) and HA tag ( bottom ) of parental Mavs - / - MEFs and two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ; see Fig 4A ) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA - tagged wild - type ( HA - mAgo2 WT ) or catalytic mutant ( HA - mAgo2 D597A ) versions of mouse Ago2 . β - actin served as loading control . The membrane was first analysed for Ago2 and β - actin , stripped and then analysed for HA tag . ( B ) Parental non - transduced ( NT ) Mavs - / - MEFs and complemented Mavs - / - Ago2 - / - MEFs as described in ( A ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Western blots for Ago2 (top) and HA tag (bottom) of parental Mavs-/- MEFs and two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3; see Fig 4A) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA-tagged wild-type (HA-mAgo2 WT) or catalytic mutant (HA-mAgo2 D597A) versions of mouse Ago2. β-actin served as loading control. The membrane was first analysed for Ago2 and β-actin, stripped and then analysed for HA tag.(B) Parental non-transduced (NT) Mavs-/- MEFs and complemented Mavs-/- Ago2-/- MEFs as described in (A) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


Figure 6 ( A ) Ifnar1 - / - MEFs were transfected with the indicated Cy5 - labeled dsRNA and the next day were infected with SFV - Rluc at the indicated multiplicities of infection ( MOI ) . Renilla luciferase activity was measured 24 hours later . Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold - reduction in viral activity achieved with dsRNA - RL vs . dsRNA - GFP treatment . ( B ) As in ( a ) but SFV - Rluc virus titre was measured by plaque assay and expressed as plaque forming units ( PFU ) per ml of supernatant at 24 h post - infection ( MOI = 0 . 1 ) . ( C ) As in ( a ) but with Ifnar1 - / - or Mavs - / - MEFs and a single dose of SFV - Rluc ( MOI = 0 . 1 ) . ( D ) As in ( a ) but with two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ) individually complemented to express wild - type ( HA - mAgo2 WT ) or a catalytic mutant ( HA - mAgo2 D597A ) version of mAgo2 or transduced with a control vector ( as described in Fig 5 ) . SFV - Rluc was used at an MOI = 0 . 1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment.(B) As in (a) but SFV-Rluc virus titre was measured by plaque assay and expressed as plaque forming units (PFU) per ml of supernatant at 24 h post-infection (MOI=0.1).(C) As in (a) but with Ifnar1-/- or Mavs-/- MEFs and a single dose of SFV-Rluc (MOI=0.1).(D) As in (a) but with two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3) individually complemented to express wild-type (HA-mAgo2 WT) or a catalytic mutant (HA-mAgo2 D597A) version of mAgo2 or transduced with a control vector (as described in Fig 5). SFV-Rluc was used at an MOI=0.1.


Figure 1A Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole ‐ cell lysates of MDA ‐ MB ‐ 231 cells followed by immunoblotting to examine the co ‐ immunoprecipitatedPIPKIγi5 and LAPTM4B . B PIPKIγi5 specifically interacts with LAPTM4B , but not LAPTM4A or LAPTM5 . Top : schematic diagram of all three LAPTM members . Bottom : each Flag ‐ tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc ‐ tagged PIPKIγi5 and empty vector or Flag ‐ tagged LAPTM , and the co ‐ immunoprecipitatedPIPKIγi5 was examined by immunoblotting . C LAPTM4B selectively associates with PIPKIγi5 . Top : schematic diagram of four PIPKI isoforms . Bottom : Myc ‐ tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag ‐ tagged LAPTM4B and each isoform of PIPKI , followed by immunoblotting to examine the co ‐ immunoprecipitatedLAPTM4B . D Kinase activity of PIPKIγi5 is required for LAPTM4B association . HEK293 cells expressing LAPTM4B and wild ‐ type ( WT ) or kinase ‐ dead ( KD ) PIPKIγi5 were starved overnight , stimulated or not with 100 ng / ml EGF for 15 min , and harvested for immunoprecipitation with anti ‐ myc . The co ‐ immunoprecipitatedLAPTM4B was detected by immunoblotting . E Endogenous LAPTM4B is targeted to both early and late endosomes . MDA ‐ MB ‐ 231 cells were fixed and costained for endogenous LAPTM4B ( red ) and EEA1 or LAMP1 ( green ) . Boxes are selected regions for magnified view . Note : non ‐ specific nuclear staining by the LAPTM4B anti ‐ sera . Scale bar : 10 μm . F Quantification of LAPTM4B colocalization with EEA1 and LAMP1 ( mean + SD ; n ≥ 4 ) . H LAPTM4B partially colocalizes with PIPKIγi5 . MDA ‐ MB ‐ 231 cells expressing Myc ‐ tagged PIPKIγi5 were stained with LAPTM4B anti ‐ sera ( red ) , anti ‐ Myc ( green ) , and DAPI . Box is selected region for magnified view . Scale bar : 10 μm . I MDA ‐ MB ‐ 231 cells stably expressing Flag ‐ LAPTM4B were stained with LAPTM4B anti ‐ sera followed by silver ‐ enhanced immuno ‐ electron microscopy . The early and late MVEs were defined by the number of intraluminal vesicles . N , nucleus ; M , mitochondria ; MVE , multi ‐ vesicular endosome ; and PM , plasma membrane . Scale bars : 2 μm ( left ) ; 200 nm ( middle and right ) .

O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole‐cell lysates of MDA‐MB‐231 cells followed by immunoblotting to examine the co‐immunoprecipitatedPIPKIγi5 and LAPTM4B.B PIPKIγi5 specifically interacts with LAPTM4B, but not LAPTM4A or LAPTM5. Top: schematic diagram of all three LAPTM members. Bottom: each Flag‐tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc‐tagged PIPKIγi5 and empty vector or Flag‐tagged LAPTM, and the co‐immunoprecipitatedPIPKIγi5 was examined by immunoblotting.C LAPTM4B selectively associates with PIPKIγi5. Top: schematic diagram of four PIPKI isoforms. Bottom: Myc‐tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag‐tagged LAPTM4B and each isoform of PIPKI, followed by immunoblotting to examine the co‐immunoprecipitatedLAPTM4B.D Kinase activity of PIPKIγi5 is required for LAPTM4B association. HEK293 cells expressing LAPTM4B and wild‐type (WT) or kinase‐dead (KD) PIPKIγi5 were starved overnight, stimulated or not with 100 ng/ml EGF for 15 min, and harvested for immunoprecipitation with anti‐myc. The co‐immunoprecipitatedLAPTM4B was detected by immunoblotting.E Endogenous LAPTM4B is targeted to both early and late endosomes. MDA‐MB‐231 cells were fixed and costained for endogenous LAPTM4B (red) and EEA1 or LAMP1 (green). Boxes are selected regions for magnified view. Note: non‐specific nuclear staining by the LAPTM4B anti‐sera. Scale bar: 10 μm.F Quantification of LAPTM4B colocalization with EEA1 and LAMP1 (mean + SD; n ≥ 4).H LAPTM4B partially colocalizes with PIPKIγi5. MDA‐MB‐231 cells expressing Myc‐tagged PIPKIγi5 were stained with LAPTM4B anti‐sera (red), anti‐Myc (green), and DAPI. Box is selected region for magnified view. Scale bar: 10 μm.I MDA‐MB‐231 cells stably expressing Flag‐LAPTM4B were stained with LAPTM4B anti‐sera followed by silver‐enhanced immuno‐electron microscopy. The early and late MVEs were defined by the number of intraluminal vesicles. N, nucleus; M, mitochondria; MVE, multi‐vesicular endosome; and PM, plasma membrane. Scale bars: 2 μm (left); 200 nm (middle and right).


Figure 2A MDA ‐ MB ‐ 231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng / ml EGF for indicated time periods , followed by whole ‐ cell lysate harvest for immunoblotting analysis of EGFR levels . B - D Quantification of the levels of EGFR ( B ) , pEGFR ( Y1068 ) ( C ) , and pAKT ( S473 ) ( D ) from the analysis in ( A ) ( mean ± SD ; n = 3 ) . E Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 cells were starved , pulsed with 25 ng / ml Alexa ‐ 555 ‐ EGF for 3 min , washed , and chased for indicated time periods followed by fixation , DAPI staining , and fluorescence microscopy . Scale bar : 10 μm . F Quantification of the relative amounts of Alexa ‐ 555 ‐ EGF internalized in the indicated conditions ( mean + SD , n = 3 ) . G Quantification of the Alexa ‐ 555 ‐ EGF degradation in control and LAPTM4B knockdown cells in ( E ) ( mean ± SD , n = 3 ) . H Control or LAPTM4B ‐ overexpressing MDA ‐ MB ‐ 231 cells were starved and then stimulated with 100 ng / ml EGF for 1 - 4 h . EGFR degradation and signaling were analyzed by Western blot . Specific antibodies recognizing pEGFR ( Y1068 ) and pAKT ( S473 ) were used . I - K Quantification for the levels of EGFR ( I ) and pEGFR ( J ) normalized to actin and pAKT ( K ) normalized to AKT in control or LAPTM4B ‐ overexpressing cells ( mean ± SD , n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A MDA‐MB‐231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng/ml EGF for indicated time periods, followed by whole‐cell lysate harvest for immunoblotting analysis of EGFR levels.B-D Quantification of the levels of EGFR (B), pEGFR (Y1068)(C), and pAKT (S473)(D) from the analysis in (A) (mean ± SD; n = 3).E Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pulsed with 25 ng/ml Alexa‐555‐EGF for 3 min, washed, and chased for indicated time periods followed by fixation, DAPI staining, and fluorescence microscopy. Scale bar: 10 μm.F Quantification of the relative amounts of Alexa‐555‐EGF internalized in the indicated conditions (mean + SD, n = 3).G Quantification of the Alexa‐555‐EGF degradation in control and LAPTM4B knockdown cells in (E) (mean ± SD, n = 3).H Control or LAPTM4B‐overexpressing MDA‐MB‐231 cells were starved and then stimulated with 100 ng/ml EGF for 1-4 h. EGFR degradation and signaling were analyzed by Western blot. Specific antibodies recognizing pEGFR (Y1068) and pAKT (S473) were used.I-K Quantification for the levels of EGFR (I) and pEGFR (J) normalized to actin and pAKT (K) normalized to AKT in control or LAPTM4B‐overexpressing cells (mean ± SD, n = 3).


Figure 3A - D Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 cells were starved , pretreated or not with chloroquine for 2 h , stimulated with 100 ng / ml EGF for 15 min , washed , and chased for indicated time periods before fixation for costaining of EGFR ( red ) with EEA1 ( A , green ) or LAMP1 ( C , green ) . Quantification of the average percentages of EGFR signals colocalized with EEA1 ( B ) and LAMP1 ( D ) at indicated time points ; mean + SD ; n = 3 ; * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , one ‐ tailed t ‐ test . E MDA ‐ MB ‐ 231 cells were starved , stimulated with 100 ng / ml EGF for 15 or 120 min , fixed , and costained for EGFR and LAPTM4B , followed by fluorescence microscopy . Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads , respectively . F The amounts of total EGFR staining in individual cells in ( E ) were plotted against LAPTM4B levels at 15 min and 120 min , respectively . Note : for LAPTM4B quantification , the non ‐ specific nuclear staining was not included . Trend lines and Pearson ' s correlation coefficients are shown . AU , arbitrary unit .

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Figure 3A-D Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green). Quantification of the average percentages of EGFR signals colocalized with EEA1 (B) and LAMP1 (D) at indicated time points; mean + SD; n = 3; *P 0.05, **P 0.01, ***P 0.001, one‐tailed t‐test.E MDA‐MB‐231 cells were starved, stimulated with 100 ng/ml EGF for 15 or 120 min, fixed, and costained for EGFR and LAPTM4B, followed by fluorescence microscopy. Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads, respectively.F The amounts of total EGFR staining in individual cells in (E) were plotted against LAPTM4B levels at 15 min and 120 min, respectively. Note: for LAPTM4B quantification, the non‐specific nuclear staining was not included. Trend lines and Pearson's correlation coefficients are shown. AU, arbitrary unit.


Figure 4MDA ‐ MB ‐ 231 cells expressing GFP ‐ Rab5Q79L were starved and stimulated with 100 ng / ml Alexa ‐ 555 ‐ EGF for 90 min followed by fixation and immunostaining for LAPTM4B ( blue ) . The percentages of luminal Alexa ‐ 555 ‐ EGF in individual endosomes were plotted against endosomal LAPTM4B levels . Trend lines and Pearson ' s correlation coefficients are shown . Data are representative for three independent experiments . C Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were starved overnight , cell surface EGFR was labeled with immuno ‐ gold on ice . Cells were then stimulated with EGF for 1 h at 37 ° C and fixed for the EM study . Scale bar , 200 nm . See Materials and Methods for details . D Relative amounts of immuno ‐ gold ‐ labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified . Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification ( mean + SD ; * * * P 0 . 001 , one ‐ tailed t ‐ test ) . MDA ‐ MB ‐ 231 cells were transfected with GFP ‐ Rab5Q79L and Flag ‐ LAPTM4B , starved , and stimulated with Alexa ‐ 555 ‐ EGF for 90 min and followed by intraluminal sorting analysis . See Materials and Methods for details . Quantification of EGF localization in LAPTM4B ‐ positive and LAPTM4B ‐ negative endosomes in ( E ) ( mean + SD ; n = 3 ; * * * P 0 . 0007 , one ‐ tailed t ‐ test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4MDA‐MB‐231 cells expressing GFP‐Rab5Q79L were starved and stimulated with 100 ng/ml Alexa‐555‐EGF for 90 min followed by fixation and immunostaining for LAPTM4B (blue). The percentages of luminal Alexa‐555‐EGF in individual endosomes were plotted against endosomal LAPTM4B levels. Trend lines and Pearson's correlation coefficients are shown. Data are representative for three independent experiments.C Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved overnight, cell surface EGFR was labeled with immuno‐gold on ice. Cells were then stimulated with EGF for 1 h at 37°C and fixed for the EM study. Scale bar, 200 nm. See Materials and Methods for details.D Relative amounts of immuno‐gold‐labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified. Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification (mean + SD; ***P 0.001, one‐tailed t‐test).MDA‐MB‐231 cells were transfected with GFP‐Rab5Q79L and Flag‐LAPTM4B, starved, and stimulated with Alexa‐555‐EGF for 90 min and followed by intraluminal sorting analysis. See Materials and Methods for details. Quantification of EGF localization in LAPTM4B‐positive and LAPTM4B‐negative endosomes in (E) (mean + SD; n = 3; ***P 0.0007, one‐tailed t‐test).


Figure 5A Co ‐ immunoprecipitation ( co ‐ IP ) of full ‐ length or N ‐ terminus ‐ deleted LAPTM4B with PIPKIγi5 in HEK293 cells . B GST ‐ tagged LAPTM4B N ‐ terminus ( 93 amino acids ) and T7 ‐ tagged PIPKIγi5 C ‐ terminus ( 223 amino acids ) were purified from E . coli for in vitro GSTpull ‐ down assay . C Co ‐ IP of full ‐ length or N ‐ terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells . ∆ 25 : amino acids 1 - 25 deleted . E Coomassie Brilliant Blue staining of purified wild ‐ type and mutated LAPTM4B N ‐ termini . F Purified LAPTM4B N ‐ termini from ( E ) were used in PIP strips assay . LPA , lysophosphatidic acid ; LPC , lysophosphocholine ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine 1 ‐ phosphate ; PI3 , 4 , 5P3 , PtdIns ( 3 , 4 , 5 ) P3 , phosphatidylinositol ( 3 , 4 , 5 ) ‐ trisphosphate ; PA , phosphatidic acid ; PS , phosphatidylserine . GST pull ‐ down assay of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ termini with PIPKIγi5 C ‐ terminus . H Co ‐ IP of wild ‐ type or PMB ‐ mutated LAPTM4B with wild ‐ type or kinase dead PIPKIγi5 in HEK293 cells . I Top : GSTpull ‐ down assay of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with increased concentration of PtdIns ( 4 , 5 ) P2 addition . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean ± SD ; n = 4 ) . J LAPTM4B N ‐ terminus binds multiple phosphoinositides including PtdIns ( 4 , 5 ) P2 in liposome ‐ binding assay . K Top : GSTpull ‐ down assay of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of different phosphoinositides . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) . L Top : GSTpull ‐ down assay of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of PtdIns ( 4 , 5 ) P2 . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Co‐immunoprecipitation (co‐IP) of full‐length or N‐terminus‐deleted LAPTM4B with PIPKIγi5 in HEK293 cells.B GST‐tagged LAPTM4B N‐terminus (93 amino acids) and T7‐tagged PIPKIγi5 C‐terminus (223 amino acids) were purified from E. coli for in vitro GSTpull‐down assay.C Co‐IP of full‐length or N‐terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells. ∆25: amino acids 1-25 deleted.E Coomassie Brilliant Blue staining of purified wild‐type and mutated LAPTM4B N‐termini.F Purified LAPTM4B N‐termini from (E) were used in PIP strips assay. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1‐phosphate; PI3,4,5P3, PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)‐trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.GST pull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐termini with PIPKIγi5 C‐terminus.H Co‐IP of wild‐type or PMB‐mutated LAPTM4B with wild‐type or kinase dead PIPKIγi5 in HEK293 cells.I Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with increased concentration of PtdIns(4,5)P2 addition. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean ± SD; n = 4).J LAPTM4B N‐terminus binds multiple phosphoinositides including PtdIns(4,5)P2 in liposome‐binding assay.K Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of different phosphoinositides. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).L Top: GSTpull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of PtdIns(4,5)P2. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).


Figure 6A Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were starved and stimulated with 100 ng / ml EGF for 30 min , and whole ‐ cell lysates were subject to co ‐ immunoprecipitation ( co ‐ IP ) assay . B Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were transfected with His ‐ tagged ubiquitin , starved , and stimulated with 100 ng / ml EGF for 30 min . Total ubiquitinated proteins were purified from whole ‐ cell lysates by Ni ‐ NTA agarose and analyzed by Western blot . C Control or LAPTM4B ‐ WT / 6RQ ‐ overexpressing MDA ‐ MB ‐ 231 cells were transfected with His ‐ tagged ubiquitin and myc ‐ tagged Hrs , starved , and stimulated with 100 ng / ml EGF for 30 min before whole ‐ cell lysate harvest . Total ubiquitinated proteins were purified by Ni ‐ NTA agarose and analyzed by Western blot . D Quantification of Hrs ubiquitination levels from the Western blot in ( C ) ( mean + SD ; n = 5 ) . E The co ‐ IP of Myc ‐ Hrs with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant in HEK293 cells . F The co ‐ IP between Hrs and Nedd4 with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression in HEK293 cells . G The effects of LAPTM4B ‐ 6RQ mutant overexpression on EGF ‐ stimulated EGFR degradation and signaling . Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . Cells with low levels of expression were selected as polyclonal pools for comparison . Control or overexpressing cells were starved and stimulated with 100 ng / ml EGF for indicated time periods and whole ‐ cell lysates were analyzed by Western blot . H Quantification of EGFR and pAKT levels from the Western blot in ( G ) ( mean ± SD ; n = 5 ) . I The PY motif mutant LAPTM4B ‐ 2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins . J The effects of LAPTM4B ‐ 2PA mutant overexpression on EGF ‐ stimulated EGFR degradation and signaling . Overexpression of Flag ‐ LAPTM4B ‐ WT or 2PA mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . Cells with high expression of Flag ‐ LAPTM4B were selected as polyclonal pools for comparison . Cells were starved and stimulated with 100 ng / ml EGF for the indicated time periods , and whole ‐ cell lysates were analyzed by Western blot . K Quantification of EGFR degradation from Western blot in ( J ) ( mean ± SD ; n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved and stimulated with 100 ng/ml EGF for 30 min, and whole‐cell lysates were subject to co‐immunoprecipitation (co‐IP) assay.B Control or LAPTM4B knockdown MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin, starved, and stimulated with 100 ng/ml EGF for 30 min. Total ubiquitinated proteins were purified from whole‐cell lysates by Ni‐NTA agarose and analyzed by Western blot.C Control or LAPTM4B‐WT/6RQ‐overexpressing MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin and myc‐tagged Hrs, starved, and stimulated with 100 ng/ml EGF for 30 min before whole‐cell lysate harvest. Total ubiquitinated proteins were purified by Ni‐NTA agarose and analyzed by Western blot.D Quantification of Hrs ubiquitination levels from the Western blot in (C) (mean + SD; n = 5).E The co‐IP of Myc‐Hrs with Flag‐LAPTM4B‐WT or 6RQ mutant in HEK293 cells.F The co‐IP between Hrs and Nedd4 with Flag‐LAPTM4B‐WT or 6RQ mutant overexpression in HEK293 cells.G The effects of LAPTM4B‐6RQ mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Flag‐LAPTM4B‐WT or 6RQ mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with low levels of expression were selected as polyclonal pools for comparison. Control or overexpressing cells were starved and stimulated with 100 ng/ml EGF for indicated time periods and whole‐cell lysates were analyzed by Western blot.H Quantification of EGFR and pAKT levels from the Western blot in (G) (mean ± SD; n = 5).I The PY motif mutant LAPTM4B‐2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins.J The effects of LAPTM4B‐2PA mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Overexpression of Flag‐LAPTM4B‐WT or 2PA mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with high expression of Flag‐LAPTM4B were selected as polyclonal pools for comparison. Cells were starved and stimulated with 100 ng/ml EGF for the indicated time periods, and whole‐cell lysates were analyzed by Western blot.K Quantification of EGFR degradation from Western blot in (J) (mean ± SD; n = 3).


Figure 7A - C Co ‐ immunoprecipitation ( co ‐ IP ) of wild ‐ type ( WT ) or C ‐ terminal deletion mutants of HA ‐ PIPKIγi5 with SNX5 ( A ) or LAPTM4B ( B and C ) in HEK293 cells cotransfected with indicated proteins . Δ675 indicates a deletion from amino acid 675 to the C ‐ terminus . D PIPKIγi5 promotes SNX5 association with LAPTM4B . HEK293 cells cotransfected with indicated proteins were harvested for co ‐ IP to assay the interaction between Myc ‐ SNX5 and Flag ‐ LAPTM4B . E SNX5 inhibits Hrs association with LAPTM4B . HEK293 cells cotransfected with indicated proteins were harvested for co ‐ IP to assay the interaction between Myc ‐ Hrs and Flag ‐ LAPTM4B .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-C Co‐immunoprecipitation (co‐IP) of wild‐type (WT) or C‐terminal deletion mutants of HA‐PIPKIγi5 with SNX5 (A) or LAPTM4B (B and C) in HEK293 cells cotransfected with indicated proteins. Δ675 indicates a deletion from amino acid 675 to the C‐terminus.D PIPKIγi5 promotes SNX5 association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐SNX5 and Flag‐LAPTM4B.E SNX5 inhibits Hrs association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐Hrs and Flag‐LAPTM4B.


Figure 1SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of ste11 + ( A , D ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 ( A , B ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 for A , B ) SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of mei2 + ( B , E ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 ( A , B ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 for A , B ) ( C , F ) Cells were grown to mid - log phase either in rich medium or shifted for 8 hours to starvation medium . Zygotes and tetrads , which correspond to differentiated cells , were counted under a light microscope . Cells of the following genotypes were analyzed : WT isogenic controls , gcn5Η , tsc1Η , gcn5Η tsc1Η , rhb1 - DA4 , gcn5Η rhb1 - DA4 , gad8Η , and gcn5Η gad8Η . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 4 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . White arrowheads indicate zygotes . Scale bar , 10 μm . SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of ste11 + ( A , D ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 3 ( D , E ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 for D , E ) . SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of mei2 + ( B , E ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 3 ( D , E ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 for D , E ) . ( C , F ) Cells were grown to mid - log phase either in rich medium or shifted for 8 hours to starvation medium . Zygotes and tetrads , which correspond to differentiated cells , were counted under a light microscope . Cells of the following genotypes were analyzed : WT isogenic controls , gcn5Η , tsc1Η , gcn5Η tsc1Η , rhb1 - DA4 , gcn5Η rhb1 - DA4 , gad8Η , and gcn5Η gad8Η . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 4 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . White arrowheads indicate zygotes . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 (A,B)independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4 for A,B)SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 (A,B) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4 for A,B)(C,F) Cells were grown to mid-log phase either in rich medium or shifted for 8 hours to starvation medium. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Cells of the following genotypes were analyzed: WT isogenic controls, gcn5Η, tsc1Η, gcn5Η tsc1Η, rhb1-DA4, gcn5Η rhb1-DA4, gad8Η, and gcn5Η gad8Η. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).(C,F) Cells were grown to mid-log phase either in rich medium or shifted for 8 hours to starvation medium. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Cells of the following genotypes were analyzed: WT isogenic controls, gcn5Η, tsc1Η, gcn5Η tsc1Η, rhb1-DA4, gcn5Η rhb1-DA4, gad8Η, and gcn5Η gad8Η. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.


Figure 2SAGA is phosphorylated in response to nutrient starvation . ( A , B ) 4 - 20 % gradient SDS - polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium ( R ) or starved for 45 minutes ( S ) . SAGA was purified using endogenously TAP - tagged Ada1 . A fraction of each eluate was loaded and either stained with silver , to detect all proteins ( A ) , . A strain without any TAP tag is shown as a negative control for the purification . The graph on the left of the gel in B shows the fluorescence intensity of the phospho - specific stain , which was quantified along the left lane in blue ( rich ) and the middle lane in red ( starved ) , using ImageJ . The area of one peak , which corresponds to the bands marked with arrowheads and was coined p55 , is 1 . 8 - fold larger in SAGA purified from starved cells , as compared to rich conditions . Below is an anti - HA immuno - blot ( IB ) of each eluate , to reveal the amount of Ada1 - HA bait recovered . Shown are gels that are representative of 3 independent experiments . A . U . , arbitrary units . SAGA is phosphorylated in response to nutrient starvation . ( A , B ) 4 - 20 % gradient SDS - polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium ( R ) or starved for 45 minutes ( S ) . SAGA was purified using endogenously TAP - tagged Ada1 . A fraction of each eluate was loaded and either stained with Pro - Q ® Diamond , which stains phosphorylated proteins ( B ) . A strain without any TAP tag is shown as a negative control for the purification . The graph on the left of the gel in B shows the fluorescence intensity of the phospho - specific stain , which was quantified along the left lane in blue ( rich ) and the middle lane in red ( starved ) , using ImageJ . The area of one peak , which corresponds to the bands marked with arrowheads and was coined p55 , is 1 . 8 - fold larger in SAGA purified from starved cells , as compared to rich conditions . Below is an anti - HA immuno - blot ( IB ) of each eluate , to reveal the amount of Ada1 - HA bait recovered . Shown are gels that are representative of 3 independent experiments . A . U . , arbitrary units .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with silver, to detect all proteins (A),. A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with Pro-Q® Diamond, which stains phosphorylated proteins (B). A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.


Figure 3 ( D ) FLAG - tagged Taf12 was purified from cells grown in rich medium ( R ) or shifted for 45 minutes to starvation medium ( S ) . Anti - FLAG immuno - precipitations ( IP ) were incubated with and without λ - phosphatase or its inhibitor and immuno - blotted ( IB ) , together with 1 % of whole cell extracts ( WCE ) , using an anti - FLAG antibody . ( E ) Phospho - Taf12 ( P - Taf12 ) , total Taf12 , and tubulin levels were quantified from cells grown in rich medium ( R ) or starved for 45 minutes ( S ) . P - Taf12 levels were normalized to those of total Taf12 , while total Taf12 levels were normalized to those of tubulin . Data points were individually plotted on the graph and overlaid with the mean and SE . Signal intensities were quantified from IBs of 7 independent experiments . Statistical significance was determined using a t - test ( n = 7 , unpaired , two - tailed ) . ( F ) P - Taf12 and total Taf12 were followed in WT , taf12 - T283A and taf12 - 5A mutants , grown in rich medium ( R ) or starved for 45 minutes ( S ) . ( G , H ) SAGA and TFIID were tandem affinity - purified using endogenously TAP - tagged Spt7 ( G ) from strains containing FLAG - tagged Taf12 , grown in rich medium or starved for 45 minutes . TAP - tagged Spt7 and Taf4 were eluted using the TEV protease , releasing a shorter form of each bait ( Spt7 - CBP or Taf4 - CBP ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of either whole cell extracts ( WCE ) or IgG - sepharose flow - through ( FT ) . Shown are IBs that are representative of 2 independent experiments . ( G , H ) SAGA and TFIID were tandem affinity - purified using endogenously TAP - tagged Taf4 ( H ) , from strains containing FLAG - tagged Taf12 , grown in rich medium or starved for 45 minutes . TAP - tagged Spt7 and Taf4 were eluted using the TEV protease , releasing a shorter form of each bait ( Spt7 - CBP or Taf4 - CBP ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of either whole cell extracts ( WCE ) or IgG - sepharose flow - through ( FT ) . Shown are IBs that are representative of 2 independent experiments . ( I ) P - Taf12 , total Taf12 , and Ser546 - phosphorylated Gad8AKT were followed in WT cells , grown in rich conditions or over a time - course after a shift to starvation medium . Gad8AKT phosphorylation at Ser546 is a proxy of TORC2 activity in S . pombe . ( J ) P - Taf12 and total Taf12 were followed in WT cells , grown in rich conditions or shifted to different starvation media for 30 minutes . These include both the removal of the nitrogen source , ammonium chloride ( NH4Cl ) , and the reduction of the carbon source , glucose , from a concentration of 2 % to 0 . 5 % . Alternatively , cells were either only deprived of NH4Cl or only exposed to reduced glucose levels ( 2 % to 0 . 5 % ) . ( K ) P - Taf12 and total Taf12 were followed in WT cells , grown in rich conditions or deprived of NH4Cl for one hour . Then , NH4Cl was added back to the medium for 30 or 60 minutes . Both short and long exposures of the FLAG IBs are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . Anti - tubulin IBs are shown as controls for loading between samples . Shown are star that are representative of at least 3 independent experiments . The number sign ( # ) symbol identifies antibody heavy chains and the star ( * ) symbol labels an unspecific band detected by the anti - FLAG antibody in S . pombe .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) FLAG-tagged Taf12 was purified from cells grown in rich medium (R) or shifted for 45 minutes to starvation medium (S). Anti-FLAG immuno-precipitations (IP) were incubated with and without λ-phosphatase or its inhibitor and immuno-blotted (IB), together with 1% of whole cell extracts (WCE), using an anti-FLAG antibody.(E) Phospho-Taf12 (P-Taf12), total Taf12, and tubulin levels were quantified from cells grown in rich medium (R) or starved for 45 minutes (S). P-Taf12 levels were normalized to those of total Taf12, while total Taf12 levels were normalized to those of tubulin. Data points were individually plotted on the graph and overlaid with the mean and SE. Signal intensities were quantified from IBs of 7 independent experiments. Statistical significance was determined using a t-test (n = 7, unpaired, two-tailed).(F) P-Taf12 and total Taf12 were followed in WT, taf12-T283A and taf12-5A mutants, grown in rich medium (R) or starved for 45 minutes (S).(G,H) SAGA and TFIID were tandem affinity-purified using endogenously TAP-tagged Spt7 (G) from strains containing FLAG-tagged Taf12, grown in rich medium or starved for 45 minutes. TAP-tagged Spt7 and Taf4 were eluted using the TEV protease, releasing a shorter form of each bait (Spt7-CBP or Taf4-CBP). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of either whole cell extracts (WCE) or IgG-sepharose flow-through (FT). Shown are IBs that are representative of 2 independent experiments.(G,H) SAGA and TFIID were tandem affinity-purified using endogenously TAP-tagged Taf4 (H), from strains containing FLAG-tagged Taf12, grown in rich medium or starved for 45 minutes. TAP-tagged Spt7 and Taf4 were eluted using the TEV protease, releasing a shorter form of each bait (Spt7-CBP or Taf4-CBP). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of either whole cell extracts (WCE) or IgG-sepharose flow-through (FT). Shown are IBs that are representative of 2 independent experiments.(I) P-Taf12, total Taf12, and Ser546-phosphorylated Gad8AKT were followed in WT cells, grown in rich conditions or over a time-course after a shift to starvation medium. Gad8AKT phosphorylation at Ser546 is a proxy of TORC2 activity in S. pombe.(J) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or shifted to different starvation media for 30 minutes. These include both the removal of the nitrogen source, ammonium chloride (NH4Cl), and the reduction of the carbon source, glucose, from a concentration of 2% to 0.5%. Alternatively, cells were either only deprived of NH4Cl or only exposed to reduced glucose levels (2% to 0.5%).(K) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or deprived of NH4Cl for one hour. Then, NH4Cl was added back to the medium for 30 or 60 minutes. Both short and long exposures of the FLAG IBs are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. Anti-tubulin IBs are shown as controls for loading between samples. Shown are star that are representative of at least 3 independent experiments. The number sign (#) symbol identifies antibody heavy chains and the star (*) symbol labels an unspecific band detected by the anti-FLAG antibody in S. pombe.


Figure 4 ( A , B ) Expression of ste11 + ( A ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , ppa2Η , par1Η , pab1Η , tsc1Η , and pab1Η tsc1Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) . ( A , B ) Expression of mei2 + ( B ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , ppa2Η , par1Η , pab1Η , tsc1Η , and pab1Η tsc1Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) . ( C ) Cells were grown to mid - log phase either in rich medium or starved for 8 hours . Zygotes and tetrads , which correspond to differentiated cells , were counted under a light microscope . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 4 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . White arrowheads indicate zygotes . Scale bar , 10 μm . ( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and pab1Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - Rpb1 IB is shown as a control for loading . The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Ratios of P - Taf12 to total Taf12 were calculated from 6 independent experiments and individually plotted in a graph below the IBs , together with the mean and SE . Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and strains . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ; * p < 0 . 01 ; # p < 0 . 05 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . ( E ) FLAG - tagged Taf12 and TAP - tagged Pab1 were affinity purified separately from cells grown in rich conditions ( upper panels ) . TAP - tagged Pab1 was cleaved off from beads using the Tobacco Etch Virus ( TEV ) protease , releasing a shorter form of Pab1 ( CBP - Pab1 ) in the eluate ( E ) . CBP - Pab1 was then mixed with beads containing FLAG - Taf12 IPs and incubated in a phosphatase buffer , with and without 0 . 5 µM microcystin . Each reaction was then analyzed by IB and simultaneously probed with anti - FLAG or anti - CBP antibodies ( lower panel ) . ( F ) Exponentially growing cells were treated for 1 hour with Torin1 , which was added at increasing concentrations , 7 and 21 µM , to rich medium . Dimethylsulphoxide ( DMSO ) was used as the vehicle and added as a negative control . ( G ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and tor2 - ts10 strains , grown in rich conditions at 25 ° C or shifted to 30 ° C for 4 and 6 hours . ( F , G ) Anti - tubulin IBs are shown as a control for loading between samples . Shown are IBs that are representative of 3 independent experiments . The star ( * ) symbol labels unspecific bands detected by the anti - FLAG or anti - CBP antibodies in S . pombe .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A,B) Expression of ste11+ (A) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), ppa2Η, par1Η, pab1Η, tsc1Η, and pab1Η tsc1Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).(A,B) Expression of mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), ppa2Η, par1Η, pab1Η, tsc1Η, and pab1Η tsc1Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).(C) Cells were grown to mid-log phase either in rich medium or starved for 8 hours. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and pab1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Ratios of P-Taf12 to total Taf12 were calculated from 6 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6; *p < 0.01; #p < 0.05). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal.(E) FLAG-tagged Taf12 and TAP-tagged Pab1 were affinity purified separately from cells grown in rich conditions (upper panels). TAP-tagged Pab1 was cleaved off from beads using the Tobacco Etch Virus (TEV) protease, releasing a shorter form of Pab1 (CBP-Pab1) in the eluate (E). CBP-Pab1 was then mixed with beads containing FLAG-Taf12 IPs and incubated in a phosphatase buffer, with and without 0.5 µM microcystin. Each reaction was then analyzed by IB and simultaneously probed with anti-FLAG or anti-CBP antibodies (lower panel).(F) Exponentially growing cells were treated for 1 hour with Torin1, which was added at increasing concentrations, 7 and 21 µM, to rich medium. Dimethylsulphoxide (DMSO) was used as the vehicle and added as a negative control.(G) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor2-ts10 strains, grown in rich conditions at 25°C or shifted to 30°C for 4 and 6 hours. (F,G) Anti-tubulin IBs are shown as a control for loading between samples. Shown are IBs that are representative of 3 independent experiments. The star (*) symbol labels unspecific bands detected by the anti-FLAG or anti-CBP antibodies in S. pombe.


Figure 5 ( A ) Igo1 phosphorylation ( P - Igo1 ) was followed by anti - MYC IB of protein extracts from WT , ppk18Η , and igo1 - S64A strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . Igo1 migration was analyzed by electrophoresis of both a 12 % SDS - polyacrylamide gel containing the Phos - tag ™ molecule ( right panel ) and a standard 10 % SDS - polyacrylamide gel ( left panel ) . Arrowheads indicate the various phosphorylated isoforms of Igo1 - MYC in each strain and condition . Shown is an IB representative of 2 independent experiments . ( B , C ) Expression of ste11 + ( C ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , pab1Η , gcn5Η ppk18Η , pab1Η , pab1Η ppk18Η , igo1Η , igo1 - S64A , gcn5Η igo1 - S64A and pab1Η igo1 - S64A . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) . ( B , C ) Expression of mei2 + ( D ) using igo1 of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , ppk18Η , gcn5Η ppk18Η , pab1Η , pab1Η ppk18Η , igo1Η , igo1 - S64A , gcn5Η igo1 - S64A and pab1Η igo1 - S64A . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) . ( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT , S . pombeΗ , and igo1Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - MYC IB is shown as a control for loading . ( D , E ) The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Shown below each blot are average measurements of 3 independent experiments ( n = 3 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . The star ( * ) symbol labels an unspecific band detected by the FLAG antibody in S . pombe . ( E ) P - Taf12 was followed in WT , igoS64A , pab1Η , and pab1Η igoS64A strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - Rpb1 IB is shown as a control for loading . ( D , E ) The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Shown below each blot are average measurements of 3 independent experiments ( n = 3 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . The star ( * ) symbol labels an unspecific band detected by the S . pombe antibody in S . pombe .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Igo1 phosphorylation (P-Igo1) was followed by anti-MYC IB of protein extracts from WT, ppk18Η, and igo1-S64A strains, grown in rich conditions (R) or starved for 45 minutes (S). Igo1 migration was analyzed by electrophoresis of both a 12% SDS-polyacrylamide gel containing the Phos-tag™ molecule (right panel) and a standard 10% SDS-polyacrylamide gel (left panel). Arrowheads indicate the various phosphorylated isoforms of Igo1-MYC in each strain and condition. Shown is an IB representative of 2 independent experiments.(B,C) Expression of ste11+ (C) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, pab1Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).(B,C) Expression of mei2+ (D) using igo1 of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, ppk18Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, S. pombeΗ, and igo1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-MYC IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the FLAG antibody in S. pombe.(E) P-Taf12 was followed in WT, igoS64A, pab1Η, and pab1Η igoS64A strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the S. pombe antibody in S. pombe.


Figure 6 ( A ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT , tor1Η , and gad8Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - tubulin IB is shown as a control for loading . The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . P - Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs , together with the mean and SE . Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 ) . ( B ) P - Taf12 was followed by anti - FLAG IB of protein extracts from sty1Η cells ( left panel ) or ssp2Η cells ( right panel ) , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - tubulin IB is shown as a control for loading . The star ( * ) symbols label an unspecific band detected by the anti - FLAG antibody in S . pombe . Shown are IBs that are representative of 2 independent experiments . ( C ) TAP - tagged Gad8 was immuno - precipitated using IgG - sepharose beads ( IP ) from strains containing FLAG - tagged Taf12 , grown either in rich medium ( left panels ) or starved for 45 minutes ( right panels ) . TAP - tagged Gad8 was eluted from beads using the TEV protease , releasing a shorter form of Gad8 ( CBP - Gad8 ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of whole cell extracts ( WCE ) , to detect Taf12 co - precipitation with Gad8 . A strain which does not contain any TAP tag is shown as a negative control for the IP . Shown is an IB representative of two independent experiments . ( A , C ) The star ( * ) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S . pombe . ( D ) Endogenous Gad8 - HA was affinity purified from starved cells , in order to activate its kinase activity ( left and middle panels ) , and mixed with recombinant , purified GST - Taf12 , GST - Taf12 - 5A , or GST - Fkh2 proteins ( right panels ) in a kinase assay buffer . Each assay was then analyzed by IB and probed with an anti - phospho - AKT substrate antibody .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, tor1Η, and gad8Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. P-Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3).(B) P-Taf12 was followed by anti-FLAG IB of protein extracts from sty1Η cells (left panel) or ssp2Η cells (right panel), grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The star (*) symbols label an unspecific band detected by the anti-FLAG antibody in S. pombe. Shown are IBs that are representative of 2 independent experiments.(C) TAP-tagged Gad8 was immuno-precipitated using IgG-sepharose beads (IP) from strains containing FLAG-tagged Taf12, grown either in rich medium (left panels) or starved for 45 minutes (right panels). TAP-tagged Gad8 was eluted from beads using the TEV protease, releasing a shorter form of Gad8 (CBP-Gad8). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of whole cell extracts (WCE), to detect Taf12 co-precipitation with Gad8. A strain which does not contain any TAP tag is shown as a negative control for the IP. Shown is an IB representative of two independent experiments. (A,C) The star (*) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S. pombe.(D) Endogenous Gad8-HA was affinity purified from starved cells, in order to activate its kinase activity (left and middle panels), and mixed with recombinant, purified GST-Taf12, GST-Taf12-5A, or GST-Fkh2 proteins (right panels) in a kinase assay buffer. Each assay was then analyzed by IB and probed with an anti-phospho-AKT substrate antibody.


Figure 7 ( A , B ) Expression of quantitative RT - PCR + ( A ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) . mei2 + ( B ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) . ( C , E ) Zygotes and tetrads , which correspond to differentiated cells , were counted from cultures of homothallic cells grown to mid - log phase in rich medium ( t0 ) and shifted to starvation medium for up to 24 hours ( C ) Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) . ( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and tor1 - 1972A strains , grown in rich medium and shifted to starved conditions for 60 minutes . An anti - Tubulin IB is shown as a control for loading . Shown is an IB which is representative of 2 independent experiments . ( C , E ) taf12 and tetrads , which correspond to differentiated cells , were counted from heterothallic cells mixed and incubated for 2 days on mating medium ( E ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A,B) Expression of quantitative RT-PCR+ (A) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).(C,E) Zygotes and tetrads, which correspond to differentiated cells, were counted from cultures of homothallic cells grown to mid-log phase in rich medium (t0) and shifted to starvation medium for up to 24 hours (C) Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor1-1972A strains, grown in rich medium and shifted to starved conditions for 60 minutes. An anti-Tubulin IB is shown as a control for loading. Shown is an IB which is representative of 2 independent experiments.(C,E) taf12 and tetrads, which correspond to differentiated cells, were counted from heterothallic cells mixed and incubated for 2 days on mating medium (E). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).