Figure 2A ) Evaluation of RB mRNA levels , normalised by GAPDH , in an MDM2 dose - dependent manner . The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress . Endogenous levels ( upper panel ) ; exogenous levels ( middle panel ) ; endogenous levels of RB but using the MDM2 ( S395D ) under normal conditions ( lower panel ) . B ) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h . The cells were treated with cichlohexamide ( CHX ) was added to stop translation , under normal conditions ( upper panel ) and treated with doxorubicin ( lower panel ) . The cells were transfected with MDM2 . C ) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence ( upper panel ) or in absence ( lower panel ) of CHX . D ) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX .

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Figure 2A) Evaluation of RB mRNA levels, normalised by GAPDH, in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Endogenous levels (upper panel); exogenous levels (middle panel); endogenous levels of RB but using the MDM2(S395D) under normal conditions (lower panel).B) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h. The cells were treated with cichlohexamide (CHX) was added to stop translation, under normal conditions (upper panel) and treated with doxorubicin (lower panel). The cells were transfected with MDM2.C) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence (upper panel) or in absence (lower panel) of CHX.D) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX.


Figure 3Different constructs of RB were transfected in H1299 - RBKO treated with doxorubicin 16 h to induce genotoxic stress ; the RB constructs levels of expression were evaluated using HA antibody . The bar diagrams show the quantification of the western blots data . A ) Evaluation of the small pocket expression in an MDM2 dose - dependent manner . B ) Cells were transfected with the RB large pocket construct . Different constructs of RB were transfected in H1299 - RBKO treated with doxorubicin 16 h to induce genotoxic stress ; the RB constructs levels of expression were evaluated using HA antibody . The bar diagrams show the quantification of the western blots data . C ) Cells were transfected with RB full length without the 5 ´ URT ( named RB - HA ) . D ) Cells were transfected with the RB full - length plus 260 nt of its own 5 ´ UTR ( named RB + 5 ´ UTR ) . E ) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP . Cells were treated with doxorubicin ( 1 µM for 16 h ) or DMSO . F ) RT - qPCR of RB and GAPDH of the MDM2 - immunoprecipitation from the polysomal fractionation ( see EV2D ) . Data are representative of three similar independent experiments . G ) PLA assay using anti - RB and anti - MDM2 antibodies , H1299 cell were treated or not with etoposide to induce stress , the endogenous proteins were visualised . Cell nuclei were visualised with DAPI blue . The red dots show the interaction between the two proteins . The scale bar corresponds to 20 μm . H ) RNA - PLA assay using rabbit anti - MDM2 and a mouse biotin - antibody . H1299 - RBKO was stable transfected with RB - HA or RB + 5 ´ UTR . Cell nuclei were visualised with DAPI blue . The red dots show the interaction between the protein and the mRNA . The scale bar corresponds to 20 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 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 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 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 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

Figure 3Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. A) Evaluation of the small pocket expression in an MDM2 dose-dependent manner. B) Cells were transfected with the RB large pocket construct. Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. C) Cells were transfected with RB full length without the 5´URT (named RB-HA). D) Cells were transfected with the RB full-length plus 260 nt of its own 5´ UTR (named RB+5´UTR). E) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP. Cells were treated with doxorubicin (1 µM for 16 h) or DMSO.F) RT-qPCR of RB and GAPDH of the MDM2-immunoprecipitation from the polysomal fractionation (see EV2D). Data are representative of three similar independent experiments.G) PLA assay using anti-RB and anti-MDM2 antibodies, H1299 cell were treated or not with etoposide to induce stress, the endogenous proteins were visualised. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the two proteins. The scale bar corresponds to 20 μm.H) RNA-PLA assay using rabbit anti-MDM2 and a mouse biotin-antibody. H1299- RBKO was stable transfected with RB-HA or RB+5´UTR. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the protein and the mRNA. The scale bar corresponds to 20 μm.


Figure 4A ) Cell cycle analysis ( FACS ) of C33 cell line after treatment with etoposide . The DNA contents of the cells were evaluated by propidium iodide staining , ( PE - A ; Phycoerythrin ) . The inset shows the % of the cells in each cell cycle phase . B ) The cell cycle analysis of synchronised C33 cell line during the time . C ) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide , ( EV ; empty vector ) . D ) The same as in ( C ) but under normal cellular conditions . E ) RB levels of the cells after 14 h of the release of nocodazole treatment . Etoposide was used to induce genotoxic stress . F ) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment . Data information : Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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 B-PANEL_START 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 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

Figure 4A) Cell cycle analysis (FACS) of C33 cell line after treatment with etoposide. The DNA contents of the cells were evaluated by propidium iodide staining, (PE-A; Phycoerythrin). The inset shows the % of the cells in each cell cycle phase.B) The cell cycle analysis of synchronised C33 cell line during the time.C) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide, (EV; empty vector). D) The same as in (C) but under normal cellular conditions.E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress.F) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment. Data information: Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).


Figure 5A ) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2 / M cell cycle phases under stress conditions . B ) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2 ( S395D ) . C ) In vitro RB ubiquitination by MDM2 ( S395D ) in presence or absence of RB mRNA . D ) A representative clonogenic assay . Increasing concentrations of MDM2 were transfected in H1299 - RBKO stable transfected with RB - HA or RB + 5 ´ UTR . The cells were treated with doxorubicin to induce genotoxic stress conditions . Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 ) . E ) Evaluation of cyclin E mRNA levels in an MDM2 dose - dependent manner . The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress . Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 , * * * p < 0 . 001 ) .

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 B-PANEL_START 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 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 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

Figure 5A) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2/M cell cycle phases under stress conditions.B) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2(S395D).C) In vitro RB ubiquitination by MDM2(S395D) in presence or absence of RB mRNA.D) A representative clonogenic assay. Increasing concentrations of MDM2 were transfected in H1299-RBKO stable transfected with RB-HA or RB+5´UTR. The cells were treated with doxorubicin to induce genotoxic stress conditions. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05).E) Evaluation of cyclin E mRNA levels in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, ***p< 0.001).


Figure 1The response of cytosolic and mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( C ) probes to boli of exogenous H2O2 at the indicated concentrations . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium . The response of cytosolic and mitochondrial matrix - localized Grx1 - roGFP2 ( D ) probes to boli of exogenous H2O2 at the indicated concentrations . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium . The response of cytosolic and mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( E ) probes to 10 µM Complex antimycin A ( a complex III inhibitor ) . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium . The response of cytosolic and mitochondrial matrix - localized Grx1 - roGFP2 ( F ) probes to 10 µM Complex antimycin A ( a complex III inhibitor ) . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium . ( G ) The response of mitochondrial matrix - localized ( left panel ) and cytosolic ( right panel ) roGFP2 - Tsa2ΔCR probes expressed in BY4742 wild - type and Δtsa1Δtsa2 cells , to the addition of exogenous H2O2 at the indicated concentrations . Cells were grown to exponential phase in SGal ( - Leu ) medium . The lighter colored curves are controls , showing the probe response upon the addition of water . ( H ) The response of mitochondrial matrix - localized ( left panel ) and cytosolic ( right panel ) roGFP2 - Tsa2ΔCR probes , expressed in wild - type and Δtsa1Δtsa2 cells , to the addition of 10 µM antimycin A . Cells were grown to exponential phase in SGal ( - Leu ) medium . The lighter colored curves are controls showing the probe responses upon addition of 0 . 1 % ( v / v ) ethanol . ( I ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δpor1 cells , to boli at exogenous H2O2 at the indicated concentrations . Cells were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing the probe response upon the addition of water . ( J ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δpor1 cells to the addition of 10 µM antimycin A . Cells were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing the probe response upon the addition of 0 . 1 % ( v / v ) ethanol .

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 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 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 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 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 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

Figure 1The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (C) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (D) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (E) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (F) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.(G) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes expressed in BY4742 wild-type and Δtsa1Δtsa2 cells, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls, showing the probe response upon the addition of water.(H) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes, expressed in wild-type and Δtsa1Δtsa2 cells, to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls showing the probe responses upon addition of 0.1% (v/v) ethanol.(I) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells, to boli at exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of water.(J) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of 0.1% (v/v) ethanol.


Figure 2 ( A ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δprx1 , to the addition of exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Lighter colored curves are controls showing the probe response to the addition of water . ( B , C ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , expressed in wild - type , Δtrx3 and Δprx1 cells , to the addition of 0 . 1 mM ( B ) or 1 mM ( C ) exogenous H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . ( D ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid , to the addition of 0 . 1 mM exogenous H2O2 . Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase . The light grey curve in both panels represents the wild - type control , treated with the same concentration of exogenous H2O2 . ( E ) The profile of mRNA expression of the yeast redox or redox - related enzymes in Δprx1 + empty vector cells , compared to Δprx1 + Prx1 - WT cells , both grown to an early exponential phase in SGal ( - Ura ) medium ( n = 3 biological replicates , with cells obtained from 3 independent cultures ) . ( F ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1 - WT plasmid to the addition of 0 . 1 mM exogenous H2O2 . Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential

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 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 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 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 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

Figure 2(A) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δprx1, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Lighter colored curves are controls showing the probe response to the addition of water.(B,C) The response of a cytosolic roGFP2-Tsa2ΔCR probe, expressed in wild-type, Δtrx3 and Δprx1 cells, to the addition of 0.1 mM (B) or 1 mM (C) exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.(D) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid, to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase. The light grey curve in both panels represents the wild-type control, treated with the same concentration of exogenous H2O2.(E) The profile of mRNA expression of the yeast redox or redox-related enzymes in Δprx1+empty vector cells, compared to Δprx1+Prx1-WT cells, both grown to an early exponential phase in SGal (-Ura) medium (n = 3 biological replicates, with cells obtained from 3 independent cultures).(F) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1-WT plasmid to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential phase


Figure 3 ( A ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtsa1Δtsa2 , Δpor1 , Δtrr2 , Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . ( B ) Left panel : The response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtrr2 , Δtrx3 or Δprx1 cells to a bolus of 2 . 5 mM H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Right panel : Scheme depicting Prx1 and its peroxidatic cysteine at position 91 . ( C ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe to exogenous 2 . 5 mM H2O2 . The probe was expressed in wild - type cells transformed with an empty vector or in ∆ prx1 cells transformed with either an empty vector , a vector encoding wild - type Prx1 or a vector encoding Prx1 - C91A , grown in SGal ( - Leu , - Ura ) . ( D ) The response to of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in either wild - type cells transformed with an empty vector or in ∆ prx1 cells transformed with either an empty vector , a Prx1 - WT or a Prx1 - C91A plasmid to exogenous H2O2 at a concentration of 2 . 5 mM . Cells were grown to SGal ( - Leu , - Ura ) medium and harvested at early exponential phase .

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 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 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

Figure 3(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtsa1Δtsa2, Δpor1, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.(B) Left panel: The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of 2.5 mM H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: Scheme depicting Prx1 and its peroxidatic cysteine at position 91.(C) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe to exogenous 2.5 mM H2O2. The probe was expressed in wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a vector encoding wild-type Prx1 or a vector encoding Prx1-C91A, grown in SGal (-Leu, -Ura). (D) The response to of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in either wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a Prx1-WT or a Prx1-C91A plasmid to exogenous H2O2 at a concentration of 2.5 mM. Cells were grown to SGal (-Leu, -Ura) medium and harvested at early exponential phase.


Figure 4 ( B ) ' Acute stress - washout ' assay . Δprx1 cells containing a Prx1 - WT plasmid and expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested and treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to the addition of 1 mM H2O2 was measured . The middle panel is an enlargement of the left panel . The graph in the right panel shows the steady state Grx1 - roGFP2 oxidation following H2O2 pre - treatment as well as the maximum Grx1 - roGFP2 oxidation in response to the subsequent second H2O2 treatment . ' Acute stress - washout ' assay . Δprx1 cells expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested and treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . ( C ) The same experiment as in ( B ) performed with Δprx1 cells transformed with a Prx1 - C91A plasmid . ( D ) ∆ prx1 cells transformed with a vector encoding wild - type Prx1 , a vector encoding the matrix - targeted D - aminooxidase ( su9 - DAO ) and expressing a mitochondrial matrix - localized Grx1 - roGFP2 were grown to exponential phase in SGal ( - Leu , - Ura , - His ) and then pre - treated with 0 . 15 M D - alanine for 0 , 0 . 5 , 1 . 5 hours . Afterwards , the cells were washed and the response of Grx1 - roGFP2 to the addition of 1 mM H2O2 was measured . ( E ) Left panel : the response of mitochondrial matrix - localized roGFP2 - Prx1 and roGFP2 probes expressed in wild - type cells to exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Right panel : model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2 - Prx1 sensor leads to a roGFP2 - like behavior . ( H ) Redox shift assays of Prx1 . Δprx1 cells transformed with a plasmid encoding wild - type Prx1 were grown to early exponential phase in SGal ( - Ura ) medium . Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins . Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP . ( J ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 , expressed in wild - type cells grown to the addition of exogenous 1 mM H2O2 . Cells were grown in SGal ( - Leu ) and harvested at early exponential phase . Left panel , the pre - treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH . Right panel , newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix .

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 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 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 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 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

Figure 4(B) 'Acute stress-washout' assay. Δprx1 cells containing a Prx1-WT plasmid and expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix-localized Grx1-roGFP2 probe to the addition of 1 mM H2O2 was measured. The middle panel is an enlargement of the left panel. The graph in the right panel shows the steady state Grx1-roGFP2 oxidation following H2O2 pre-treatment as well as the maximum Grx1-roGFP2 oxidation in response to the subsequent second H2O2 treatment.'Acute stress-washout' assay. Δprx1 cells expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. (C) The same experiment as in (B) performed with Δprx1 cells transformed with a Prx1-C91A plasmid.(D) ∆prx1 cells transformed with a vector encoding wild-type Prx1, a vector encoding the matrix-targeted D-aminooxidase (su9-DAO) and expressing a mitochondrial matrix-localized Grx1-roGFP2 were grown to exponential phase in SGal (-Leu, -Ura, -His) and then pre-treated with 0.15 M D-alanine for 0, 0.5, 1.5 hours. Afterwards, the cells were washed and the response of Grx1-roGFP2 to the addition of 1 mM H2O2 was measured.(E) Left panel: the response of mitochondrial matrix-localized roGFP2-Prx1 and roGFP2 probes expressed in wild-type cells to exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2-Prx1 sensor leads to a roGFP2-like behavior.(H) Redox shift assays of Prx1. Δprx1 cells transformed with a plasmid encoding wild-type Prx1 were grown to early exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins. Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP.(J) The response of a mitochondrial matrix-localized Grx1-roGFP2, expressed in wild-type cells grown to the addition of exogenous 1 mM H2O2. Cells were grown in SGal (-Leu) and harvested at early exponential phase. Left panel, the pre-treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH. Right panel, newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix.


Figure 5 ( B ) The response of a mitochondrial matrix - localized roGFP2 - Prx1 - P233stop probe expressed in wild - type cells to the indicated concentrations of exogenous H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) . ( C ) Maleimide - based gel - shift assay to assess Prx1 cysteine redox state . Wild - type cells transformed with an empty plasmid or ∆ prx1 cells with either an empty plasmid or a plasmid encoding wild - type Prx1 , the hyperoxidation - resistant Prx1 - P233stop or the inactive Prx1 - C91A variants , were grown to exponential phase in SGal ( - Ura ) medium . Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24 . ( D ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 probe , expressed in Δprx1 cells containing a plasmid encoding either wild - type Prx1 , Prx1 - P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2 . Cells had been pre - treated with either 10 mM H2O2 or with water as a control . For these experiments , cells were grown in SGal ( - Leu , - Ura ) medium and harvested at early exponential phase . OxD refers to the degree of sensor oxidation . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) . ( E ) Hydrogen peroxide ' acute stress ' assay . Δprx1 cells co - transformed with empty vector or with a vector encoding either wild - type Prx1 , the P233stop or the C91A variants were grown in SD ( - Ura ) and treated with the indicated concentrations of H2O2 for 30 mins . Afterwards , the cells were diluted and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM treatment . Error bars represent standard deviation ( n = 5 - 8 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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 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 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 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 5(B) The response of a mitochondrial matrix-localized roGFP2-Prx1-P233stop probe expressed in wild-type cells to the indicated concentrations of exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).(C) Maleimide-based gel-shift assay to assess Prx1 cysteine redox state. Wild-type cells transformed with an empty plasmid or ∆prx1 cells with either an empty plasmid or a plasmid encoding wild-type Prx1, the hyperoxidation-resistant Prx1-P233stop or the inactive Prx1-C91A variants, were grown to exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24.(D) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe, expressed in Δprx1 cells containing a plasmid encoding either wild-type Prx1, Prx1-P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2. Cells had been pre-treated with either 10 mM H2O2 or with water as a control. For these experiments, cells were grown in SGal (-Leu, -Ura) medium and harvested at early exponential phase. OxD refers to the degree of sensor oxidation. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).(E) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with empty vector or with a vector encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SD (-Ura) and treated with the indicated concentrations of H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 5-8 biological replicates, with cells taken from independent cultures for each individual biological replicate).


Figure 6 ( A ) Hydrogen peroxide ' acute stress ' assay . Δprx1 cells co - transformed with an empty plasmid or a plasmid encoding Prx1 , Tsa1 , PRDX3 , PRDX5 , PRDX6 , PfAOP or PfAOP - L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre - treated with 0 , 1 , 5 , 10 , 25 mM H2O2 for 30 mins . Afterwards , the cells were diluted and a fixed volume was plated on YPD plates . The number of viable colonies was counted after 2 days of growth at 30 ° C , here represented as a percentage relative to the 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 - 8 biological replicates , with cells taken from independent cultures for each individual biological replicate ) . Significance was assessed with a Student ' s 2 - tailed , unpaired , t - test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ( B ) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress . Data from viability and matrix Grx1 - roGFP2 responses during acute H2O2 stress were correlated .

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 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

Figure 6(A) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with an empty plasmid or a plasmid encoding Prx1, Tsa1, PRDX3, PRDX5, PRDX6, PfAOP or PfAOP-L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre-treated with 0, 1, 5, 10, 25 mM H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume was plated on YPD plates. The number of viable colonies was counted after 2 days of growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3-8 biological replicates, with cells taken from independent cultures for each individual biological replicate). Significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001.(B) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress. Data from viability and matrix Grx1-roGFP2 responses during acute H2O2 stress were correlated.


Figure 7 ( A ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to 1 mM H2O2 . in BY4742 wild - type cells with an empty vector , in Δglr1 cells transformed either with an empty vector or a vector encoding wild - type Glr1 or the cytosolic form of Glr1 , where the MTS - encoding region was removed , or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1 . Cells were grown to exponential phase in SGal ( - Leu , - Ura ) medium . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) . ( B ) Growth curve of wild - type , Δprx1 , Δglr1 and Δglr1Δprx1 cells in SD medium complemented with all amino acids ( n = 3 biological replicates ) . Significance for the difference in the time the cultures reach 50 % of their maximal OD600 was assessed with the t - test . Error bars ( as ribbon ) represent the standard deviation ( n = 4 biological replicates , with cells taken from independent cultures for each individual biological replicate ) . ( C ) H2O2 ' acute stress ' assay . Wild - type , Δprx1 , Δglr1 and Δglr1Δprx1 cells pre - grown in SGal medium complemented with all amino acids to early exponential phase . Cells were treated with H2O2 at the indicated concentrations for 30 mins . Subsequently cells were diluted , and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 biological replicates , with cells taken from independent cultures for each individual biological replicate ) . An inset with a different scale on the y - axis is presented for the 1 and 5 mM concentration to allow better interpretation . ( D ) H2O2 ' acute stress ' assay . Δprx1Δglr1 cells co - transformed with an empty plasmid or with a plasmid encoding either wild - type Prx1 , the P233stop or the C91A variants were grown in SGal ( - Ura ) medium to early exponential phase . Cells were treated with the indicated amounts of H2O2 for 30 mins . Subsequently , the cells were diluted , and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM treatment . Error bars represent standard deviation ( n = 3 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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 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 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 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

Figure 7(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe to 1 mM H2O2. in BY4742 wild-type cells with an empty vector, in Δglr1 cells transformed either with an empty vector or a vector encoding wild-type Glr1 or the cytosolic form of Glr1, where the MTS-encoding region was removed, or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1. Cells were grown to exponential phase in SGal (-Leu, -Ura) medium. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).(B) Growth curve of wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells in SD medium complemented with all amino acids (n = 3 biological replicates). Significance for the difference in the time the cultures reach 50% of their maximal OD600 was assessed with the t-test. Error bars (as ribbon) represent the standard deviation (n = 4 biological replicates, with cells taken from independent cultures for each individual biological replicate).(C) H2O2 'acute stress' assay. Wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells pre-grown in SGal medium complemented with all amino acids to early exponential phase. Cells were treated with H2O2 at the indicated concentrations for 30 mins. Subsequently cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate). An inset with a different scale on the y-axis is presented for the 1 and 5 mM concentration to allow better interpretation.(D) H2O2 'acute stress' assay. Δprx1Δglr1 cells co-transformed with an empty plasmid or with a plasmid encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SGal (-Ura) medium to early exponential phase. Cells were treated with the indicated amounts of H2O2 for 30 mins. Subsequently, the cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate).


Figure 1c , Knockdown of NetB in the eye disc of GMR - RasV12 flies enhances survival . d , NetB heterozygous mutant flies survive better over Rasv12 - induced oncogenic stress . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Dunnett ' s multiple comparisons test ( c ) and two - tailed unpaired t - test d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . f , qRT - PCR with mRNA from the eye disc confirms higher expression of NetB in GMR - RasV12 flies Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by two - tailed unpaired t - test * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . g - h , Oncogenic Ras expression in the eye disc induces NetB protein , which was detected by GFP signal using CPTI - 000748 , a protein trap of NetB . Single confocal z - section images are shown . i , Quantification of GFP signals in g and h . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by two - tailed unpaired t - test * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . Scale bar , 50 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 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 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

Figure 1c, Knockdown of NetB in the eye disc of GMR-RasV12 flies enhances survival. d, NetB heterozygous mutant flies survive better over Rasv12-induced oncogenic stress. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test (c) and two-tailed unpaired t-test d, *P < 0.05, **P <0.01, ****P <0.0001.f, qRT-PCR with mRNA from the eye disc confirms higher expression of NetB in GMR-RasV12 flies Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by two-tailed unpaired t-test *P < 0.05, **P <0.01, ****P <0.0001.g-h, Oncogenic Ras expression in the eye disc induces NetB protein, which was detected by GFP signal using CPTI-000748, a protein trap of NetB. Single confocal z-section images are shown. i, Quantification of GFP signals in g and h. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by two-tailed unpaired t-test *P < 0.05, **P <0.01, ****P <0.0001. Scale bar, 50 μm.


Figure 2a - b , The amount of NetB protein increases in the fat body GMR - RasV12 ( a ) compared to control flies ( b ) . CPTI - 000748 , a protein trap of NetB , labels endogenous NetB . Data information : Scale bar , 20 μm ( a - be - h , Ectopic expression of GFP - tagged NetB in the eye disc induces high levels GFP - NetB protein in the fat body . Single confocal z - section are shown images ( e and g ) . Data information : Scale bar , 20 μm f - h 50 μmj - k , Ectopic expression of GFP - tagged NetB in the eye disc leads to detection of GFP signals in the hemolymph . Data information : Scale bar , 20 μm j - k )

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Figure 2a-b, The amount of NetB protein increases in the fat body GMR-RasV12 (a) compared to control flies (b). CPTI-000748, a protein trap of NetB, labels endogenous NetB. Data information: Scale bar, 20 μm (a-be-h, Ectopic expression of GFP-tagged NetB in the eye disc induces high levels GFP-NetB protein in the fat body. Single confocal z-section are shown images (e and g). Data information: Scale bar, 20 μm f-h 50 μmj-k, Ectopic expression of GFP-tagged NetB in the eye disc leads to detection of GFP signals in the hemolymph. Data information: Scale bar, 20 μm j-k)


Figure 3a , Knockdown of InR ( InR ) or expressing a dominant - negative form of InR ( InR - DN ) in the fat body using CG - Gal4 increases survival over oncogenic Ras expression in the imaginal disc . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Dunnett ' s multiple comparisons test ( a NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . c , qRT - PCR demonstrates that TMLHE expression in the fat body is reduced by Ras expression in the eye disc and reversed by insulin inhibition in the fat body . d , qRT - PCR demonstrates that NetB knockdown in the eye disc increases TMLHE expression in the fat body . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Tukey ' s multiple comparison test ( c and d ) NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . e , Knockdown of unc - 5 , but not other NetB receptors ( Fra or Dscam1 ) , in the fat body increases TMLHE expression . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Dunnett ' s multiple comparisons test e ) . NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Figure 3a, Knockdown of InR (InR) or expressing a dominant-negative form of InR (InR-DN) in the fat body using CG-Gal4 increases survival over oncogenic Ras expression in the imaginal disc. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test (a NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.c, qRT-PCR demonstrates that TMLHE expression in the fat body is reduced by Ras expression in the eye disc and reversed by insulin inhibition in the fat body. d, qRT-PCR demonstrates that NetB knockdown in the eye disc increases TMLHE expression in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test (c and d) NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.e, Knockdown of unc-5, but not other NetB receptors (Fra or Dscam1), in the fat body increases TMLHE expression. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test e). NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


Figure 4b , Local expression of oncogenic Ras in the eye disc decreases the amount of carnitine in the fat body . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by two - tailed unpaired t - test NS , not significant , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . c , TMLHE knockdown in the fat body aggravates organismal survival over the oncogenic stress . d , In the absence of GMR - RasV12 , inhibition of TMLHE in the fat body is not sufficient to induce organismal death . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by two - tailed unpaired t - test NS , not significant , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . g , Feeding of acetyl - CoA precursor ( acetate ) makes GMR - RasV12 flies survive even with TMLHE knockdown in the fat body . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by two - tailed unpaired t - test NS , not significant , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Figure 4b, Local expression of oncogenic Ras in the eye disc decreases the amount of carnitine in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.c, TMLHE knockdown in the fat body aggravates organismal survival over the oncogenic stress. d, In the absence of GMR-RasV12, inhibition of TMLHE in the fat body is not sufficient to induce organismal death. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.g, Feeding of acetyl-CoA precursor (acetate) makes GMR-RasV12 flies survive even with TMLHE knockdown in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.


Figure 5e , qRT - PCR with mRNA from the adult gut confirms higher expression of NetB in esg ts > RasV12 flies after oncogenic Ras induction for 10 days . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The statistical significance was determined by two - tailed unpaired t - test NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . f - g , Oncogenic Ras expression in the adult gut induces NetB protein , which was detected by GFP signal using CPTI - 000748 , a protein trap of NetB . Ras was induced for 10 days . Single confocal z - section images are shown . h , Quantification of GFP signals in f and g . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The statistical significance was determined by two - tailed unpaired t - test NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Scale bar , 50 μm . i , Knockdown of NetB in the adult gut of esg ts > RasV12 flies enhances survival . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The experiments were repeated independently at least twice with similar results The statistical significance was determined by log - rank ( Mantel - Cox ) test NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . l , Knockdown of TMLHE in the fat body of adult flies shortened lifespan of esgts > RasV12 flies but not of control flies . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and n represents the number of flies that were analyzed . The experiments were repeated independently at least twice with similar results The statistical significance was determined by log - rank ( Mantel - Cox ) test l ) . NS , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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 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 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

Figure 5e, qRT-PCR with mRNA from the adult gut confirms higher expression of NetB in esg ts>RasV12 flies after oncogenic Ras induction for 10 days. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The statistical significance was determined by two-tailed unpaired t-test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.f-g, Oncogenic Ras expression in the adult gut induces NetB protein, which was detected by GFP signal using CPTI-000748, a protein trap of NetB. Ras was induced for 10 days. Single confocal z-section images are shown. h, Quantification of GFP signals in f and g. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The statistical significance was determined by two-tailed unpaired t-test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001. Scale bar, 50 μm.i, Knockdown of NetB in the adult gut of esg ts>RasV12 flies enhances survival. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The experiments were repeated independently at least twice with similar results The statistical significance was determined by log-rank (Mantel-Cox) test NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.l, Knockdown of TMLHE in the fat body of adult flies shortened lifespan of esgts>RasV12 flies but not of control flies. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and n represents the number of flies that were analyzed. The experiments were repeated independently at least twice with similar results The statistical significance was determined by log-rank (Mantel-Cox) test l). NS, not significant, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.


Figure 1Figure 1 - The origin of N - terminal proteoforms . A . 2 , 578 proteoforms were identified including 2 , 135 dbTIS and 443 aTIS . For the majority of proteins we found a unique translation initiation site ( grey ) . Additionally , 335 N - terminal peptides pointed to proteins with multiple N - termini created by alternative translation initiation ( blue ) , whereas 81 N - termini were generated by alternative iMet processing ( green ) . B - C . Proteomics - derived translation initiation events were verified at the transcript level using Ribo - Seq data obtained in Jurkat cells and HCT 116 cells ( data unpublished ) . Although in this study , the degree of in vivo Nt - acetylation could not be directly determined , all retained N - termini were compliant with the rules of N - terminal processing from previous experiments ( Van Damme et al , 2014 ) . In the case N - termini were found to be in vivo Nt - acetylated ( previous MS / MS - based evidence ( Van Damme et al , 2014 ) ) this information is indicated . dbTIS and aTIS peptides were further mapped to protein N - termini annotated in Swiss - Prot Isoform , UniProt TrEMBL and Ensembl databases and neo - N - termini ( proteolytic products ) of the knowledgebase TopFind 3 . 0 . The number of matching N - termini is indicated in dark grey . N - terminal peptides supported by at least one source of metadata pointing to translation ( Nt - acetylation , Ribo - Seq , Swiss - Prot Isoforms , TrEMBL or Ensembl ) were classified as highly confident TIS ( indicated in green ) . D . Using experimental and metadata we assigned the most likely origin of aTIS peptides and confirmed that alternative translation initiation within the same transcript ( leaky scanning ) contributes to the majority of alternative TIS identified .

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Figure 1Figure 1 - The origin of N-terminal proteoforms. A. 2,578 proteoforms were identified including 2,135 dbTIS and 443 aTIS. For the majority of proteins we found a unique translation initiation site (grey). Additionally, 335 N-terminal peptides pointed to proteins with multiple N-termini created by alternative translation initiation (blue), whereas 81 N-termini were generated by alternative iMet processing (green). B-C. Proteomics-derived translation initiation events were verified at the transcript level using Ribo-Seq data obtained in Jurkat cells and HCT 116 cells (data unpublished). Although in this study, the degree of in vivo Nt-acetylation could not be directly determined, all retained N-termini were compliant with the rules of N-terminal processing from previous experiments (Van Damme et al, 2014). In the case N-termini were found to be in vivo Nt-acetylated (previous MS/MS-based evidence (Van Damme et al, 2014)) this information is indicated. dbTIS and aTIS peptides were further mapped to protein N-termini annotated in Swiss-Prot Isoform, UniProt TrEMBL and Ensembl databases and neo-N-termini (proteolytic products) of the knowledgebase TopFind 3.0. The number of matching N-termini is indicated in dark grey. N-terminal peptides supported by at least one source of metadata pointing to translation (Nt-acetylation, Ribo-Seq, Swiss-Prot Isoforms, TrEMBL or Ensembl) were classified as highly confident TIS (indicated in green). D. Using experimental and metadata we assigned the most likely origin of aTIS peptides and confirmed that alternative translation initiation within the same transcript (leaky scanning) contributes to the majority of alternative TIS identified.


Figure 2Figure 2 - Examples of translation initiation events confirmed by Ribo - Seq in Jurkat cells . A - B . Alternative translation initiation due to leaky scanning . C . Alternative splicing . D . The preferential expression of an N - terminally truncated proteoform confirmed by Ribo - Seq data . E - F . Evidence for alternative translation initiation occurring at two consecutive AUG codons .

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Figure 2Figure 2 - Examples of translation initiation events confirmed by Ribo-Seq in Jurkat cells. A-B. Alternative translation initiation due to leaky scanning. C. Alternative splicing. D. The preferential expression of an N-terminally truncated proteoform confirmed by Ribo-Seq data. E-F. Evidence for alternative translation initiation occurring at two consecutive AUG codons.


Figure 3A . Jurkat cells were pre - labeled with light or medium L - Arg isotopes . A label swap was performed of cells growing in medium Arg , whereas light cells were cultured in the same medium . Cells were harvested at different time points and equal proteome amounts were mixed , followed by N - terminal COFRADIC fractionation and LC - MS / MS analysis . C . The distribution of turnover times ( N = 1 , 894 ) in our experiment was not unimodal , with a sizable number of quickly turned over proteins , with the median protein turnover rate being 21 . 6 h . The doubling time of Jurkat cells ( 24 h ) is indicated in red .

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Figure 3A. Jurkat cells were pre-labeled with light or medium L-Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N-terminal COFRADIC fractionation and LC-MS/MS analysis.C. The distribution of turnover times (N=1,894) in our experiment was not unimodal, with a sizable number of quickly turned over proteins, with the median protein turnover rate being 21.6 h. The doubling time of Jurkat cells (24 h) is indicated in red.


Figure 6A . Degradation profiles of AIMP2 , MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N - terminal proteoforms that differ in stability . B . MS spectra of the N - termini of two proteoforms originating from the aminoacyl tRNA synthase complex - interacting multifunctional protein 2 ( AIMP2 ) : a dbTIS indicative N - terminus with processed iMet and starting at the second amino acid Pro , an aTIS indicative peptide starting at position 3 and retaining its iMet . As evident from the MS spectra shown , these N - terminal proteoforms displayed largely different turnover rates .

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Figure 6A. Degradation profiles of AIMP2, MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N-terminal proteoforms that differ in stability.B. MS spectra of the N-termini of two proteoforms originating from the aminoacyl tRNA synthase complex-interacting multifunctional protein 2 (AIMP2): a dbTIS indicative N-terminus with processed iMet and starting at the second amino acid Pro, an aTIS indicative peptide starting at position 3 and retaining its iMet. As evident from the MS spectra shown, these N-terminal proteoforms displayed largely different turnover rates.


Figure 8A . Jurkat cells were treated with 100 µg / ml CHX for 0 , 0 . 5 , 1 . 5 , 4 , 8 , 12 or 24 hours . Protein degradation was monitored by Western blotting and stabilities of several short - lived endogenous proteins were confirmed using antibodies ( including lamin B , securin , β - catenin , GCIP interacting protein p29 ) and compared to stable proteins ( such as GAPDH and actin ) . Proteoform - specific bands were used to calculate protein half - lives which were in a good agreement with turnover times obtained from pSILACB - C . Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E . Selective C - terminal V5 - tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse - chase experiments were performed as described for Jurkat cells . Degradation of overexpressed proteoforms was monitored by an anti - V5 antibody and compared to the turnover of stable proteins such as actin and tubulin .

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Figure 8A. Jurkat cells were treated with 100 µg/ml CHX for 0, 0.5, 1.5, 4, 8, 12 or 24 hours. Protein degradation was monitored by Western blotting and stabilities of several short-lived endogenous proteins were confirmed using antibodies (including lamin B, securin, β-catenin, GCIP interacting protein p29) and compared to stable proteins (such as GAPDH and actin). Proteoform-specific bands were used to calculate protein half-lives which were in a good agreement with turnover times obtained from pSILACB-C. Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E. Selective C-terminal V5-tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse-chase experiments were performed as described for Jurkat cells. Degradation of overexpressed proteoforms was monitored by an anti-V5 antibody and compared to the turnover of stable proteins such as actin and tubulin.


Figure 1 ( A , B ) Overexpression of wildtype full - length human tau ( hTau , also termed tau441 or tau40 or tau2N4R ) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II , in which 96 transcription factors ( Appendix Table S1 and S2 ) were monitored . The empty vector was transfected as a control ( Ctrl ) . ( C - F ) Expression of hTau ( probed by HT7 ) increased total and the phosphorylated STAT1 at Tyr701 ( pY - STAT1 ) in cell whole extracts ( C , D ) and the nuclear fraction ( E , F ) measured by Western blotting ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . * , p < 0 . 05 vs Ctrl . ( G ) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control ( eGFP ) ( n = 5 ) . Scale bar , 10 μm . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . , * * , p < 0 . 01 vs Ctrl . ( H ) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction ( Nu ) measured by Western blotting . ( I ) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . * , p < 0 . 05 , * * * , p < 0 . 001 vs Ctrl . ( J ) Overexpression of hTau increased STAT1 - DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay ( EMSA ) . * , indicates STAT1 / DNA complex .

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 O O O O O O 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 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 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 1(A, B) Overexpression of wildtype full-length human tau (hTau, also termed tau441 or tau40 or tau2N4R) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II, in which 96 transcription factors (Appendix Table S1 and S2) were monitored. The empty vector was transfected as a control (Ctrl).(C-F) Expression of hTau (probed by HT7) increased total and the phosphorylated STAT1 at Tyr701 (pY-STAT1) in cell whole extracts (C, D) and the nuclear fraction (E, F) measured by Western blotting (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 vs Ctrl.(G) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control (eGFP) (n=5). Scale bar, 10 μm. Data information: Data were presented as mean ± SD (Mann-Whitney test). , **, p<0.01 vs Ctrl.(H) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction (Nu) measured by Western blotting.(I) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 , ***, p<0.001 vs Ctrl.(J) Overexpression of hTau increased STAT1-DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay (EMSA). *, indicates STAT1/DNA complex.


Figure 2 ( A , B ) AAV - hTau - eGFP ( AAV - hTau ) or the empty vector AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , the increased levels of STAT1 and pY - STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting ( n = 6 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl . ( C , D ) The increased STAT1 and pY - STAT1 in hippocampal total extracts and the nuclear fraction of 12 - month - old hTau transgenic mice measured by Western blotting ( n = 4 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl . ( E , F ) The representative images of STAT1 and pY - STAT1 in the brain of AD patients probed by co - immunohistochemical staining and quantitative analysis ( hematoxylin stains nuclei , purple ; DAB stains the target proteins , brown ; n = 5 - 6 slices ) . Arrowheads indicated typical nuclear staining of STAT1 / pY - STAT1 . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl . ( G ) The increased AT8 ( pS202 / pT205 ) , STAT1 and pY - STAT1 in cortex total extracts of AD patients measured by Western blotting ( n = 3 , Student ' s t test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl . ( H ) STAT1 mRNA was analyzed by qPCR in AAV - hTau or AAV - eGFP infected hippocampal tissues ( n = 6 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

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 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 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 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

Figure 2(A, B) AAV-hTau-eGFP (AAV-hTau) or the empty vector AAV-eGFP (1.13×1013 v.g./ml) was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, the increased levels of STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting (n=6, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.(C, D) The increased STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction of 12-month-old hTau transgenic mice measured by Western blotting (n=4, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.(E, F) The representative images of STAT1 and pY-STAT1 in the brain of AD patients probed by co-immunohistochemical staining and quantitative analysis (hematoxylin stains nuclei, purple; DAB stains the target proteins, brown; n=5-6 slices). Arrowheads indicated typical nuclear staining of STAT1/pY-STAT1. Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.(G) The increased AT8 (pS202/pT205), STAT1 and pY-STAT1 in cortex total extracts of AD patients measured by Western blotting (n=3, Student's t test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.(H) STAT1 mRNA was analyzed by qPCR in AAV-hTau or AAV-eGFP infected hippocampal tissues (n=6, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


Figure 3AAV - Cre ( 5 × 1012 v . g . / ml ) mixed with AAV - hTau or AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) were stereotaxically injected into the hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by immunohistochemical staining . AAV - Cre ( 5 × 1012 v . g . / ml ) mixed with AAV - hTau or AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) were stereotaxically injected into the hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by Western blotting ( C ) Downregulation of STAT1 ameliorated hTau - induced spatial learning deficit shown by the decreased escape latency during 5 consecutive days training in Morris water maze ( MWM ) test ( n = 9 - 11 for each group ) . Data information : Data were presented as mean ± s . e . m . ( two - way repeated measures analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for C * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau . ( D - G ) Downregulation of STAT1 ameliorated hTau - induced spatial memory deficit shown by the decreased latency to reach the platform quadrant ( D ) , the increased crossing time in the platform site ( E ) and time spent in the target quadrant ( F ) measured at day 6 by removed the platform in MWM test ; no motor dysfunction was seen ( G ) ( n = 9 - 11 for each group ) . Data information : Data were presented as mean ± s . e . m . , one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau . ( H ) Downregulation of STAT1 ameliorated hTau - induced contextual memory deficits measured at 24 h during contextual fear conditioning test ( n = 8 each group ) . Data information : Data were presented as mean ± s . e . m . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau . ( I - L ) One month after the virus infection , paired - pulse ratio ( PPR ) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice ( I ) . The IO curve of fEPSP recorded on acute hippocampal slices ( J ) . The slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice ( K ) . LTP magnitude was calculated as the average ( normalized to baseline ) of the responses recorded 40 - 60 min after conditioning stimulation . ( L ) . ( n = 5 slices from 4 mice for each group ) . Data information : Data were presented as mean ± SD two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for I - K , one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau . ( M - P ) One month after the virus infection , whole cell patch clamp was used to measure the function of NMDA ( at + 40 mV ) and AMPA ( at - 70 mV ) receptors on acute brain slices ( 400 μm ) . The insets show representative sample traces of EPSCs in virus infected neurons ( M ) . The reduced NMDA and unchanged AMPA currents with a reduced NMDA / AMPA ratio were seen in hTau infected neurons , while knockdown of STAT1 restored the hTau - induced NMDA currents ( N - P ) . ( n = 12 neurons from 4 animals for eGFP group ; n = 11 neurons from 4 animals for hTau group ; n = 13 neurons from 4 animals for hTau + CRE group ) . Data information : Data were presented as mean ± SD one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

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 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 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 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 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 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 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

Figure 3AAV-Cre (5×1012 v.g./ml) mixed with AAV-hTau or AAV-eGFP (1.13×1013 v.g./ml) were stereotaxically injected into the hippocampal CA3 of 3-month-old STAT1flox/flox mice. One month later, downregulation of STAT1 was confirmed by immunohistochemical staining.AAV-Cre (5×1012 v.g./ml) mixed with AAV-hTau or AAV-eGFP (1.13×1013 v.g./ml) were stereotaxically injected into the hippocampal CA3 of 3-month-old STAT1flox/flox mice. One month later, downregulation of STAT1 was confirmed by Western blotting(C) Downregulation of STAT1 ameliorated hTau-induced spatial learning deficit shown by the decreased escape latency during 5 consecutive days training in Morris water maze (MWM) test (n=9-11 for each group). Data information: Data were presented as mean ± s.e.m. (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni's post hoc test for C *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.(D-G) Downregulation of STAT1 ameliorated hTau-induced spatial memory deficit shown by the decreased latency to reach the platform quadrant (D), the increased crossing time in the platform site (E) and time spent in the target quadrant (F) measured at day 6 by removed the platform in MWM test; no motor dysfunction was seen (G) (n=9-11 for each group). Data information: Data were presented as mean ± s.e.m. , one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.(H) Downregulation of STAT1 ameliorated hTau-induced contextual memory deficits measured at 24 h during contextual fear conditioning test (n=8 each group). Data information: Data were presented as mean ± s.e.m. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.(I-L) One month after the virus infection, paired-pulse ratio (PPR) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice (I). The IO curve of fEPSP recorded on acute hippocampal slices (J). The slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice (K). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (L). (n=5 slices from 4 mice for each group). Data information: Data were presented as mean ± SD two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for I-K, one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.(M-P) One month after the virus infection, whole cell patch clamp was used to measure the function of NMDA (at +40 mV) and AMPA (at -70 mV) receptors on acute brain slices (400 μm). The insets show representative sample traces of EPSCs in virus infected neurons (M). The reduced NMDA and unchanged AMPA currents with a reduced NMDA/AMPA ratio were seen in hTau infected neurons, while knockdown of STAT1 restored the hTau-induced NMDA currents (N-P). (n=12 neurons from 4 animals for eGFP group; n=11 neurons from 4 animals for hTau group; n=13 neurons from 4 animals for hTau+CRE group). Data information: Data were presented as mean ± SD one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


Figure 4 ( A , B ) Overexpression of AAV - hTau decreased the protein levels of GluN1 , GluN2A and GluN2B detected by Western blotting in the hippocampal CA3 of C57 mice , compared with the AAV - eGFP vector control . ( C , D ) Simultaneous downregulation of STAT1 by infusing AAV - Cre in hippocampal CA3 of STAT1flox / flox mice abolished the hTau - induced inhibition in expression of NMDARs protein . Data information : Data were presented as mean ± SD ( n = 4 ; Mann - Whitney test ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau . ( E , F ) Overexpression of AAV - hTau or simultaneous downregulation of STAT1 changed the mRNA levels of GluN1 , GluN2A and GluN2B detected by qRT - PCR in the hippocampal CA3 . Data information : Data were presented as mean ± SD ( n = 4 ; Mann - Whitney test ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau .

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 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

Figure 4(A, B) Overexpression of AAV-hTau decreased the protein levels of GluN1, GluN2A and GluN2B detected by Western blotting in the hippocampal CA3 of C57 mice, compared with the AAV-eGFP vector control. (C, D) Simultaneous downregulation of STAT1 by infusing AAV-Cre in hippocampal CA3 of STAT1flox/flox mice abolished the hTau-induced inhibition in expression of NMDARs protein. Data information: Data were presented as mean ± SD (n=4; Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau.(E, F) Overexpression of AAV-hTau or simultaneous downregulation of STAT1 changed the mRNA levels of GluN1, GluN2A and GluN2B detected by qRT-PCR in the hippocampal CA3. Data information: Data were presented as mean ± SD (n=4; Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau.


Figure 5 ( A ) Overexpression of AAV - hTau increased binding of STAT1 to the promoter regions of GluN1 , GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay ( ChIP ) ( n = 3 from three independent experiments ) . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters . ( B ) Overexpression of hTau or wildtype STAT1 ( WT - STAT1 ) inhibits the transcription activity of NMDARs compared with the empty vector control ( Ctrl ) measured by luciferase activity assay in HEK293 cells ( n = 3 from three independent experiments ) . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters . Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN1 ( C ) The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters . Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN2B ( E ) The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters . Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN2A ( G , I ) . The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overexpression of AAV-hTau increased binding of STAT1 to the promoter regions of GluN1, GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay (ChIP) (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.(B) Overexpression of hTau or wildtype STAT1 (WT-STAT1) inhibits the transcription activity of NMDARs compared with the empty vector control (Ctrl) measured by luciferase activity assay in HEK293 cells (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN1 (C) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2B (E) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2A (G, I). The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.