Figure 5A . Luciferasereporter assay of wild - type ( wt ) and mutant ( mut ) ITPKB 3 ' UTR in HEK293 cells co - transfected with a synthetic miR - 132 ( miR - 132 ) or a negative control ( Neg Ctr ) oligonucleotide . B . ELISA of Aβ40 and Aβ42 levels in HEK293 - APPswecells transfected with a miR - 132 antisense oligonucleotide ( miR - 132 inh ) , an siRNA against ITPKB ( ITPKB siRNA ) or both . The assays in A and B were performed in three independent experiments , each in triplicates . C . Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB . Sample size , n = 6 per group . Values were normalized to control - injected group ( Ctr ) and presented as mean ± SEM . D . ELISA of insoluble ( Formic acid - soluble ) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA - and control - injected animals at three months of age . Sample size , n = 6 per group . Values were normalized to control group and presented as mean ± SEM . E , F . Western blot analysis of ITPKB levels upon miR - 132 down - ( E ) or up - regulation ( F ) at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to control groups and presented as mean ± SEM .

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 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 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 O O O O O O 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 5A. Luciferasereporter assay of wild-type (wt) and mutant (mut) ITPKB 3'UTR in HEK293 cells co-transfected with a synthetic miR-132 (miR-132) or a negative control (Neg Ctr) oligonucleotide.B. ELISA of Aβ40 and Aβ42 levels in HEK293-APPswecells transfected with a miR-132 antisense oligonucleotide (miR-132 inh), an siRNA against ITPKB (ITPKB siRNA) or both. The assays in A and B were performed in three independent experiments, each in triplicates.C. Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB. Sample size, n=6 per group. Values were normalized to control-injected group (Ctr) and presented as mean ± SEM.D. ELISA of insoluble (Formic acid-soluble) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM.E, F. Western blot analysis of ITPKB levels upon miR-132 down- (E) or up-regulation (F) at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to control groups and presented as mean ± SEM.


Figure 6Western blot analysis of CTFβ ( A ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used . Western blot analysis of sAPPβ - swe ( B ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used . Western blot analysis of phosphorylated ERK1 / 2 ( pERK1 / 2 ) ( C ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

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 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 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

Figure 6Western blot analysis of CTFβ (A) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.Western blot analysis of sAPPβ-swe (B) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.Western blot analysis of phosphorylated ERK1/2 (pERK1/2) (C) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Figure 7A . Double immunofluorescence staining of amyloid plaques ( 6E10 ) and ITPKB in AD prefrontal cortex . Scale bar , 50 μm . Arrowheads indicate ITPKB immunopositivity . B . miR - 132FISH coupled with double immunofluorescence against hyperphosphorylated TAU ( AT8 ) - containing neurofibrillary tangles ( NFTs ) and ITPKB in AD prefrontal cortex . miR - 124 was used as a control . Scale bar , 50 μm . C . Quantification of miR - 132 , ITPKB and hyperphosphorylated TAU ( pTAU ) signal in single neurons . Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed ( see Materials and Methods ) . Sample size ( AD patients ) , n = 3 . Values are presented as mean ± SEM . Two - way Anova was used . Quantifications are summarized in the table provided . Arrow directions refer to the comparison of each normalized signal to the same signal in the other group ( in the " low miR - 132 " group comparisons are made to the " high miR - 132 " group and vice versa ) .

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 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 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

Figure 7A. Double immunofluorescence staining of amyloid plaques (6E10) and ITPKB in AD prefrontal cortex. Scale bar, 50 μm. Arrowheads indicate ITPKB immunopositivity.B. miR-132FISH coupled with double immunofluorescence against hyperphosphorylated TAU (AT8)-containing neurofibrillary tangles (NFTs) and ITPKB in AD prefrontal cortex. miR-124 was used as a control. Scale bar, 50 μm.C. Quantification of miR-132, ITPKB and hyperphosphorylated TAU (pTAU) signal in single neurons. Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed (see Materials and Methods). Sample size (AD patients), n=3. Values are presented as mean ± SEM. Two-way Anova was used. Quantifications are summarized in the table provided. Arrow directions refer to the comparison of each normalized signal to the same signal in the other group (in the "low miR-132" group comparisons are made to the "high miR-132" group and vice versa).


Figure 8Semi - quantitative PCR of miR - 132 and western blot analysis of ITPKB , phosphorylated ( pERK1 / 2 ) and total ERK1 / 2 , phosphorylated ( AT8 , AT270 ) and total TAU , phosphorylated sphingosine kinase 1 ( pSphK1 ) , BACE1 , full length APP ( flAPP ) , APP CTFs and sAPPβ in human AD hippocampi ( AD ) compared to non demented control samples ( ND ) . Sample size , n = 39 for AD and n = 15 for ND . Values were normalized to ND and presented as mean ± SEM . The Student ' s t - test was used .

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

Figure 8Semi-quantitative PCR of miR-132 and western blot analysis of ITPKB, phosphorylated (pERK1/2) and total ERK1/2, phosphorylated (AT8 , AT270) and total TAU, phosphorylated sphingosine kinase 1 (pSphK1), BACE1, full length APP (flAPP), APP CTFs and sAPPβ in human AD hippocampi (AD) compared to non demented control samples (ND). Sample size, n=39 for AD and n=15 for ND. Values were normalized to ND and presented as mean ± SEM. The Student's t-test was used.


Figure 1A Dose - response binary food choice assay of saponin ( n ≥ 4 ) . B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla ( n ≥ 8 ) . C Sample traces of saponin - induced action potentials in ( B ) . D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla ( n ≥ 8 ) . E Sample traces from ( D ) . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis as a post hoc test to compare two sets of data ( * * P < 0 . 01 ) . The error bars represent SEMs .

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

Figure 1A Dose-response binary food choice assay of saponin (n ≥ 4).B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla (n ≥ 8).C Sample traces of saponin-induced action potentials in (B).D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla (n ≥ 8). E Sample traces from (D). The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (**P < 0.01). The error bars represent SEMs.


Figure 2A Binary food choice assay with 26 Gr mutants for 5 % saponin ( n ≥ 4 ) . The error bars represent SEMs . B Concentration - dependent avoidance of saponin in control , Gr28bMi and Gr28b Df / Gr28bMi ( n ≥ 4 ) . C Amount of ingestion calculated at the indicated time points ( 15 , 30 , 60 , and 240 min ) using spectrophotogram analysis for control and 5 % saponin - containing food ( n = 8 ) . D , E PER assay for control and Gr28bMi flies in the ( D ) legs or ( E ) proboscis . Flies were initially given 2 % sucrose , then 2 % sucrose in combination with 5 % saponin ( n = 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis as a post hoc test to compare two sets of data ( * 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2A Binary food choice assay with 26 Gr mutants for 5% saponin (n ≥ 4). The error bars represent SEMs.B Concentration-dependent avoidance of saponin in control, Gr28bMi and Gr28b Df/ Gr28bMi (n ≥ 4).C Amount of ingestion calculated at the indicated time points (15, 30, 60, and 240 min) using spectrophotogram analysis for control and 5% saponin-containing food (n = 8).D, E PER assay for control and Gr28bMi flies in the (D) legs or (E) proboscis. Flies were initially given 2% sucrose, then 2% sucrose in combination with 5% saponin (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (*P < 0.05, **P < 0.01).


Figure 3A Mapping analysis of all the sensilla following stimulation with 5 % saponin in control and Gr28bMi flies ( we followed Tanimura ' s nomenclature ) ( n ≥ 8 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) . B Sample traces of saponin - induced action potentials on S6 sensilla . C Tip recording analysis of S6 sensilla from control and Gr28bMi flies . Stimuli used are 0 . 2 % DEET , 1 . 0 mM coumarin , 0 . 5 mM quinine , 10 mM umbelliferone , 10 mM caffeine , 0 . 3 mM strychnine , 0 . 3 mM lobeline , 0 . 1 mM chloroquine , 0 . 2 mM denatonium , 0 . 5 mM papaverine , 0 . 1 mM berberine and 0 . 1 % SDS ( n ≥ 7 ) . The error bars represent SEMs . D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene ( hid ) , under Gr33a - GAL4 or Gr66a - GAL4 . All heterozygote controls ( UAS - hid / + , Gr33a - GAL4 / + , and Gr66a - GAL4 / + ) are shown ( n ≥ 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) . E Tip recording analysis of the indicated S - type sensilla and genotypes ( n ≥ 7 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * 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 O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Mapping analysis of all the sensilla following stimulation with 5% saponin in control and Gr28bMi flies (we followed Tanimura's nomenclature) (n ≥ 8). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).B Sample traces of saponin-induced action potentials on S6 sensilla.C Tip recording analysis of S6 sensilla from control and Gr28bMi flies. Stimuli used are 0.2% DEET, 1.0 mM coumarin, 0.5 mM quinine, 10 mM umbelliferone, 10 mM caffeine, 0.3 mM strychnine, 0.3 mM lobeline, 0.1 mM chloroquine, 0.2 mM denatonium, 0.5 mM papaverine, 0.1 mM berberine and 0.1% SDS (n ≥ 7). The error bars represent SEMs.D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr33a-GAL4 or Gr66a-GAL4. All heterozygote controls (UAS-hid/+, Gr33a-GAL4/+, and Gr66a-GAL4/+) are shown (n ≥ 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).E Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


Figure 4B RT - PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control . From left to right , the band of tubulin of wild - type , tubulin of Gr28bMi , DNA ladder , Gr28b of wild - type , and Gr28b of Gr28bMi . Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . The asterisks indicate significant differences from that of the Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s analysis ( * * P < 0 . 01 ) . C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4 / UAS systems . ( n ≥ 4 ) . D Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . The asterisks indicate significant differences from that of the Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s analysis ( * * P < 0 . 01 ) . D Saponin - induced action potential frequencies from the indicated transgenic flies on S6 sensilla ( n = 7 ) .

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

Figure 4B RT-PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control. From left to right, the band of tubulin of wild-type, tubulin of Gr28bMi, DNA ladder, Gr28b of wild-type, and Gr28b of Gr28bMi.Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4/UAS systems. (n ≥ 4).D Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). D Saponin-induced action potential frequencies from the indicated transgenic flies on S6 sensilla (n = 7).


Figure 5A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene ( hid ) , under Gr28b . c - GAL4 ( n = 4 ) . B Tip recording analysis of the indicated S - type sensilla and genotypes ( n ≥ 7 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * * P < 0 . 01 ) . C Expression of the Gr28b . c - GAL4 reporter in subsets of Gr66a - positive GRNs . First panel shows that all bitter responsive GRNs are labeled by Gr66a - I - GFP ( anti - GFP , green ) . Second panel shows the expression of the Gr28b . c - GAL4 , crossed with UAS - DsRed ( anti - DsRed , red ) . Third panel shows merged image of first and second panels . Forth panel shows a schematic diagram of sensilla arrangement . Red - marked sensilla indicate the expression of Gr28b . c - GAL4 reporter in second panel . Scale bars represent 40 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 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 O O O O 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 Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr28b.c-GAL4 (n = 4).B Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (**P < 0.01). C Expression of the Gr28b.c-GAL4 reporter in subsets of Gr66a-positive GRNs. First panel shows that all bitter responsive GRNs are labeled by Gr66a-I-GFP (anti-GFP, green). Second panel shows the expression of the Gr28b.c-GAL4, crossed with UAS-DsRed (anti-DsRed, red). Third panel shows merged image of first and second panels. Forth panel shows a schematic diagram of sensilla arrangement. Red-marked sensilla indicate the expression of Gr28b.c-GAL4 reporter in second panel. Scale bars represent 40 μm.


Figure 6A , B Survival rate of ( A ) control and ( B ) Gr28bMi adult raised on control and the indicated concentrations of saponin - containing cornmeal diet ( n = 4 ) . C , D Survival rate based on binary food choice . control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM ( C ) or 200 mM fructose ( D ) mixed with 5 % or 7 . 5 % saponin . E , F Cumulative pupation rate of ( E ) control and ( F ) Gr28bMi larvae raised on the indicated saponin - containing food as well as control food ( n = 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from normal diet fed which were detected by a single - factor ANOVA with Scheffe ' s analysis ( * 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B Survival rate of (A) control and (B) Gr28bMi adult raised on control and the indicated concentrations of saponin-containing cornmeal diet (n = 4).C, D Survival rate based on binary food choice. control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM (C) or 200 mM fructose (D) mixed with 5% or 7.5% saponin.E, F Cumulative pupation rate of (E) control and (F) Gr28bMi larvae raised on the indicated saponin-containing food as well as control food (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from normal diet fed which were detected by a single-factor ANOVA with Scheffe's analysis (*P < 0.05, **P < 0.01).


Figure 1 ( A ) miRNA abundance in RNA - sequencing samples from murine , porcine , and bovine oocytes Each graph shows on the y - axis reads per million of 19 - 32 nt reads for miRNAs and on the x - axis miRNAs ordered by name . ( C ) qPCR quantification of ssc - miR - 205 and bta - miR - 10b miRNAs in murine ( M . m . ) , porcine ( S . s . ) , and bovine ( B . t . ) oocytes , respectively . Shown are a mean values from three independent experiments . Error bars = SD . Detailed information concerning miRNA quantification is provided in the Methods section .

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

Figure 1(A) miRNA abundance in RNA-sequencing samples from murine, porcine, and bovine oocytes Each graph shows on the y-axis reads per million of 19-32 nt reads for miRNAs and on the x-axis miRNAs ordered by name.(C) qPCR quantification of ssc-miR-205 and bta-miR-10b miRNAs in murine (M.m.), porcine (S.s.), and bovine (B.t.) oocytes, respectively. Shown are a mean values from three independent experiments. Error bars = SD. Detailed information concerning miRNA quantification is provided in the Methods section.


Figure 2 ( A ) bta - miR - 10b and ssc - miR - 205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary ( 1x perfect ) miRNA binding site or four partially complementary ( bulged ) miRNA binding sites . Error bars = SD . Each data point represents a value obtained from five microinjected oocytes . Data were collected in two microinjection sessions , each microinjection session used a new batch of in vitro transcribed reporter mRNAs . Data information : asterisks indicate statistical significance ( p - value ) of one - tailed t - test , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . ( B ) De - repression of predicted ssc - miR - 205 targets . In three independent microinjection experiments , ssc - miR - 205 was inhibited by a microinjected antisense oligonucleotide inhibitor and target levels were assessed 24 hours after microinjection by RT - qPCR . Error bars = SD . Data information : asterisks indicate statistical significance ( p - value ) of one - tailed t - test , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . ( C ) Inhibition of ssc - miR - 205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to the blastocyst stage . Shown is development of microinjected oocytes to the blastocyst stage relative to water - injected oocytes . Five independent experiments ( represented by individual data points ) were performed , ~ 50 porcine oocytes were microinjected in each group in each experiment . Difference between let - 7a and miR - 205 inhibitors effects is statistically significant ( two - tailed paired t - test p - value = 0 . 016 ) . Error bars = SD . Data information : asterisks indicate statistical significance ( p - value ) of one - tailed t - test , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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 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 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

Figure 2(A) bta-miR-10b and ssc-miR-205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary (1x perfect) miRNA binding site or four partially complementary (bulged) miRNA binding sites. Error bars = SD. Each data point represents a value obtained from five microinjected oocytes. Data were collected in two microinjection sessions, each microinjection session used a new batch of in vitro transcribed reporter mRNAs. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).(B) De-repression of predicted ssc-miR-205 targets. In three independent microinjection experiments, ssc-miR-205 was inhibited by a microinjected antisense oligonucleotide inhibitor and target levels were assessed 24 hours after microinjection by RT-qPCR. Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).(C) Inhibition of ssc-miR-205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to the blastocyst stage. Shown is development of microinjected oocytes to the blastocyst stage relative to water-injected oocytes. Five independent experiments (represented by individual data points) were performed, ~50 porcine oocytes were microinjected in each group in each experiment. Difference between let-7a and miR-205 inhibitors effects is statistically significant (two-tailed paired t-test p-value = 0.016). Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).


Figure 4 ( A ) ssc - miR - 205 turnover . Fully - grown transcriptionally quiescent oocytes were cultured for indicated periods of time and selected miRNAs were quantified by RT - qPCR in four ( ssc - let - 7a ) , five ( ssc - miR - 22 ) or six ( ssc - mir10b and ssc - miR - 205 ) independent experiments . Error bars = SD . ( B ) Comparison of mean relative changes of miRNA level at 20 h of culture ssc - miR - 205 in oocytes is less than ssc - miR - 10b , ssc - miR - 22 , or ssc - let - 7a . Error bars = SD . Asterisks indicate statistical significance ( p - value ) of one - tailed paired t - test , ( * p < 0 . 05 , * * p < 0 . 01 ) . ( C ) Treatment by sodium periodate followed by qPCR does not support 2 ' - OH modification of either ssc - miR - 205 or bta - miR - 10b miRNA . Shown are relative levels of the sodium periodate - treated samples to non - treated samples , which were set to one . The experiment was performed three times . Error bars = SD . Efficiency of periodate treatment was confirmed using non - methylated and methylated miR - 221 RNA oligonucleotides ( D ) 3 ′ tailing of small RNAs in murine , bovine , and porcine oocytes Shown are percentages of miRNAs tailed at 3 ′ end with mono - and oligonucleotides .

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 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

Figure 4(A) ssc-miR-205 turnover. Fully-grown transcriptionally quiescent oocytes were cultured for indicated periods of time and selected miRNAs were quantified by RT-qPCR in four (ssc-let-7a), five (ssc-miR-22) or six (ssc-mir10b and ssc-miR-205) independent experiments. Error bars = SD.(B) Comparison of mean relative changes of miRNA level at 20 h of culture ssc-miR-205 in oocytes is less than ssc-miR-10b, ssc-miR-22, or ssc-let-7a. Error bars = SD. Asterisks indicate statistical significance (p-value) of one-tailed paired t-test, (*p < 0.05, **p < 0.01).(C) Treatment by sodium periodate followed by qPCR does not support 2'-OH modification of either ssc-miR-205 or bta-miR-10b miRNA. Shown are relative levels of the sodium periodate-treated samples to non-treated samples, which were set to one. The experiment was performed three times. Error bars = SD. Efficiency of periodate treatment was confirmed using non-methylated and methylated miR-221 RNA oligonucleotides(D) 3′ tailing of small RNAs in murine, bovine, and porcine oocytes Shown are percentages of miRNAs tailed at 3′ end with mono- and oligonucleotides.


Figure 1 ( A ) In vivo fundusimage of an AAV - injected rd1mouse expressing ReaChR - mCitrine driven by the hSyn promoter ( AAV2 - hSyn : ReaChR - mCitrine ) ; transduced RGCs are visible as green puncta . ( B ) Live 2 - photon image showing that ReaChR - mCitrine expression is restricted to RGC - membranes and their dendritic processes . ( C ) Confocal image of a vertical retinal section expressing ReaChR - mCitrine . Endogenous mCitrine ( mCit ) fluorescence , is localized to RGCmembranes as well as RGCdendritic processes ( see also Fig EV1A ) ; blue : DAPI ( to visualize retinalnuclei ) ; inner nuclear layer ( INL ) ; inner plexiform layer ( IPL ) and ganglion cell layer ( GCL ) ; scale bars = 10 µm .

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

Figure 1(A) In vivo fundusimage of an AAV-injected rd1mouse expressing ReaChR-mCitrine driven by the hSyn promoter (AAV2-hSyn:ReaChR-mCitrine); transduced RGCs are visible as green puncta.(B) Live 2-photon image showing that ReaChR-mCitrine expression is restricted to RGC-membranes and their dendritic processes.(C) Confocal image of a vertical retinal section expressing ReaChR-mCitrine. Endogenous mCitrine (mCit) fluorescence, is localized to RGCmembranes as well as RGCdendritic processes (see also Fig EV1A); blue: DAPI (to visualize retinalnuclei); inner nuclear layer (INL); inner plexiform layer (IPL) and ganglion cell layer (GCL); scale bars = 10 µm.


Figure 2 ( A ) Light sensitivity : Photocurrents of a ReaChR - expressing RGC stimulated with varying light intensities at 590 nm ( ~ 1013 − 1018 photons cm - 2 s - 1 ) . ( B ) ReaChR - induced photocurrents as a function of light intensity ( 590 nm , n = 7 cells , mean photocurrent amplitude at 4 . 2 × 1017 photons cm - 2 s - 1 was − 349 ± 192 pA ) . At the bottom , the sensitivity range for wild - type cones ( Nikonov et al , 2006 ) is shown in comparison to the sensitivity range of ReaChR expressing RGCs . The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm , respectively ( EU - Directive , 2006 ; ICNIRP , 2013 ) . ( C ) Photocurrent action spectrum of a ReaChR - expressing RGC ( 1 . 2 × 1016 photons cm - 2 s - 1 ) . ( D ) Normalized action spectrums of ReaChR - RGCs . Solid line represents patch - clamp data ( n = 8 cells ; 1 . 2 × 1016 photons cm - 2 s - 1 ) . The mean photocurrent amplitude at 550 nm was − 324 ± 97 pA . Dashed line represents multi - electrode array data ( n = 168 cells ; ~ 1016 photons cm - 2 s - 1 ) . ( E ) Temporal properties . Modulation of ReaChR - induced photocurrents at increasing stimulation frequencies ( patch - clamp recording from a ReaChR - expressing RGC ; at 22 Hz magnified trace is shown ; 1 . 3 × 1016 photons cm - 2 s - 1 ) . ( F ) Spiking modulation ratio of ReaChR - RGCs quantified from multi - electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz ( 297 cells , 1 . 3 × 1016 photons cm - 2 s - 1 ) .

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

Figure 2(A) Light sensitivity: Photocurrents of a ReaChR-expressing RGC stimulated with varying light intensities at 590 nm (~1013 − 1018 photons cm-2 s-1).(B) ReaChR-induced photocurrents as a function of light intensity (590 nm, n = 7 cells, mean photocurrent amplitude at 4.2 × 1017 photons cm-2 s-1 was −349 ± 192 pA). At the bottom, the sensitivity range for wild-type cones (Nikonov et al, 2006) is shown in comparison to the sensitivity range of ReaChR expressing RGCs. The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm, respectively (EU-Directive, 2006; ICNIRP, 2013).(C) Photocurrent action spectrum of a ReaChR-expressing RGC (1.2 × 1016 photons cm-2 s-1). (D) Normalized action spectrums of ReaChR-RGCs. Solid line represents patch-clamp data (n = 8 cells; 1.2 × 1016 photons cm-2 s-1). The mean photocurrent amplitude at 550 nm was −324 ± 97 pA. Dashed line represents multi-electrode array data (n = 168 cells; ~1016 photons cm-2 s-1).(E) Temporal properties. Modulation of ReaChR-induced photocurrents at increasing stimulation frequencies (patch-clamp recording from a ReaChR-expressing RGC; at 22 Hz magnified trace is shown; 1.3×1016 photons cm-2 s-1). (F) Spiking modulation ratio of ReaChR-RGCs quantified from multi-electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz (297 cells, 1.3 × 1016 photons cm-2 s-1).


Figure 3 ( A ) Examples of local field potential ( LFP ) traces in response to light flashes at 595 nm ( light stimulus is shown as orange bar ; 1 . 25 × 1017 photons cm - 2 s - 1 ) . ( B ) Spike raster plots in response to 200 repetitions of the same light stimulus . ( C ) Averaged spike responses shown as peristimulus time histograms ( PSTH , bin size : 5 ms ) based on the spike raster plots . Top - row : WT - mice , middle row : rd1 control mice , bottom - row : ReaChR - treated rd1 mice . ( D ) ReaChR - induced cortical responses ( spikes and LFP ) as a function of light intensity at ( 595 nm ) demonstrating that ReaChR - treated rd1 mice ( n = 4 ) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina . Data presented as mean ± SEM .

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

Figure 3(A) Examples of local field potential (LFP) traces in response to light flashes at 595 nm (light stimulus is shown as orange bar; 1.25 × 1017 photons cm-2 s-1). (B) Spike raster plots in response to 200 repetitions of the same light stimulus. (C) Averaged spike responses shown as peristimulus time histograms (PSTH, bin size: 5 ms) based on the spike raster plots. Top-row: WT-mice, middle row: rd1 control mice, bottom-row: ReaChR-treated rd1 mice. (D) ReaChR-induced cortical responses (spikes and LFP) as a function of light intensity at (595 nm) demonstrating that ReaChR-treated rd1 mice (n = 4) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina. Data presented as mean ± SEM.


Figure 4 ( A ) Locomotory behavior of a representative rd1 and ReaChR - treated mouse during illumination with light at 590 nm in an open field arena ( irradiance measured at the level of the mice ' s eyes was ~ 1015 photons cm - 2 s - 1 , cumulative activity trajectories are shown as red traces after 2 min of illumination ) , see also EV Movie 1 and EV Movie 2 . ( B ) Mean velocities of the ReaChR - treated and rd1mice analyzed over a duration of 2 min at light onset . The mean velocity of ReaChR - treated mice were significantly reduced than rd1mice ( P = 0 . 006 , one - tailed t - test ) upon illumination . Mean ± SEM are shown for each group . ReaChR - treated rd1 ( n = 11 ) , rd1 ( n = 12 ) . ( C ) Light / dark box test . Percentage of time mice spent in the light compartment as a function of light intensity ( WT , n = 9 ; rd1 , n = 9 ; ReaChR - treated n = 7 ) . Mean ± SEM are shown for each group . ( D ) Percentage of time rd1 ( n = 9 ) , WT ( n = 9 ) and ReaChR - treated ( n = 7 ) mice spent in the light compartment for an illumination intensity of 2 . 11 × 1015 photons cm - 2 s - 1 . Both WT as well as ReaChR - treated mice spent significantly less time in the light compartment than the rd1mice ( P < 0 . 0001 , ordinary one - way ANOVA ) . * * * and * * * * indicates significance levels determined by Tukey ' s multiple comparisons test for rd1 vs . WT and rd1 vs . ReaChR - treated . See also EV Movie 3 for the response of a representative ReaChR - treated rd1 mouse plotted in ( D ) .

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 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

Figure 4(A) Locomotory behavior of a representative rd1 and ReaChR-treated mouse during illumination with light at 590 nm in an open field arena (irradiance measured at the level of the mice's eyes was ~1015 photons cm-2 s-1, cumulative activity trajectories are shown as red traces after 2 min of illumination), see also EV Movie 1 and EV Movie 2. (B) Mean velocities of the ReaChR-treated and rd1mice analyzed over a duration of 2 min at light onset. The mean velocity of ReaChR-treated mice were significantly reduced than rd1mice (P = 0.006, one-tailed t-test) upon illumination. Mean ± SEM are shown for each group. ReaChR-treated rd1 (n = 11), rd1 (n = 12).(C) Light/dark box test. Percentage of time mice spent in the light compartment as a function of light intensity (WT, n = 9; rd1, n = 9; ReaChR-treated n = 7). Mean ± SEM are shown for each group. (D) Percentage of time rd1 (n = 9), WT (n = 9) and ReaChR-treated (n = 7) mice spent in the light compartment for an illumination intensity of 2.11 × 1015 photons cm-2 s-1. Both WT as well as ReaChR-treated mice spent significantly less time in the light compartment than the rd1mice (P < 0.0001, ordinary one-way ANOVA). *** and **** indicates significance levels determined by Tukey's multiple comparisons test for rd1 vs. WT and rd1 vs. ReaChR-treated. See also EV Movie 3 for the response of a representative ReaChR-treated rd1 mouse plotted in (D).


Figure 5 ( A ) Vertical section of macaqueretinal explant transfected with AAV2 hSyn : ReaChR - mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL . ( B ) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F - CAG : ReaChR - GFP after 3 days of incubation . GFP expression was primarily observed in the cell bodies but also in processes of RGCs ; green : endogenous GFP fluorescence ; blue : DAPI ; outer nuclear layer ( ONL ) . scale bars = 10 µm . ( C ) Averaged spike responses obtained from multi - electrode array recordings shown as PSTH of 4 , 8 and 2 cells respectively of three different macaques ( 580 nm ; 1 . 24 × 1017 photons cm - 2 s - 1 ) . PSTHs are presented as mean ± SEM ( dashed lines , calculated over responding cells ) . Raster plots of a representative cell from each individual macaque are shown below ( 10 stimulus - repetitions ) . ( D ) Action spectrum of ReaChR in primate RGCs from multi - electrode array recordings . Data is shown as mean ± SEM of the normalized firing rate , averaged from three individual macaques ( 28 cells , error bars calculated over cells ) , see also Fig EV2B for action spectrums of three individual macaques obtained from multi - electrode array recordings . ( E ) Responses ( Hz ) of a ReaChR - expressing RGC ( patch clamp recording , in cell - attached mode ) to flicker stimulations at increasing frequencies ( 2 − 22 Hz ; 1 . 3 × 1016 photons cm - 2 s - 1 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Vertical section of macaqueretinal explant transfected with AAV2 hSyn:ReaChR-mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL. (B) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F-CAG:ReaChR-GFP after 3 days of incubation. GFP expression was primarily observed in the cell bodies but also in processes of RGCs; green: endogenous GFP fluorescence; blue: DAPI; outer nuclear layer (ONL). scale bars = 10 µm.(C) Averaged spike responses obtained from multi-electrode array recordings shown as PSTH of 4, 8 and 2 cells respectively of three different macaques (580 nm; 1.24 × 1017 photons cm-2 s-1). PSTHs are presented as mean ± SEM (dashed lines, calculated over responding cells). Raster plots of a representative cell from each individual macaque are shown below (10 stimulus-repetitions).(D) Action spectrum of ReaChR in primate RGCs from multi-electrode array recordings. Data is shown as mean ± SEM of the normalized firing rate, averaged from three individual macaques (28 cells, error bars calculated over cells), see also Fig EV2B for action spectrums of three individual macaques obtained from multi-electrode array recordings. (E) Responses (Hz) of a ReaChR-expressing RGC (patch clamp recording, in cell-attached mode) to flicker stimulations at increasing frequencies (2−22 Hz; 1.3 × 1016 photons cm-2 s-1).


Figure 6 ( B ) Brightfield image of the retinal explant showing the macula ( yellow pigmented ) with the approximate location of the multi - electrode array chip indicated . ( C ) Infrared image of the explant pressed against the multi - electrode array ; the black dashed line depicts the ' electrophysiological boundary ' of the para - fovea based on the absence of RGC - spike trains ( scale bar : 100 µm ; see also Fig EV3A and B ) . Voltage trace of a para - foveal RGC stimulated with orange light ( 600 nm ; 1 . 1 × 1017 photons cm - 2 s - 1 ) is shown on the right . Individual electrodes that recorded ReaChR - responding cells are indicated in green . ( D ) Action spectrum of ReaChR constructed from ( 6 cells , mean ± SEM , error bars calculated over cells ) exhibiting peak activity at 550 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) similar to what was observed in mice and in the macaqueretinae . ( E ) Peristimulus time histograms constructed for wavelengths 400 to 650 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) demonstrate ReaChR - evoked spike trains ( under L - AP4 ) in the humanretina ( 6 cells , mean ± SEM , error bars calculated over cells ) , bin size : 100 ms ; Bottom : Raster plots of the light responses of a representative spike - sorted cell .

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 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 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 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

Figure 6(B) Brightfield image of the retinal explant showing the macula (yellow pigmented) with the approximate location of the multi-electrode array chip indicated.(C) Infrared image of the explant pressed against the multi-electrode array; the black dashed line depicts the 'electrophysiological boundary' of the para-fovea based on the absence of RGC-spike trains (scale bar: 100 µm; see also Fig EV3A and B). Voltage trace of a para-foveal RGC stimulated with orange light (600 nm; 1.1 × 1017 photons cm-2 s-1) is shown on the right. Individual electrodes that recorded ReaChR-responding cells are indicated in green.(D) Action spectrum of ReaChR constructed from (6 cells, mean ± SEM, error bars calculated over cells) exhibiting peak activity at 550 nm (1.1 × 1017 photons cm-2 s-1) similar to what was observed in mice and in the macaqueretinae.(E) Peristimulus time histograms constructed for wavelengths 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate ReaChR-evoked spike trains (under L-AP4) in the humanretina (6 cells, mean ± SEM, error bars calculated over cells), bin size: 100 ms; Bottom: Raster plots of the light responses of a representative spike-sorted cell.


Figure 7 ( A ) Confocal image of the central retina , exhibiting endogenous ReaChR - mCitrine fluorescence ( mCit ) in ganglion cells , located within ~ 500 µm from the center of the foveal pit , a part of the humanretina where midget cell density is estimated to be ~ 95 % of total RGC population ( Dacey , 1993 ) ( arrowhead : soma ; arrow : dendritic arbor ) . The dashed line illustrates the beginning of the foveal slope leading towards the ' cone - only ' foveal pit . ( B ) Confocal image of the same explant at a higher retinal eccentricity ( 3 - 4 mm from the foveal center ) showing ReaChR - mCitrine fluorescence in RGCs with larger dendritic arbors . The endogenous mCitrine was not amplified . Scale bars : 10 µm . PR : Photoreceptors , GC : Ganglion cells , DAPI ( blue ) .

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

Figure 7(A) Confocal image of the central retina, exhibiting endogenous ReaChR-mCitrine fluorescence (mCit) in ganglion cells, located within ~500 µm from the center of the foveal pit, a part of the humanretina where midget cell density is estimated to be ~95% of total RGC population (Dacey, 1993) (arrowhead: soma; arrow: dendritic arbor). The dashed line illustrates the beginning of the foveal slope leading towards the 'cone-only' foveal pit. (B) Confocal image of the same explant at a higher retinal eccentricity (3-4 mm from the foveal center) showing ReaChR-mCitrine fluorescence in RGCs with larger dendritic arbors. The endogenous mCitrine was not amplified. Scale bars: 10 µm. PR: Photoreceptors, GC: Ganglion cells, DAPI (blue).


Figure 8 ( A ) Humanretinal explant , prepared from the far periphery , infected with an AAV8 Y447 733F - CAG : ReaChR - GFP , placed on a multi - electrode array chip ( left ) ; GFP epifluorescence of the same explant following 12 days of incubation is shown on the right . Scale bars : 100 µm . The location of two cells that responded to repeated 2 s full field stimulations of orange light ( 600 nm ) are highlighted . ( B ) The corresponding voltage traces show that the responses in the far peripheral humanretina are more transient , compared to the experiments from the human para - fovea ( see Fig 6C and E , Fig EV3B ) where only sustained light responses were evoked . The light response of cell ( 2 ) was restricted to a burst of action potentials fired within the initial ~ 300 ms in response to a 2 s full field 600 nm light stimulus . ( C ) Peristimulus time histograms constructed for wavelengths from 400 to 650 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) demonstrate AAV - ReaChR - evoked spike trains ( mean ± SEM , error bars calculated over 3 - 4 cells , bin size : 100 ms ) Bottom : Raster plots of the light responses of a representative spike - sorted cell . All recordings were performed under pharmacological blockade of photoreceptor input ( 50 µM L - AP4 ) .

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 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

Figure 8(A) Humanretinal explant, prepared from the far periphery, infected with an AAV8 Y447 733F-CAG :ReaChR-GFP, placed on a multi-electrode array chip (left); GFP epifluorescence of the same explant following 12 days of incubation is shown on the right. Scale bars: 100 µm. The location of two cells that responded to repeated 2 s full field stimulations of orange light (600 nm) are highlighted.(B) The corresponding voltage traces show that the responses in the far peripheral humanretina are more transient, compared to the experiments from the human para-fovea (see Fig 6C and E, Fig EV3B) where only sustained light responses were evoked. The light response of cell (2) was restricted to a burst of action potentials fired within the initial ~300 ms in response to a 2 s full field 600 nm light stimulus.(C) Peristimulus time histograms constructed for wavelengths from 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate AAV-ReaChR-evoked spike trains (mean ± SEM, error bars calculated over 3-4 cells, bin size: 100 ms) Bottom: Raster plots of the light responses of a representative spike-sorted cell. All recordings were performed under pharmacological blockade of photoreceptor input (50 µM L-AP4).


Figure 2 ( A ) ELISA titration curves ( OD at 450nm ) of Fab3 ( blue ; left panel ) and Fab71 ( blue ; right panel ) against mouse recPrP23 ‑ 231 compared to their best affinity - matured versions , Fab83 and Fab101 Curves were fitted by non - linear least - squares regression analysis for statistical analysis : log ( agonist ) vs . response . ( B - E ) FT - peptide competition ELISA to map the epitopes of the indicated Fabs . Peptides that strongly inhibit the binding of the Fabs to recPrP23 ‑ 231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes . Positive control : recPrP23 ‑ 231 ; negative controls : Neu , BSA , and the secondary antibody ( 2 ° Ab ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA titration curves (OD at 450nm) of Fab3 (blue; left panel) and Fab71 (blue; right panel) against mouse recPrP23‑231 compared to their best affinity-matured versions, Fab83 and Fab101 Curves were fitted by non-linear least-squares regression analysis for statistical analysis: log(agonist) vs. response.  (B-E) FT-peptide competition ELISA to map the epitopes of the indicated Fabs. Peptides that strongly inhibit the binding of the Fabs to recPrP23‑231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes. Positive control: recPrP23‑231; negative controls: Neu, BSA, and the secondary antibody (2° Ab).


Figure 3 ( A ) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 ( CC123 ‑ 50 ) , Fab71 ( OR51 ‑ 91 ) or Fab1 ( GD ) for 45 days . Scale bar : 500 μm . ( B ) Same as ( A ) , but in the presence of the affinity - maturated antibodies . ( C ) NeuN immunofluorescence coverage of prion - infected slices cultured in the presence of the Fabs . Antibodies targeting the CC123 ‑ 50 and the OR51 ‑ 91 afforded protection against neurodegeneration . NBH : non - infectious brain homogenate . Each dot represents a cerebellar slice for all treatment groups . ( D ) Western blot analysis of COCS lysates . Prion - infected slices treated with Fab100 , but not with Fab83 and Fab25 showed reduced levels of PrPSc . n = 3 biological replicates .

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

Figure 3 (A) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 (CC123‑50), Fab71 (OR51‑91) or Fab1 (GD) for 45 days. Scale bar: 500 μm. (B) Same as (A), but in the presence of the affinity-maturated antibodies. (C) NeuN immunofluorescence coverage of prion-infected slices cultured in the presence of the Fabs. Antibodies targeting the CC123‑50 and the OR51‑91 afforded protection against neurodegeneration. NBH: non-infectious brain homogenate. Each dot represents a cerebellar slice for all treatment groups.  (D) Western blot analysis of COCS lysates. Prion-infected slices treated with Fab100, but not with Fab83 and Fab25 showed reduced levels of PrPSc. n = 3 biological replicates.


Figure 4 ( C ) Left : Heat map showing the binding specificities ( Z - scores ) of selected Fab71 human analogous antibodies to human recPrP23 ‑ 230 compared to the negative controls ( BSA and Neu ) . Right : Heat map representing the reactivity ( - logEC50 ) obtained from dose - dependent ELISA binding curves of the analogous antibodies to human recPrP23 ‑ 230 . Red : low reactivity ; blue : high reactivity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity.


Figure 5 ( C ) Boxplot with half - violin plot displaying the distribution of autoantibody reactivity in 48 , 718 plasma samples . Twenty - seven samples over the reactivity threshold of - log ( EC50 ) ≥ 2 and fitting error < 20 % were considered as hits in the primary screen . Black dots correspond to single samples . The boxplot divides the data set into three quartiles . The box extends from the first to the third quartile values of the data set , with a line at the median . Whiskers show the range of the data ( minimum and maximum ) . ( E ) Heat map representation for the validation of the hits from the primary screen ( 21 out of 27 hits ) . Confirmed hits displayed selective reactivity against human recPrP23 ‑ 230 . Controls : Patients # 11 - 13 , ApoE4 and BSA . Red : high reactivity ; blue : low reactivity . ( F ) Heat maps representing autoantibody clonality of hits analyzed by ELISA . Autoantibodies were mostly constituted by lambda light chains ( λ ) , while the kappa light chains ( κ ) showed prevalence in the total IgG fraction . ( G ) Determination of antibody reactivity ( - log ( EC50 ) ) for three individuals at two different time points . Individuals were selected by displaying strong anti - PrP IgG reactivity in the first test ( Time 1 ) . Reactivity was maintained for even more than one year ( Time 2 ) . ( H ) Purified IgGs from patients with strong anti - PrP reactivity were tested against PrP variants and BSA ( negative control ) in dose - dependent ELISAs . Patient # 5 and # 6 showed binding mostly to the human recPrP121 ‑ 230 . Patient # 1 displayed a polyreactive pattern with binding to the human recPrP121 ‑ 230 and mouse recPrP23 - 110 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C) Boxplot with half-violin plot displaying the distribution of autoantibody reactivity in 48,718 plasma samples. Twenty-seven samples over the reactivity threshold of -log(EC50) ≥ 2 and fitting error < 20% were considered as hits in the primary screen. Black dots correspond to single samples. The boxplot divides the data set into three quartiles. The box extends from the first to the third quartile values of the data set, with a line at the median. Whiskers show the range of the data (minimum and maximum).  (E) Heat map representation for the validation of the hits from the primary screen (21 out of 27 hits). Confirmed hits displayed selective reactivity against human recPrP23‑230. Controls: Patients #11-13, ApoE4 and BSA. Red: high reactivity; blue: low reactivity.  (F) Heat maps representing autoantibody clonality of hits analyzed by ELISA. Autoantibodies were mostly constituted by lambda light chains (λ), while the kappa light chains (κ) showed prevalence in the total IgG fraction.  (G) Determination of antibody reactivity (-log(EC50)) for three individuals at two different time points. Individuals were selected by displaying strong anti-PrP IgG reactivity in the first test (Time 1). Reactivity was maintained for even more than one year (Time 2).  (H) Purified IgGs from patients with strong anti-PrP reactivity were tested against PrP variants and BSA (negative control) in dose-dependent ELISAs. Patient #5 and #6 showed binding mostly to the human recPrP121‑230. Patient #1 displayed a polyreactive pattern with binding to the human recPrP121‑230 and mouse recPrP23-110.


Figure 1RT - qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF - β for 16h normalized to MoDC . Data represent mean values ± SD of triplicates from two donors . Subcellular localization analysis of S100A9 and S100A8 in MoLC , MoDC and MoDC - TGF - β . S100A9 mean fluorescence intensities ( MFI ) were normalized to MoLC and represented in the upper graph . The same was done for S100A9 cytoplasmic / nuclear MFI and shown in the lower graph . Scale bar : 5µm . Graphs show normalized percentages ± SD of data from four different donors for MoLC ( n = 4 ) and MoDC ( n = 4 ) and three different donors for TGF - β - treated MoDC ( n = 3 ) . When applicable , student t - test was performed to determine statistical significance * * 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 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 1RT-qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF-β for 16h normalized to MoDC. Data represent mean values ± SD of triplicates from two donors.Subcellular localization analysis of S100A9 and S100A8 in MoLC, MoDC and MoDC-TGF-β. S100A9 mean fluorescence intensities (MFI) were normalized to MoLC and represented in the upper graph. The same was done for S100A9 cytoplasmic/nuclear MFI and shown in the lower graph. Scale bar: 5µm. Graphs show normalized percentages ± SD of data from four different donors for MoLC (n=4) and MoDC (n=4) and three different donors for TGF-β-treated MoDC (n=3). When applicable, student t-test was performed to determine statistical significance **P<0.01).


Figure 2MoLC from six different donors ( n = 6 ) were left untreated ( NT ) or incubated with LPS for 48h and analysed for S100A9 expression by RT - qPCR ( left graph ) . MoLC treated as before were then transduced with HIV - GFP vectors for 48h and GFP + cells were analysed by flow cytometry and data were graphically represented as fold relative to non - treated MoLC ( right graph ) . Black horizontal line correspond to the mean . When applicable , student t - test was performed to determine statistical significance ( * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).


Figure 3Cells transfected as above were pre - treated or not with nevirapine and transduced with HIV - GFP vectors for 48h . The percentage of GFP + MoLC was analysed by flow cytometry . Data represent means values ± SD from two different donors .

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

Figure 3Cells transfected as above were pre-treated or not with nevirapine and transduced with HIV-GFP vectors for 48h. The percentage of GFP+ MoLC was analysed by flow cytometry. Data represent means values ± SD from two different donors.


Figure 4MoLC , dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis . Scale bar : 5µm . Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV - Luc . When indicated cells were pre - treated with 10μM Nevirapine for 30 min before viral challenge . Luciferase activities measured in duplicates for each condition from two ( infection with 20 ng P24 ) or three ( infection with 200 ng P24 ) different skin samples were represented on a graph as means of relative luminescence units ( RLU ) ± SD .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4MoLC, dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis. Scale bar: 5µm.Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.


Figure 5The experiment shown is representative of four donors for HIV - 1 - R5 ( n = 4 ) and three donors for HIV - 1 primary isolates ( n = 3 ) . Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV - 1 - R5 and HIV - 1 primary isolates . Nevirapine ( Nev ) ( 10μM ) was used as control when indicated and data were represented as means ± SD of HIV - Gag + cells from at least 2 ( for siS100A9 - MoLC infected by HIV - 1 primary isolates ) to 3 donors . When applicable , student t - test was performed to determine statistical significance ( * P < 0 . 1 ; * * P < 0 . 01 ; * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV-1-R5 and HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).


Figure 6E2C - and S100A9 - expressing HEK293T cells were infected with the indicated MOI of HIV - GFP for 48h and GFP + cells were analysed by flow cytometry . Mean values ± SD from four independent experiments ( n = 4 ) were normalized to the E2C - HEK293T control and represented in graph .

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

Figure 6E2C- and S100A9-expressing HEK293T cells were infected with the indicated MOI of HIV-GFP for 48h and GFP+ cells were analysed by flow cytometry. Mean values ± SD from four independent experiments (n=4) were normalized to the E2C-HEK293T control and represented in graph.


Figure 7The percentage of HIV - Gag p24 + cells from four independent experiments for CEM ( n = 4 ) and two for Jurkat ( n = 2 ) analysed by flow cytometry were graphically represented as means of HIV - Gag + cells ± SD . When applicable , student t - test was performed to determine statistical significance ( * * P < 0 . 01 ; * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7The percentage of HIV-Gag p24+ cells from four independent experiments for CEM (n=4) and two for Jurkat (n=2) analysed by flow cytometry were graphically represented as means of HIV-Gag+ cells ± SD. When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).


Figure 8E2C - and S100A9 - expressing CHO cells were challenged with indicated MOI of MMLV - YFP vectors for 24h or 48h . The percentages of YFP + CHO infected cells analysed by flow cytometry and data were represented on graphs .

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

Figure 8E2C- and S100A9-expressing CHO cells were challenged with indicated MOI of MMLV-YFP vectors for 24h or 48h. The percentages of YFP+ CHO infected cells analysed by flow cytometry and data were represented on graphs.


Figure 9E2C - or S100A9 - expressing THP1 were left uninfected ( / ) or infected for 3 h with the indicated p24 amounts of HIV - 1 NL4 - 3 / Nef - IRES - Renilla viruses containing BlaM - VPR . Cells were processed for Blam - Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry . The mock condition corresponds to unstained E2C - expressing cells . Dot - plots from a representative experiment are shown .

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

Figure 9E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown.


Figure 10Schematic description of fate - of - capsid assay is displayed above . HEK293T expressing E2C or S100A9 were transduced with HIV - Luc ( 10ng p24 ) for 4 h . Cells were then subjected to the fate of capsid assay ( see methods ) in order to pull - down intact cores ( CA cores ) ( recovered in the pellet ) . Equal volumes from the soluble ( top fraction ) and pelleted ( down fraction ) materials were analyzed by western blotting in order to evaluate the presence of p24 ( CA ) , CypA , S100A9 and E2C proteins .

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

Figure 10Schematic description of fate-of-capsid assay is displayed above. HEK293T expressing E2C or S100A9 were transduced with HIV-Luc (10ng p24) for 4 h. Cells were then subjected to the fate of capsid assay (see methods) in order to pull-down intact cores (CA cores) (recovered in the pellet). Equal volumes from the soluble (top fraction) and pelleted (down fraction) materials were analyzed by western blotting in order to evaluate the presence of p24 (CA), CypA, S100A9 and E2C proteins.


Figure 11Colorimetric HIV - 1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations . Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C - containing conditions and shown on graphs .

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

Figure 11Colorimetric HIV-1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations. Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C-containing conditions and shown on graphs.


Figure 3TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast . Growth of yap1Δahp1Δ cells carrying empty vector ( ev ) , wild type peroxiredoxin gene ( AHP1 ) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R . In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 and Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R .

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

Figure 3TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast. Growth of yap1Δahp1Δ cells carrying empty vector (ev), wild type peroxiredoxin gene (AHP1) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.


Figure 7TBH cytotoxicity assay in vivo . Basic growth and the synthetic effect in wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH . H2S production using Biggy agar plates - Wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , and cys4Δ urm1Δ strains were grown on Biggy medium , and H2S production was recordedIn vivo Ahp1 conjugation analyses with protein extracts obtained from the indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 and Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control . Northern - blot analysis for thiolation levels of tRNA < sub > UUC < / sub > < sup > Glu < / sup > in the genetic background of the indicated yeast strains . Total tRNA was resolved on denaturing PAGE supplemented with APM to retard the migration of thiolated tRNAUUCGlu . APM : ( [ N - Acryloyl - amino ] phenyl ) mercuric chloride . ( bottom ) Quantified thiolation levels from 3 independent biological replicates .

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

Figure 7TBH cytotoxicity assay in vivo. Basic growth and the synthetic effect in wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH.H2S production using Biggy agar plates- Wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains were grown on Biggy medium, and H2S production was recordedIn vivo Ahp1 conjugation analyses with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control.Northern-blot analysis for thiolation levels of tRNAUUCGlu in the genetic background of the indicated yeast strains. Total tRNA was resolved on denaturing PAGE supplemented with APM to retard the migration of thiolated tRNAUUCGlu. APM: ([N-Acryloyl-amino] phenyl) mercuric chloride. (bottom) Quantified thiolation levels from 3 independent biological replicates.


Figure 1Ferrozine - based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points . * P < 0 . 05 ( unpaired Student ' s t - test versus each time points with FeSO4 100 µM ) . Representative confocal microscope images of L6 cells stained with IP - 1 ( Iron Probe 1 ) or transfected with IRE - CFP ( Iron Regulatory Element ) reporter after iron treatment ( FeSO4 , 250 µM ) for 24 h . Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment ( 250 µM , 24 h ) with iron chelator DPD ( 500 µM ) . Relative gene expressions - ferritin heavy chain ( FTH ) , ferritin light chain ( FTL ) , ferroportin ( SLC40A1 , transferrin receptor 1 ( tfr1 , TFRC ) - normalized to 18sr RNA expression after iron treatment ( FeSO4 , 250 µM ) for 24 h . * P < 0 . 05 ( unpaired Student ' s t - test versus Basal ) . Representative western blot images and quantification of ferritin ( heavy chain ) and tfr1 ( indicated with arrowhead ) over GAPDH after iron treatment ( 250 µM , 24 h ) . Glucose uptake of L6 cells with insulin stimulation ( 10 nM or 100 nM , 20 min ) after iron treatment ( 250 µM , 1 h or 24 h ) . Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) over GAPDH with insulin stimulation ( 10 nM or 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) .

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 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 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 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 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

Figure 1Ferrozine-based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points. *P < 0.05 (unpaired Student's t-test versus each time points with FeSO4 100 µM).Representative confocal microscope images of L6 cells stained with IP-1 (Iron Probe 1) or transfected with IRE-CFP (Iron Regulatory Element) reporter after iron treatment (FeSO4, 250 µM) for 24 h.Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment (250 µM, 24 h) with iron chelator DPD (500 µM).Relative gene expressions - ferritin heavy chain (FTH), ferritin light chain (FTL), ferroportin (SLC40A1, transferrin receptor 1 (tfr1, TFRC) - normalized to 18sr RNA expression after iron treatment (FeSO4, 250 µM) for 24 h. *P < 0.05 (unpaired Student's t-test versus Basal).Representative western blot images and quantification of ferritin (heavy chain) and tfr1 (indicated with arrowhead) over GAPDH after iron treatment (250 µM, 24 h).Glucose uptake of L6 cells with insulin stimulation (10 nM or 100 nM, 20 min) after iron treatment (250 µM, 1 h or 24 h).Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) over GAPDH with insulin stimulation (10 nM or 100 nM, 5 min) after iron treatment (250 µM, 24 h).


Figure 2Representative confocal microscope image of L6 cells stably expressing tandem fluorescent - eGFP - mCherry LC3B treated with iron ( FeSO4 , 250 µM ) for the indicated time points ( 4 h , 8 h , 24 h ) . Quantification of mean LC3B puncta per cell from ( A ) . Experiments were repeated three times , and one representative experiment is presented here . Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment ( FeSO4 , 250 µM ) at multiple time points . Representative western blot images and quantification of LC3B - II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine ( CQ , 30 µM ) . * Representative western blot images and quantification of LC3B - II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM . Representative confocal microscope z - stack image of mCherry - LC3B L6 cells after iron treatment ( 250 µM , 24 h ) . Quantification of LC3B puncta size from ( F ) . Experiments were performed three times , and one representative experiment is presented here . Quantification of autophagosomes motility from live cell microscopy ( Supplementary Movie 1 ) from mCherry - LC3B L6 cells under basal , iron ( 250 µM , 24 h ) and starvation ( amino acid free ) condition . Experiments were performed three times , and one representative experiment is presented here . * P < 0 . 05 ( unpaired Student ' s t - test compared to Iron 24h ) . Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment ( 250 µM , 24 h ) . Representative confocal microscope images of eGFP - mCherry - LC3B L6 cells with Lysotracker DeepRed after iron treatment ( 250 µM , 24 h ) .

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 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 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 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 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

Figure 2Representative confocal microscope image of L6 cells stably expressing tandem fluorescent-eGFP-mCherry LC3B treated with iron (FeSO4, 250 µM) for the indicated time points (4 h, 8 h, 24 h). Quantification of mean LC3B puncta per cell from (A). Experiments were repeated three times, and one representative experiment is presented here.Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment (FeSO4, 250 µM) at multiple time points.Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine (CQ, 30 µM). * Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM.Representative confocal microscope z-stack image of mCherry-LC3B L6 cells after iron treatment (250 µM, 24 h).Quantification of LC3B puncta size from (F). Experiments were performed three times, and one representative experiment is presented here.Quantification of autophagosomes motility from live cell microscopy (Supplementary Movie 1) from mCherry-LC3B L6 cells under basal, iron (250 µM, 24 h) and starvation (amino acid free) condition. Experiments were performed three times, and one representative experiment is presented here. *P < 0.05 (unpaired Student's t-test compared to Iron 24h).Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment (250 µM, 24 h).Representative confocal microscope images of eGFP-mCherry-LC3B L6 cells with Lysotracker DeepRed after iron treatment (250 µM, 24 h).


Figure 3Representative western blot images of phospho - ULK1 ( S757 ) , phospho - S6Kp70 ( T389 ) , total S6K , GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points . Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points . Representative epi - immunofluorescent images of L6 cells transfected with myc - RHEB Q46L and immuno - stained against LC3B and myc after iron treatment ( FeSO4 , 250 µM ) for 24 h . Ferrozine - based colorimetric measurement of intracellular iron in wild - type ( wt ) L6 and RHEB - Q64L L6 cells after iron treatment ( 50 µM or 250 µM ) for 24 h . Representative confocal images of wt L6 and RHEB - Q64L L6 cells pulsed with Lysotracker Deep Red and immuno - stained against LC3B after iron treatment ( 250 µM , 24 h ) . Quantification of LC3B - free lysosome numbers in ( E ) . Experiments were repeated three times , and one representative experiment is presented here . * P < 0 . 05 ( multiple unpaired Student ' s t - test versus Basal in wt and RHEB Q64L cells ) Representative TEM images of wt L6 and RHEB - Q64L cells after iron treatment ( 250 µM , 24 h ) . Representative western blot images and quantifications of phospho - IRS1 ( Y612 , indicated by arrowhead ) and AKT ( T308 , indicated by arrowhead ) to GAPDH in wt and RHEB - Q64L L6 cells stimulated with insulin ( 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) . * P < 0 . 05 ( multiple unpaired Student ' s t - test versus Basal in wt and RHEB Q64L cells )

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

Figure 3Representative western blot images of phospho-ULK1 (S757), phospho-S6Kp70 (T389), total S6K, GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points.Representative epi-immunofluorescent images of L6 cells transfected with myc-RHEB Q46L and immuno-stained against LC3B and myc after iron treatment (FeSO4, 250 µM) for 24 h.Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h.Representative confocal images of wt L6 and RHEB-Q64L L6 cells pulsed with Lysotracker Deep Red and immuno-stained against LC3B after iron treatment (250 µM, 24 h).Quantification of LC3B-free lysosome numbers in (E). Experiments were repeated three times, and one representative experiment is presented here. *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)Representative TEM images of wt L6 and RHEB-Q64L cells after iron treatment (250 µM, 24 h).Representative western blot images and quantifications of phospho-IRS1 (Y612, indicated by arrowhead) and AKT (T308, indicated by arrowhead) to GAPDH in wt and RHEB-Q64L L6 cells stimulated with insulin (100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)


Figure 4Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment ( 250 µM , 24 h ) followed by 24 h iron withdrawal . Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal . Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) to GAPDH in L6 cells iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal . Representative epi - immunofluorescent microscope images of L6 cells immuno - stained against LC3B and LAMP1 after iron treatment ( 250 µM , 24 h ) followed by 3 h withdrawal . Quantification of autophagosome - free lysosomes in ( E ) . Experiments were performed three times , and one representative experiment is presented here . Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h or 24 h .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 4Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment (250 µM, 24 h) followed by 24 h iron withdrawal. Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment (250 µM, 24 h) followed by 24 h withdrawal.Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in L6 cells iron treatment (250 µM, 24 h) followed by 24 h withdrawal.Representative epi-immunofluorescent microscope images of L6 cells immuno-stained against LC3B and LAMP1 after iron treatment (250 µM, 24 h) followed by 3 h withdrawal.Quantification of autophagosome-free lysosomes in (E). Experiments were performed three times, and one representative experiment is presented here.Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h or 24 h.


Figure 5Representative western blot images and quantification of phospho - mTOR S2448 , total mTOR and actin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h . Representative western blot images of phospho - mTOR S2448 , total mTOR phsophpo - TSC2 T1462 , total TSC2 , phospho - AKT S473 , total AKT , actin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without MK2206 ( AKT inhibitor . ) Representative western blot images of phospho - UVRAG S550 and total UVRAG in L6 cells transfected with FLAG - UVRAG and FLAG pulldown after iron treatment ( 250 µM , 4 h and 24 h ) followed by withdrawal for 1 h and 3 h . Representative western blot images of endogenous phospho - UVRAG S550 , total UVRAG expression in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without torin1 ( 200 nM ) . Live cell imaging analysis of L6 cells transfected with LAMP1 - RFP from Movie EV2 : lysosomal number in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) . Live cell imaging analysis of L6 cells transfected with LAMP1 - RFP from Movie EV2 : speed ( F ) in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) . Representative confocal images of time - lapse captures of lysosomes at 6 sec intervals for 30 sec . White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds , and the traces of puncta were shown as lines in final panel .

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

Figure 5Representative western blot images and quantification of phospho-mTOR S2448, total mTOR and actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h.Representative western blot images of phospho-mTOR S2448, total mTOR phsophpo-TSC2 T1462, total TSC2, phospho-AKT S473, total AKT, actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without MK2206 (AKT inhibitor.)Representative western blot images of phospho-UVRAG S550 and total UVRAG in L6 cells transfected with FLAG-UVRAG and FLAG pulldown after iron treatment (250 µM, 4 h and 24 h) followed by withdrawal for 1 h and 3 h.Representative western blot images of endogenous phospho-UVRAG S550, total UVRAG expression in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without torin1 (200 nM).Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: lysosomal number in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: speed (F) in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).Representative confocal images of time-lapse captures of lysosomes at 6 sec intervals for 30 sec. White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds, and the traces of puncta were shown as lines in final panel.