Week 15 WSM COs stained for astrocytic markers GFAP S100β . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . of damage foci , marked by γH2AX ( left ) 53BP1 ( right ) , in nuclei of in VZs of week 6 WSM COs . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM F1 : n = 4 from 1 batch , WSM M2 : n = 4 from 1 batch , WSM S2 : n = 4 from 1 batch , WSM S5 : n = 4 from 1 batch , WSM S5 W - AAV3 : n = 4 from 1 batch , WSM S5 W - AAV6 : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

Week 15 WSM COs stained for astrocytic markers GFAP and S100β. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of DNA damage foci, marked by γH2AX (left) and 53BP1 (right), in the nuclei of the cells in the VZs of week 6 WSM COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=4 from 1 batch, WSM M2: n=4 from 1 batch, WSM S2: n=4 from 1 batch, WSM S5: n=4 from 1 batch, WSM S5 W-AAV3: n=4 from 1 batch, WSM S5 W-AAV6: n=4 from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


qPCR for selected Wnt target genes in week 10 WSM COs . Y - axis indicates relative expression fold change . Data are represented as mean ± . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM P : n = 6 from 1 batch , WSM S : n = 4 from 1 batch , WSM S W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analysis for selected Wnt target genes in week 10 WSM COs. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM P: n=6 from 1 batch, WSM S: n=4 from 1 batch, WSM S W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 15 WSM COs stained for cortical layers ' markers CTIP2 SATB2 . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . of presented in E . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . E

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

Week 15 WSM COs stained for the cortical layers' markers CTIP2 and SATB2. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the staining presented in E. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. E


C Calculated BRET ratios for indicated pairs . positive control PA - mCit - NL interacting fusion NL - BCL2L1 PA - mCit - BAD show high BRET ratios .

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

C Calculated BRET ratios for the indicated protein pairs. The positive control protein PA-mCit-NL and the interacting fusion proteins NL-BCL2L1 and PA-mCit-BAD show high BRET ratios.


D Calculated LuC ratios for indicated pairs . positive control PA - NL PA - mCit - NL interacting NL - BCL2L1 PA - mCit - BAD show high LuC ratios .

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

D Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA-NL and PA-mCit-NL and the interacting proteins NL-BCL2L1 and PA-mCit-BAD show high LuC ratios.


Reproducibility of LuTHy PPI mapping experiments with positive negative reference sets . scatter plots show mean cBRET ( A ) ratios from two independent experiments ( Exp 1 Exp 2 ) , calculated two - Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

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

Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cBRET (A) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


Reproducibility of LuTHy PPI mapping experiments with positive negative reference sets . scatter plots show mean cLuC ( B ) ratios from two independent experiments ( Exp 1 Exp 2 ) , calculated two - Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cLuC (B) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


C Receiver operating characteristic ( ROC ) of cBRET ( AUC = 0 . 80 ± 0 . 04 ) cLuC ( AUC = 0 . 72 ± 0 . 04 ) data determines thresholds to define true positive binary interactions .

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

C Receiver operating characteristic (ROC) analysis of cBRET (AUC=0.80±0.04) and cLuC (AUC=0.72±0.04) data determines the thresholds to define true positive binary interactions.


D Performance of BRET LuC assays in benchmarking studies with hPRS hRRS .

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D Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS.


C Recovery rates of PPIs from affinity - sub - groups with BRET LuC assays . Significance was calculated by two - sided Fisher ' s exact ; * p < 0 . 05 . D Success of PPI detection from AIRS sub - groups considering single double LuTHy readouts .

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

C Recovery rates of PPIs from affinity-based sub-groups with BRET and LuC assays. Significance was calculated by two-sided Fisher's exact test; *p<0.05. D Success of PPI detection from AIRS sub-groups considering single and double LuTHy readouts.


E Scatter plot depicting relationships between published binding affinities ( KD ) in - BRET50 measurements for 25 interactions in AIRS ; BRET50 values are mean from two independent experiments . Significance was calculated by two - Spearman correlation ; * * * p < 0 . 001 .

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

E Scatter plot depicting the relationships between published binding affinities (KD) and in-cell BRET50 measurements for 25 interactions in AIRS; BRET50 values are the mean from two independent experiments. Significance was calculated by two-tailed Spearman correlation; ***p<0.001.


A Live - bioluminescence imaging ( BLI ) of HEK293 co - producing indicated fusion . mCitrine was excited at 500 nm , emitted was detected at 535 nm . After substrate addition , short ( 460 ) long ( 535 ) band - pass ( BP ) filters in a dual - view adapter were used to detect emitted luminescence simultaneously at respective wavelengths . BRET were calculated by dividing 535 BP by 460 BP using ImageJ . Scale bar = 20 μm .

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

A Live-cell bioluminescence imaging (BLI) of HEK293 cells co-producing the indicated fusion proteins. mCitrine was excited at 500 nm, emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band-pass (BP) filters in a dual-view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm.


B of rapamycin - induced interaction between NL - FKBP12 PA - mCit - FRB . Plasmids encoding fusion were co - transfected in HEK293 24 h later treated with indicated concentrations of rapamycin . After an additional incubation for 4 h BRET was quantified upon substrate addition . After lysis in buffer with indicated rapamycin concentrations , LuC ratios were determined . EC50 values were obtained with non - linear curve fitting .

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

B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting.


Plasmids encoding fusion were co - transfected in HEK293 After an additional incubation for 4 h BRET was quantified upon substrate addition . LuC ratios were determined . EC50 values were obtained with non - linear curve fitting . C Effects of Nutlin - 3 on interaction between NL - MDM2 PA - mCit - p53 . for 6 h treatment with Nutlin - 3 .

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

Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition. LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. for 6 h treatment with Nutlin-3.


D Time resolved HSF1 oligomerization by Hsp90 inhibition . HEK293 co - producing NL - HSF1 PA - mCit - HSF1 for 24 h were transferred to 384 - well plates containing Ganetespib or Geldanamycin to reach a final of 1 µM . Luminescence was measured at indicated time points calculated BRET ratios normalized to respective untreated control . Data are mean of two biological replicates . values are mean ± . Significance was calculated by two - way ANOVA followed by Dunnett ' s multiple comparison ' s post - hoc .

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

D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test.


E Schematic depiction of a heat shock experiment . HEK293 co - producing NL - HSF1 / PA - mCit - HSF1 for 48h were subjected to a heat shock at 42 ° C for 6 h , after which were recovered at 37 ° C for 24h . Luminescence was measured at indicated time points : immediately before heat shock , during heat shock at 90 , 180 360 min after a recovery phase of 24 h . Calculated cBRET ratios were normalized to a control plate that was not heat treated . values are mean ± from three independent experiments . Significance was calculated by one - way ANOVA followed by Dunnett ' s multiple comparisons post - hoc ; * p < 0 . 05 .

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

E Schematic depiction of a heat shock experiment. HEK293 cells co-producing NL-HSF1/PA-mCit-HSF1 for 48h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180 and 360 min and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat treated. All values are mean ± sem from three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; *p<0.05.


B , C , D VCP UBX domain - containing were co - expressed as N - C - terminal NL PA - mCit fusion in eight different orientations for which cBRET ( B ) cLuC ( C ) ratios were generated . cBRET cLuC values over threshold of ≥ 0 . 01 ≥ 0 . 03 , respectively , are colored in cyan . D cBRET cLuC ratios were normalized to highest of obtained eight values to easily identify orientation among eight tested interactions with highest cBRET cLuC value .

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

B,C,D VCP and the UBX domain-containing proteins were co-expressed as N- and C-terminal NL and PA-mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥0.01 and ≥0.03, respectively, are colored in cyan. D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.


Tukey box plots of mean cBRET ( B ) ratios obtained from PPI experiments with wild - type CSPα - mCit - PA its mutant variants .

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

Tukey box plots of the mean cBRET (B) ratios obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


Tukey box plots of mean cLuC ratios ( C ) obtained from PPI experiments with wild - type CSPα - mCit - PA its mutant variants .

Tukey box plots of the mean cLuC ratios (C) obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


D Number of screened presynaptic manually annotated according to their function localization in presynaptic terminal . Number of identified with wild - type ( wt ) CSPα - mCit - PA or its mutant variants ( ΔL116 L115R ) are indicated .

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

D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal. Number of proteins identified with wild-type (wt) CSPα-mCit-PA or its mutant variants (ΔL116 and L115R) are indicated.


E Influence of mutations in CSPα ( ΔL116 , L115R ) on interactions with ZDHHC17 in hippocampal neurons . BRET LuC ratios were systematically quantified for interactions with wild - type mutant CSPα fusion .

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

E Influence of mutations in CSPα (ΔL116, L115R) on the interactions with ZDHHC17 in primary hippocampal neurons. BRET and LuC ratios were systematically quantified for interactions with wild-type and mutant CSPα fusion proteins.


F Immunoblot of SHEP transfected with plasmids encoding wild - type CSPα - mCit - PA mutant variants . Blot was developed with an anti - CSP antibody ; loading control : anti - Histone - H3 antibody . Arrowhead bracket indicate monomeric CSPα - mCit - PA SDS - insoluble , respectively .

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

F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.


G Immunofluorescence image of SHEP co - transfected with NL - ZDHHC17 CSPα - mCit - wt , - ΔL116 or - L115R constructs lacking PA - tag . NL - ZDHHC17 was stained with an anti NanoLuc antibody . Scale bar : 20 µm .

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

G Immunofluorescence image of SHEP cells co-transfected with NL-ZDHHC17 and CSPα-mCit-wt, -ΔL116 or -L115R constructs lacking the PA-tag. NL-ZDHHC17 was stained with an anti NanoLuc antibody. Scale bar: 20 µm.


A Flow cytometric of lymphocytes , monocytes , B - , natural killer ( NK ) , dendritic ( DCs ) granulocytes in peripheral venous blood samples from non - diabetic ( ND ) type - 2 diabetic ( T2D ) with COVID - 19 .

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O B-DISEASE I-DISEASE I-DISEASE O

A Flow cytometric quantification of lymphocytes, monocytes, B-cells, natural killer (NK) cells, dendritic cells (DCs) and granulocytes in peripheral venous blood samples from non-diabetic (ND) and type-2 diabetic (T2D) patients with COVID-19.


B of monocyte subpopulation as classical ( CD14Hi CD16 - ) , intermediate ( CD14Hi CD16 + ) or non - classical ( CD14Lo CD16 + ) .

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

B Quantification of monocyte subpopulation phenotypes as classical (CD14Hi CD16-), intermediate (CD14Hi CD16+) or non-classical (CD14Lo CD16+).


C Principle component of lymphocyte monocyte population frequencies in ND T2D COVID - 19 in T2D without COVID - 19 .

O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE O

C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID-19 patients and in T2D patients without COVID-19.


A of CD14 expression ( FSC ) in monocyte populations from non diabetic ( ND ) type - 2 diabetic ( T2D ) COVID - 19 .

O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE O

A Quantification of CD14 expression and size (FSC) in monocyte populations from non diabetic (ND) and type-2 diabetic (T2D) COVID-19 patients.


B Frequency of conventional ( FSCLo ) large ( FSCHi ) monocytes in ND T2D COVID - 19 .

O O O O O O O O O O O B-CELL_TYPE O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O

B Frequency of conventional (FSCLo) and large (FSCHi) monocytes in ND and T2D COVID-19 patients.


C Expression of CD16 , CD14 HLA - DR in FSCLo FSCHi monocytes from COVID - 19 .

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O B-DISEASE I-DISEASE I-DISEASE O

C Expression of CD16, CD14 and HLA-DR in FSCLo and FSCHi monocytes from COVID-19 patients.


D Proportions of classical , intermediate non - classical monocytes within FSCLo FSCHi monocytes from COVID - 19 .

O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O B-DISEASE I-DISEASE I-DISEASE O

D Proportions of classical, intermediate and non-classical monocytes within FSCLo and FSCHi monocytes from COVID-19 patients.


E Correlative analyses of IL8 expression in peripheral blood mononuclear ( PBMCs ) to monocyte FSC .

E Correlative analyses of IL8 expression in peripheral blood mononuclear cells (PBMCs) to monocyte FSC.


F IL8 , IL6 CCL2 mRNA expression in PBMCs from ND T2D COVID - 19 .

O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O

F IL8, IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID-19 patients.


G Monocyte quantified in ND T2D COVID - 19 treated in general wards ( Gen ) or in intensive care unit ( ICU ) .

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G Monocyte size quantified in ND and T2D COVID-19 patients treated in general wards (Gen) or in intensive care unit (ICU).


A of IRF5 IFNB1 mRNA expression in peripheral blood mononuclear of non - diabetic ( ND ) type 2 diabetic ( T2D ) COVID - 19 ( A ) .

O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE O O O O

A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non-diabetic (ND) and type 2 diabetic (T2D) COVID-19 patients (A).


B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples .

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

B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples.


C IRF5 median ( MFI ) in monocytes of ND T2D with COVID - 19 .

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C IRF5 median fluorescence intensity (MFI) in monocytes of ND and T2D patients with COVID-19.


D IRF5 expression overlaid onto monocyte phenotypic gating .

D IRF5 expression overlaid onto monocyte phenotypic gating.


E IRF5 expression ( MFI ) in monocyte subtypes from ND T2D COVID - 19 .

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E IRF5 expression (MFI) in monocyte subtypes from ND and T2D COVID-19 patients.


F HLA - DR IRF5 expression in monocytes from COVID - 19 .

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F HLA-DR and IRF5 expression in monocytes from COVID-19 patients.


G IRF5 expression in monocytes of ND or T2D COVID - 19 admitted to intensive care unit ( ICU ) or treated exclusively in general wards ( Gen ) .

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G IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted to intensive care unit (ICU) or treated exclusively in general wards (Gen) .


H IRF5 expression in monocytes of ND or T2D COVID - 19 admitted ICU or treated exclusively in Gen .

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H IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted ICU or treated exclusively in Gen.


B Proportions of lymphocytes monocytes in peripheral venous blood from T2D with COVID - 19 either treated in Gen or requiring ICU admission .

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B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID-19 either treated in Gen or requiring ICU admission.


C Phenotypic of lymphocyte monocyte subpopulations in peripheral venous blood from T2D COVID - 19 treated in Gen or requiring ICU treatment .

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C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID-19 patients treated in Gen or requiring ICU treatment.


a ) QF - FRL of unilamellar liposomes containing 95 mol % of phosphatidylcholine 5 mol % of a phosphoinositide , labelled with GST - PX . Only liposomes containing PtdIns ( 3 ) P were labelled intensely . GST - PXR58A , a PX - domain mutant that does not bind PtdIns ( 3 ) P , yielded virtually no labelling in PtdIns ( 3 ) P - containing liposomes . Scale bar , 0 . 2 μm .

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

a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX. Only liposomes containing PtdIns(3)P were labelled intensely. GST-PXR58A, a PX-domain mutant that does not bind PtdIns(3)P, yielded virtually no labelling in PtdIns(3)P-containing liposomes. Scale bar, 0.2 μm.


( b ) QF - FRL of unilamellar liposomes containing 0 - 5 mol % of PtdIns ( 3 ) P . Scale bar , 0 . 2 μm . labelling density increased roughly in proportion to PtdIns ( 3 ) P content of liposomes . experiment was repeated twice number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes . Values for concentrations are statistically different from those in adjacent conditions ( for example , 1 versus 2 mol % ; mean ± s . e . m . ; Student ' s t - ; P0 . 05 ) .

O O O O O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) QF-FRL of unilamellar liposomes containing 0-5 mol % of PtdIns(3)P. Scale bar, 0.2 μm. The labelling density increased roughly in proportion to the PtdIns(3)P content of the liposomes. The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes. Values for all concentrations are statistically different from those in the adjacent conditions (for example, 1 versus 2 mol%; mean±s.e.m.; Student's t-test; P0.05).


( b ) QF - FRL in wild - type yeast in log phase . PtdIns ( 3 ) P was labelled with GFP - PX marked with colloidal gold ( 10 nm ) . vacuolar PF was labelled intensely , whereas other organellar membranes did not exhibit significant labelling . Scale bar , 0 . 5 μm .

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(b) QF-FRL in wild-type yeast in log phase. PtdIns(3)P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm.


c ) of labelling of PtdIns ( 3 ) P ( mean ± s . e . m . ) . experiment was repeated three times number of colloidal gold particles per μm2 was measured in more than ten randomly selected .

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c) Quantification of the labelling intensity of PtdIns(3)P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( d , e ) Negative controls . Both wild - type yeast incubated with GST - PXR58A ( d ) vps34Δ yeast incubated with GST - PX ( e ) were virtually unlabelled . Scale bar , 0 . 5 μm .

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(d,e) Negative controls. Both wild-type yeast incubated with GST-PXR58A (d) and vps34Δ yeast incubated with GST-PX (e) were virtually unlabelled. Scale bar, 0.5 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in EF of autophagosomal membranes . A lower but still significant amount of labelling was also seen in PF of autophagosome membranes as well as in EF of vacuolar . Scale bar , 0 . 5 μm .

O O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O

(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagosomal membranes. A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane. Scale bar, 0.5 μm.


( b ) Autophagosomes in wild - type yeast cultured in S ( - NC ) for 4 h . autophagosome was identified as a smooth - surfaced double - structure , seen in two different patterns , as shown in diagrams in c . Upper panel : convex structures displaying outer EF inner PF . Lower panel : concave structures displaying outer PF inner EF . In both cases , PtdIns ( 3 ) P was labelled more intensely in EF than in PF . Scale bars , 0 . 2 μm .

O O O B-SUBCELLULAR O O O O B-ORGANISM O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(b) Autophagosomes in wild-type yeast cultured in S(-NC) for 4 h. The autophagosome was identified as a smooth-surfaced double-membrane structure, and seen in two different patterns, as shown in the diagrams in c. Upper panel: convex structures displaying the outer membrane EF and the inner membrane PF. Lower panel: concave structures displaying the outer membrane PF and the inner membrane EF. In both cases, PtdIns(3)P was labelled more intensely in the EF than in the PF. Scale bars, 0.2 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in EF of autophagic membranes in vacuolar lumen . A lower amount of labelling was also seen in PF of autophagic membranes . Scale bar , 0 . 5 μm . (

O O O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O

(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen. A lower amount of labelling was also seen in the PF of autophagic body membranes. Scale bar, 0.5 μm. (


( c ) Labelling of PtdIns ( 3 ) P in wild - type yeast cultured in S ( - NC ) for 4 h ( average ± s . e . m . ) . PtdIns ( 3 ) P was found in both leaflets of autophagosome autophagic , labelling was much stronger in EF than in PF . In vacuolar , significant labelling was found in EF , albeit at much lower than in PF . experiment was repeated three times , number of colloidal gold particles per μm2 was measured in more than ten randomly selected .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(c) Labelling intensity of PtdIns(3)P in wild-type yeast cultured in S(-NC) for 4 h (average±s.e.m.). PtdIns(3)P was found in both leaflets of the autophagosome and autophagic body membrane, and the labelling was much stronger in the EF than in the PF. In the vacuolar membrane, significant labelling was found in the EF, albeit at much lower levels than in the PF. The experiment was repeated three times, and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( a ) Huh7 expressing GFP - LC3 treated with 0 . 25 μM Torin1 0 . 4 μM bafilomycin A1 for 1 h . autophagosome was identified as IMP - poor double - structure labelled with anti - GFP antibody ( large gold , 10 nm ) . Both PtdIns ( 3 ) P ( small gold , 6 nm ) GFP - LC3 were seen exclusively in PF in both convex concave structures . small large gold particles are shown as green red dots , respectively , in figures at right . Scale bars , 0 . 1 μm . Figures displaying wider areas , as well as additional examples , are presented in Supplementary Fig . 4c . (

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

(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3)P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. (


( b ) NIH3T3 expressing Atg4C78A were incubated in Earle ' s balanced salt solution for 1 h . isolation was observed as parallel membranes with few IMPs . PtdIns ( 3 ) P was labelled only in PF . Scale bar , 0 . 1 μm . Other examples are presented in Supplementary Fig . 5 .

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

(b) NIH3T3 cells expressing Atg4C78A were incubated in Earle's balanced salt solution for 1 h. The isolation membrane was observed as parallel membranes with few IMPs. PtdIns(3)P was labelled only in the PF. Scale bar, 0.1 μm. Other examples are presented in Supplementary Fig. 5.


( c ) Labelling density of PtdIns ( 3 ) P in mammalian autophagosomal ( mean ± s . e . m . ) . experiment was repeated three times number of colloidal goldparticles per μm2 was measured in more than ten randomly selected . Some labelling was observed in EF but majority of gold particles in EF occurred near EF - PF boundary ( circles ; see Supplementary Fig . 6c for more examples ) , suggesting that many of them actually represent PtdIns ( 3 ) P in PF .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O

(c) Labelling density of PtdIns(3)P in mammalian autophagosomal membrane (mean±s.e.m.). The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images. Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF-PF boundary (white circles; see Supplementary Fig. 6c for more examples), suggesting that many of them actually represent PtdIns(3)P in the PF.


( a ) ymr1Δsjl3Δ cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled in comparable densities in PF EF of autophagosomal membranes . Scale bar , 0 . 2 μm .

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

(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3)P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm.


( b ) PF / EF of PtdIns ( 3 ) P labelling density in autophagosomal ( mean ± s . e . m . ) . was small ( that is , PF ) in WT , but close to 1 ( that is , PF ≅ EF ) in ymr1Δsjl3Δ . experiment was repeated three times number of colloidal goldparticles per μm2 was measured in more than ten randomly selected .

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

(b) The PF/EF ratio of the PtdIns(3)P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images.


( A - B ) Live third instar discs incubated in DiBAC . DiBAC is observed in wing imaginal disc in both apical ( A - A ' ) , basal ( B - B ' ) optical sections . Increased is observed in center of pouch ( arrow , B ' ) , at dorsoventral ( D - V ) compartment boundary in anterior compartment ( yellow arrow , B ' , inset , D ) .

O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE I-TISSUE O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D).


( C - E ) Comparison of DiBAC with voltage - insensitive dye FM4 - 64 . Incubation of live discs in FM4 - 64 ( E ) shows more uniform when compared to DiBAC ( D ) . Quantitative comparison of in two boxes in each panel is shown in ( C ) . N = 7 discs for each treatment , data compared using an unpaired t , * * indicates p < 0 . 001 , error bars are standard deviations . region boxed in yellow in ( D ) is shown at higher magnification to show DiBAC at D - V compartment boundary .

O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary.


( F , G ) Incubation in ouabain results in brighter more uniform DiBAC .

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(F, G) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence.


( H - H ' ' ) Live wing discs expressing UAS - RFP anterior to compartment boundary , under control of ptc - Gal4 , were incubated in DiBAC , showing that stripe of increased DiBAC coincides with posterior edge of ptc expression . arrowhead in ( H ) indicates stripe in wing pouch ; yellow arrowheads indicate stripe in dorsal ventral hinge .

O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE I-TISSUE O

(H-H'') Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge.


( I , I ' ) Early L3 discs incubated in DiBAC . Patterned depolarization is evident throughout third larval instar , with stripe of increased becoming narrower in more mature discs with developmental time ( Compare I with A , bracket in I indicates width of increased DiBAC ) .

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

(I, I') Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).


Expression of Rpk is increased anterior to A - P compartment boundary , in two rows of flanking D - V compartment boundary in A compartment .

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Expression of Rpk is increased anterior to the A-P compartment boundary, and in two rows of cells flanking the D-V compartment boundary in the A compartment.


( B - B ' ' ) Blockade of DEG / ENaC channels by incubation in amiloride abolishes stripe of increased DiBAC along A - P compartment boundary ( arrowhead ) . boxes indicate regions used to calculate average DiBAC across compartment boundary = 0 . 98 , standard deviation = 0 . 05 , n = 5 discs . Increased at D - V boundary in A compartment is still observable ( yellow arrowhead ) .

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(B-B'') Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).


( C , C ' ) Amiloride incubation does not diminish Rpk expression . arrowhead indicates approximate position of A - P compartment boundary , yellow arrowhead indicates approximate position of D - V compartment boundary .

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(C, C') Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary.


( D , E ) Expression of rpk RNAi using dpp - Gal4 results in diminished DiBAC along A - P compartment boundary ( arrowhead ) , while increased at D - V boundary in A compartment is still observable ( yellow arrowhead ) .

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(D, E) Expression of rpk RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).


( A - A ' ' ' ) Immunostaining of discs expressing ptc > RFP with antibodies to Rpk ATPα showing elevated of both anterior to compartment boundary . arrowheads indicate approximate position of A - P compartment boundary .

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(A-A''') Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary.


( B - C ' ) L3 discs that are - sensitive for hh following shift to restrictive for 12 h show loss of increased expression of Rpk ( B , B ' ) ATPα ( C , C ' ) anterior to compartment boundary . Controls shown in Figure EV2 . arrowheads indicate approximate position of A - P compartment boundary .

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(B-C') L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B') and ATPα (C, C') anterior to the compartment boundary. Controls shown in Figure EV2. White arrowheads indicate approximate position of the A-P compartment boundary.


( D - E ' ' ' ) Clones of expressing a constitutively activated form of transcription factor Ci show elevated Rpk ATPα expression in anterior compartment ( box ) as well as posterior compartment ( yellow arrowhead ) . ( E - E ' ' ' ) A single clone in A compartment is shown at higher magnification .

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(D-E''') Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E-E''') A single clone in the A compartment is shown at higher magnification.


( F - F ' ) A clone of in anterior compartment expressing ci3M following incubation in DiBAC .

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(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.


( G - G ' ) A clone of in anterior compartment expressing ptc RNAi following incubation in DiBAC . clones also express RFP .

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(G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.


( A - A ' ' ' ) Immunostaining of Smo ( red ) full - Ci ( blue ) in discs expressing en - Venus . Smo is expressed at higher in P compartment in a stripe 5 - 10 wide just anterior to A - P compartment boundary . arrowhead indicates just anterior to A - P compartment boundary .

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(A-A''') Immunostaining of Smo protein (red) and full-length Ci (light blue) in discs expressing en-Venus. Smo is expressed at higher levels in the P compartment and in a stripe 5-10 cells wide just anterior to the A-P compartment boundary. White arrowhead indicates cells just anterior to the A-P compartment boundary.


( B - C ) Effect of expressing an RNAi against ENaC channel rpk in dorsal compartment of disc using ap - Gal4 . Knockdown of Rpk in dorsal compartment results in decreased accumulation of Hh signal transducer Smo ( B , B ' ) , activator form of Ci ( B , B ' ' ) . expression of ptc , visualized using an anti - Ptc antibody ( C ) , a downstream target gene of Hh signaling , is also diminished . Rpk RNAi BL : 39053 is used in panels shown , but phenotype was validated with a second RNAi ( BL : 25847 ) .

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(B-C) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap-Gal4. Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo (B, B'), and the activator form of Ci (B, B''). The expression of ptc, visualized using an anti-Ptc antibody (C), a downstream target gene of Hh signaling, is also diminished. The Rpk RNAi line BL:39053 is used in all panels shown, but the phenotype was validated with a second RNAi line (BL:25847).


( A - C ) Localization of Smo ( blue ) following expression of 71B - Gal4 ( A ) , 71B - Gal4 UAS - NaChBac ( B ) or 71B - Gal4 UAS - Kir2 . 1 ( C ) . Expression of sodium channel NaChBac increases localization of Smo while expression of potassium channel Kir2 . 1 reduces localization , increases Smo ( B , C ) .

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(A-C) Localization of Smo protein (blue) following expression of 71B-Gal4 (A), 71B-Gal4 and UAS-NaChBac (B) or 71B-Gal4 and UAS-Kir2.1 (C). Expression of the sodium channel NaChBac increases membrane localization of Smo while expression of the potassium channel Kir2.1 reduces membrane localization, and increases cytosolic Smo (B, C).


( D ) of of Smo to cytosol . N = 7 salivary per genotype . Data were compared using ANOVA followed by Tukey for significance ( * * indicates p < 0 . 01 , * * * indicates p < 0 . 001 ) , error bars are standard deviations .

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(D) Quantification of the ratio of membrane Smo fluorescence to cytosol fluorescence. N=7 salivary glands per genotype. Data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01, *** indicates p<0.001), error bars are standard deviations.


( E , F ) Expression of Hh target gene ptc is visualized using anti - Ptc immunostaining . Compared to 71B - Gal4 ( E ) , Ptc expression is increased in expressing 71B - Gal4 UAS - NaChBac ( F ) .

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(E, F) Expression of the Hh target gene ptc is visualized using anti-Ptc immunostaining. Compared to 71B-Gal4 (E), Ptc expression is increased in glands expressing 71B-Gal4 and UAS-NaChBac (F).


( G - I ) Effect of expression activation of depolarizing channelrhodopsin ChR2 on Smo abundance . After dissection , were kept in either conditions ( G ) or exposed to 15 minutes of blue ( H ) . ( I ) of ; N = 13 , data compared using an unpaired t ( * * indicates p < 0 . 01 ) , error bars are standard deviations .

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(G-I) Effect of expression and activation of the depolarizing channelrhodopsin ChR2 on Smo protein membrane abundance. After dissection, glands were kept in either dark conditions (G) or exposed to 15 minutes of blue light (H). (I) Quantification of membrane fluorescence; N=13 glands, data compared using an unpaired t test (** indicates p<0.01), error bars are standard deviations.


( J - L ) Effect of expression activation of Cl - - selective channelrhodopsin ChloC . An overall reduction in Smo immunostaining was observed following channel activation ( K ) , as compared to control kept in ( J ) . ( L ) of ; N = 10 , data were compared using an unpaired t , ( * * * indicates p < 0 . 001 ) , error bars are standard deviations .

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(J-L) Effect of expression and activation of the Cl--selective channelrhodopsin ChloC. An overall reduction in Smo immunostaining was observed following channel activation (K), as compared to control glands kept in the dark (J). (L) Quantification of membrane fluorescence; N=10 glands, data were compared using an unpaired t test, (*** indicates p<0.001), error bars are standard deviations.


( M - Q ) Time course of change in Smo localization . Salivary expressing channelrhodopsin ChR2 were subjected to increasing intervals of activating , then fixed stained for Smo ( M - P ' ' ) . ( Q ) of - associated ; N = 7 / timepoint , data were compared using ANOVA followed by Tukey for significance ( * * indicates p < 0 . 01 ) , error bars are standard deviations .

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(M-Q) Time course of change in Smo localization. Salivary glands expressing the channelrhodopsin ChR2 were subjected to increasing intervals of activating light, then fixed and stained for Smo protein (M-P''). (Q) Quantification of membrane-associated fluorescence; N=7 glands/timepoint, data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01), error bars are standard deviations.


( A - B ' ' ) Wing discs expressing channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating in culture , then fixed stained for Smo . In ( B ' ' ) , yellow arrowhead indicates dorsoventral compartment boundary , arrowhead indicates anteroposterior compartment boundary . ( C ) of of mean Smo in 25µm2 regions in dorsal anterior compartment compared to ventral posterior compartment . N = 5 for 0min discs , n = 9 for 25min discs , data were compared using an unpaired t ( * * * p < 0 . 001 ) , error bars are standard deviations . Identical analyses were carried out comparing dorsal posterior to ventral posterior ( p = 0 . 132 ) , dorsal posterior to dorsal anterior ( p = 0 . 12 ) , ventral posterior to ventral anterior ( p = 0 . 36 ) .

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(A-B'') Wing discs expressing the channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating light in culture, then fixed and stained for Smo protein. In (B''), the yellow arrowhead indicates dorsoventral compartment boundary, and the white arrowhead indicates anteroposterior compartment boundary. (C) Quantification of the ratio of mean Smo fluorescence intensity in 25µm2 regions in the dorsal anterior compartment compared to the ventral posterior compartment. N=5 for 0min discs, n=9 for 25min discs, data were compared using an unpaired t test (***p<0.001), error bars are standard deviations. Identical analyses were carried out comparing dorsal posterior fluorescence to ventral posterior fluorescence (p=0.132), dorsal posterior fluorescence to dorsal anterior fluorescence (p=0.12), and ventral posterior fluorescence to ventral anterior fluorescence (p=0.36).


( D - E ' ) Discs expressing red - activated channelrhodopsin ReaChR under control of ap - Gal4 were raised on a 12hr / cycle , dissected stained for Smo ( D , D ' ) or Ptc ( E , E ' ) .

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(D-E') Discs expressing the red-light activated channelrhodopsin ReaChR under control of ap-Gal4 were raised on a 12hr light/dark cycle, dissected and stained for Smo protein (D, D') or Ptc protein (E, E').


ORFs for sense non - AUG - dependent translation of ( G4C2 ) n ~ 188 , DPR coding for ( GA ) , ( GP ) , ( GR ) can be detected quantified using a filter - trap binding probing for c - terminal HA tag of C9 DPR reporter construct . ( GA ) ORF is most abundantly detected DPR GR lowest in HEK293T 24 h post transient transfection of C9 DPR reporter construct . n = 5 .

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All ORFs for the sense non-AUG-dependent translation of the (G4C2)n~188, DPR coding for (GA), (GP), (GR) can be detected and quantified using a filter-trap binding assay probing for the c-terminal HA tag of the C9 DPR reporter construct. The (GA) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct. n = 5.


C9orf72 NRE non - AUG - dependent reporter constructs recapitulate localization patterns pathological features identified in C9orf72 NRE models . Dendra2 ( green ) c - terminal fusion was used to monitor localization for each DPR translated from G4C2 strand in HEK293T transiently cotransfected with an NES - mIFP - mIFP - NES ( NES - mIFP ) fusion marker .

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The C9orf72 NRE non-AUG-dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models. The Dendra2 (green) c-terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES-mIFP-mIFP-NES (NES-mIFP) fusion protein cytoplasmic marker.


C9 DPR reporter constructs show - type variability in number of that are DPR positive overall DPR . Transient cotransfections of HEK293T robustly undergo G4C2 - Dendra2 non - AUG - dependent translation compared to NSC34 or rat cortical neurons . DPR positive DPR were analyzed calculated 24 h post - transfection only in expressing co - transfected NES - mIFP construct . DPR was normalized to NES - mIFP to adjust for specific AUG - dependent translation ; represented as fold - change relative to HEK293T . HEK293T : n = 5 with m > 500 analyzed per n . NSC34 : n = 5 with m > 500 per n . Rat cortical neuron : n = 5 with m > 40 per n . ( * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 ) See also Figure EV1 - 2 .

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C9 DPR reporter constructs show cell-type variability in the number of cells that are DPR positive and the overall DPR levels. Transient cotransfections of HEK293T cells robustly undergo G4C2 -Dendra2 non-AUG-dependent translation compared to NSC34 or rat primary cortical neurons. DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post-transfection only in cells expressing the co-transfected NES-mIFP construct. DPR fluorescent intensity was normalized to NES-mIFP fluorescent intensity to adjust for cell specific AUG-dependent translation levels; represented as fold-change relative to HEK293T. HEK293T: n = 5 with m > 500 cells analyzed per n. NSC34: n = 5 with m > 500 cells per n. Rat primary cortical neuron: n = 5 with m > 40 cells per n. (***P < 0.001, **P < 0.01, *P <0.05) See also Figure EV1-2.


Quantitative microscopy imaging shows a dose - dependent increase in number of C9 DPR reporter positive with increasing concentrations of an apoptotic stimulator , staurosporine ( SSP ) . C9 DPR reporter in GP frame was utilized for these experiments . n = 4 .

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Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4.


Filter trap binding assays using DPR - specific antibodies show DPR are increased for sense - coding C9 DPR reporter when NSC34 are challenged with stress - inducing compounds . These stress - inducing compounds broadly cover distinct or overlapping pathways ( see Table EV2 ) Antibodies specific for C9orf72 NRE DPRs , poly - GA , - GP , or - GR , were used to quantify DPR normalized to GAPDH 24 h post transient transfection of C9 DPR reporter constructs . n = 4 .

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Filter trap binding assays using DPR-specific antibodies show all DPR levels are increased for the sense-coding C9 DPR reporter when NSC34 cells are challenged with stress-inducing compounds. These stress-inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2) Antibodies specific for the C9orf72 NRE DPRs, poly-GA, -GP, or -GR, were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs. n = 4.


Quantitative microscopy imaging shows that NSC34 , rat cortical neurons have increased relative DPR following compound induced stress . In NES - mIFP cotransfection positive NSC34 rat cortical neurons there is a significant increase in C9 DPR reporter 24 post treatment with most stress - inducing compounds . was not normalized to NES - mIFP . NSC34 : n = 10 with m > 500 analyzed per n . Cortical neuron : n = 6 with m > 40 per n . Endogenously expression of DPRs in iPS spinal motor neurons ( iPS sMN ) derived from C9orf72 NRE increases following induction of stress . DPR in iPS sMN was measured using DPR - specific antibodies described above . IPS sMN : n = 4 with m > 20 per n .

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Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES-mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress-inducing compounds. Fluorescent intensity was not normalized to NES-mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n. Cortical neuron: n = 6 with m > 40 cells per n. Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR-specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n.


microscopy shows Glutamate - induced ( Glut ) excitotoxic stress increases DPR in rat cortical neurons expressing C9 DPR reporter in iPS sMN derived from C9orf72 NRE . Glutamate - induced DPR are reduced by pretreating with AMPA NMDA specific antagonists NBQX MK801 , respectively . Relative demonstrates there is a significant increase in DPR when cortical neurons sMNs are challenged with excitotoxic stressors : glutamate , AMPA , NMDA , HC . Blocking either AMPA or NMDA receptors reduces DPR in presence of excitotoxic stressors . NES - mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs , respectively , for . DPR in iPS sMN was measured using DPR - specific antibodies . Changes in DPR are relative to non - stressed ( CTRL ) Cortical neuron : n = 6 with m > 40 per n . IPSC sMN : n = 4 with m > 25 per n .

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Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively. Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n.


Microscopy show increased DPR from medium high frequency stimulation of ChR2 in cortical neurons transfected with C9 DPR reporter .

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Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.


Relative shows a significant increase in DPR with increases in neuronal activity through medium high stimulation in cortical neurons . No stimulation low stimulation frequencies did not increase of DPR . C9 DPR reporter , monitoring GP DPR ( ORF2 ) , was cotransfected with ChR2 . Cortical neuron : n = 4 with m > 10 per n . ( * * * * P < 0 . 0001 ) .

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Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons. No stimulation and low stimulation frequencies did not increase levels of DPR. The C9 DPR reporter, monitoring the GP DPR (ORF2), was cotransfected with ChR2. Cortical neuron: n = 4 with m > 10 cells per n. (****P < 0.0001).


Filter trap binding assays show that ISR are enhanced by stressors . Significant increases following stress treatments are seen in of phospho - eif2α to total eif2α as well as phospho - PERK ATF4 compared to untreated controls . Changes in ISR are normalized to GAPDH shown relative to non - stressed or DMSO - only treated ( CTRL ) depending on vehicle compounds were dissolved in . n = 4 .

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Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.


measurements show of two ISR - related , PERK relative phospho - eif2α ( phospho - eif2α / total eif2α ) , are increased following different excitotoxic stress treatments in cortical neurons . Representative indicate differences for phospho - eif2α phospho - PERK 24 h post treatment with different excitotoxic stressor paradigms . Blocking of AMPA NMDA receptors using known antagonists significantly reduces ISR - related , which parallels reduced DPR ( Figure 3B ) . Changes in ISR are relative to non - stressed ( CTRL ) .

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Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).


Relative shows a significant decrease in C9 DPR reporter when stressor challenged NSC34 were treated with compounds inhibiting ISR - related or translation - targeting pathways . Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) , which inhibits eif2α - P activity , had most significant decreases in DPR production . Trazodone ( Traz ; 20 µM ) showed similar significant reduction in DPR as PERK inhibitor , GSK2606414 ( 500 nM ) .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).


Relative shows a significant decrease in C9 DPR reporter when glutamate stressed cortical neurons were treated with compounds Trazodone ( Traz ; 20 µM ) or Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) . Similar to treatments in NSC34 , 1 , 3DBM shows most significant decrease in DPR production .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.


A , Western blotting showing INSL5 in immortalized NPEC NPC lines after inhibition of secretion by BFA , β - actin was used as a loading control .

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A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.


B , ELISA showing secreted INSL5 in supernatants of NPECs NPC lines . For NPECs , n = 3 ; for NPC , n = 7 .

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B, ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n=3; for NPC, n=7.


C , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , in training cohort .

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C, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in training cohort.


E , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , in validation cohort 1 ( GZ ) . F , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , in validation cohort 2 ( ZS ) .

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E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ). F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).


A - C , Kaplan - Meier curves showing overall survival ( OS ) curves of NPC with low or high INSL5 ( A ) , low ( ≤ 4000 copy / ml ) or high ( > 4000 copy / ml ) EBV copy number ( B ) , combination of INSL5 with EBV copy number ( C ) .

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A-C, Kaplan-Meier curves showing the overall survival (OS) curves of NPC patients with low or high INSL5 concentration (A), low (≤4000 copy/ml) or high (>4000 copy/ml) EBV DNA copy number (B), and the combination of INSL5 concentration with EBV DNA copy number (C).


D - F , Kaplan - Meier curves showing disease - free survival ( DFS ) curves of NPC with low or high INSL5 ( D ) , low or high EBV copy number ( E ) , combination of INSL5 with EBV copy number ( F ) .

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D-F, Kaplan-Meier curves showing the disease-free survival (DFS) curves of NPC patients with low or high INSL5 concentration (D), low or high EBV DNA copy number (E), and the combination of INSL5 concentration with EBV DNA copy number (F).


A . Exogenous expression of INSL5 in NPC . Representative immunoblotting ( upper panel ) immunofluorescent ( lower panel ) showed stable exogenous expression of INSL5 in both CNE1 , CNE2 HK1 NPC lines . Scale bars represent 20μm .

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A. Exogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2 and HK1 NPC cell lines. Scale bars represent 20μm.B


MTT of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines ( upper panel ) either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) ( lower panel ) . n = 4 biological replicates for CNE1 CNE2 , n = 6 biological replicates for HK1 .

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MTT assay of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1.


colony formation ( C D ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( C ) for colony formation Number of colonies per field of view were plotted in D results are from three different experiments .

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colony formation (C and D) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments.


Brdu incorporation ( E F ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( E ) Brdu incorporation of Brdu positive were plotted in results are from three different experiments . Scale bars represent 20μm in ( E )

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Brdu incorporation (E and F) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (E) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments. Scale bars represent 20μm in (E)


migration assays ( G H ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( G ) for migration assays migrated per field of view were plotted in H , respectively . results are from three different experiments . Scale bars represent 100μm in ( G )

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migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (G) for migration assays migrated cells per field of view were plotted in H, respectively. The results are from three different experiments. Scale bars represent 100μm in (G)


I , J , Xenograft growth of INSL5 over expression NPC HK1 stable lines in nude mice . . ( I ) n = 11 mice per group .

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I, J, Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor size (I) n=11 mice per group.


Xenograft growth of INSL5 over expression NPC HK1 stable lines in nude mice . . of two groups . n = 11 mice per group .

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Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor weight of two groups. n=11 mice per group.


A , Gene expression profiles from CNE2 overexpressed with INSL5 or vector control . genes are shaded with green , black or red in heat map to indicate low , intermediate or high expression , respectively .

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A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively.


C , signal pathway of subset of genes involved in metabolism was assessed by Gene Set Enrichment .

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C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis.


D , of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock - down lines .

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D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines.


E , Metabolites were purified from five ( CNE1 HK1 ) or seven ( CNE2 ) independent samples of two vector INSL5 overexpressed lines analyzed by liquid chromatography - mass spectrometry ( LC - MS ) . Red : glycolytic intermediate metabolites . Green : tricarboxylic acid cycle ( TCA cycle ) intermediate metabolites . PPP : pentose phosphate pathway . color scale bar represents Z - score of metabolites ' level .

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E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.


F , extracellular acidification rate ( ECAR ) was measured in with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer . Glu , glucose ; Oligo , oligomycin .

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F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.


G , Statistical of effects of INSL5 overexpression on glycolytic activity .

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G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity.


H , CNE1 - vector CNE1 - INSL5 were grown in presence of glucose analog 2 - NBDG ( Invitrogen ) for 15min , glucose uptake was then quantified using flow cytometry ( FACS ) ( left panel ) . Statistical of glucose uptake was showed in right panel .

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H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.


glucose consumption lactate production ( I ) in CNE1 - vector CNE1 - INSL5 .

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glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells.


basic ATP level or ATP level of which was treated by 1uM oligomycin or 50uM 2 - DG for 24h ( J ) in CNE1 - vector CNE1 - INSL5 .

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basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells.


HK2 enzyme activity ( K ) in CNE1 - vector CNE1 - INSL5 .

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HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells.


L , ATP , HK2 enzyme activity lactate production in INSL5 wide - type or knock down HNE1 - EBV .

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L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.


B , phosphorylation of STAT5 under INSL5 stimulation . CNE1 were starved in FBS - free medium for 24h then treated with INSL5 peptide ( 50ng / mL ) in presence of an anti - INSL5 antibody at 100ug / ml , or an isotype - matched IgG control ( IgG ) for 30min .

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B, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min.


C , Localization of STAT5 . A confocal of STAT5 was performed using CNE1 transfected vector or INSL5 . Scale bars represent

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C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μm


D , from vector or INSL5overexpressing were separated subjected to immunoblot as indicated . marker Lamin B1 marker GAPDH were used to demonstrate purity of fractions .

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D, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions.


E , INSL5 overexpression activated AKT , ERK , JAK1 STAT5 phosphorylation , not STAT3 in HK1 . INSL5 overexpressing HK1 were treated with MK2206 ( 50uM ) , U0126 ( 20uM ) , MK2206 + U0126 , or Ruxolitinib ( 5uM ) for 24h . Western blotting was performed to evaluate effects of those inhibitors on STAT5 STAT3 phosphorylation level .

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E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.


F , ChIP - qPCR analyzed occupancy of potential E - boxes by STAT5 in genes of HK2 , Glut3 PFK1 in INSL5 - overexpressing CNE1 using IgG or anti - STAT5 antibodies .

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F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies.


G , Luciferase activity of different glycolytic gene promoter reporters in 293T transfected with STAT5 or empty vector .

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G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector.


H , Glucose uptake assessed by 2 - NBDG incubation in INSL5 overexpressed CNE1 with STAT5 knock down .

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H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.


I , Glucose uptake was assessed by 2 - BNDG incubation in INSL5 overexpressed HK1 treated with JAK kinase inhibitor ( Ruxolitinib ) .

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I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).


J , MTT of vector control or INSL5 overexpressing HK1 either treated with STAT5 siRNAs or JAK kinase inhibitor .

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J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.


A , Brdu for CNE1 stimulated with 100ng / ml INSL5 peptide in presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 20μm .

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A, Brdu assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 20μm.


B , similar to ( A ) , migration invasion for CNE1 stimulated with 100ng / ml INSL5 peptide in presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 100μm .

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B, similar to (A), migration and invasion assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 100μm.


C - E Growth curves of lines stably expressing INSL5 or vector 48 h post - treatment with indicated dose of glycolysis inhibitor , 2 - DG . CNE1 ( C ) , CNE2 ( D ) HK1 ( E ) . n = 4 biological replicates for CNE1 CNE2 , n = 6 biological replicates for

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

C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1


F , Growth curves of lines stably expressing INSL5 or vector 48 h post - treatment with indicated dose of cis - platinum ( DDP ) 2 - DG . n = 4 biological replicates per group .

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

F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.


( G ) of nude mice burdened with NPC PDX intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O

Tumor size (G) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


( H ) of nude mice burdened with NPC PDX intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

tumor weight (H) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.