A ) Real - time PCR analysis of Lamp1 transcript ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by Student ' s t test * * p = 0 . 008 , * p = 0 . 038

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

A) Real-time PCR analysis of Lamp1 transcript (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test **p=0.008, *p=0.038


B ) Anti - LAMP1 staining in rapamycin - and rilmenidine - treated WT and Cox15sm / sm muscles vs . untreated ( UT ) samples . Rapamycin , but not rilmendine , increases the number of LAMP1 - positive vesicles in Cox15sm / sm samples ( inset ) . The scale bars correspond to 30µm

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

B) Anti-LAMP1 staining in rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. Rapamycin, but not rilmendine, increases the number of LAMP1-positive vesicles in Cox15sm/sm samples (inset). The scale bars correspond to 30µm


C ) Quantification of LAMP1 - positive vesicles ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * * * p < 0 . 0001

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

C) Quantification of LAMP1-positive vesicles (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001


D ) Cathepsin L specific activity ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by Student ' s t test * p = 0 . 0133

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

D) Cathepsin L specific activity (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test *p=0.0133


( C ) Cleavage of supercoiled ( SC ) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants . Efficient full length linearization ( FLL ) of the substrate resulting from a complete double - strand break was only observed when using sgRNAs bearing the 5 ' most end of the tracrRNA ( variants 1 and 3 ) . Moreover , the terminator - like Hairpin 3 is not required for target cleavage ( variant 1 ) . OC - open circular ; FLL - full length linear ; SC - supercoiled .

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

(C) Cleavage of supercoiled (SC) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants. Efficient full length linearization (FLL) of the substrate resulting from a complete double-strand break was only observed when using sgRNAs bearing the 5' most end of the tracrRNA (variants 1 and 3). Moreover, the terminator-like Hairpin 3 is not required for target cleavage (variant 1). OC - open circular; FLL - full length linear; SC- supercoiled.


( B ) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l . wt - wildtype .

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

(B) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l. wt - wildtype.


( C ) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition . The molar ratio of RNP to substrate was kept low ( 5 : 1 ) to increase reaction stringency . Asp2Cas12l predominantly recognizes a 5 ' - CCY - 3 ' PAM and Asp3Cas12l a 5 ' - CCB - 3 ' PAM . N = 3 . Data information : In ( C ) , individual data points are plotted , where n = 4 replicates from independent experiments ( Asp2Cas12l ) and n = 3 replicates from independent experiments ( Asp3Cas12l ) . The data points were fitted to a single exponential association curve ( solid lines ) .

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

(C) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition. The molar ratio of RNP to substrate was kept low (5:1) to increase reaction stringency. Asp2Cas12l predominantly recognizes a 5'-CCY-3' PAM and Asp3Cas12l a 5'-CCB-3' PAM. N=3. Data information: In (C), individual data points are plotted, where n = 4 replicates from independent experiments (Asp2Cas12l) and n = 3 replicates from independent experiments (Asp3Cas12l). The data points were fitted to a single exponential association curve (solid lines).


( A ) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single - stranded ( ss ) DNA or dsDNA ( including PAM ) activators with target sequences complementary to the gRNA spacer . NS activator - non specific activator ( ssDNA oligonucleotide or dsDNA duplex ) with no sequence complementarity to the spacer of the gRNA .

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

(A) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single-stranded (ss) DNA or dsDNA (including PAM) activators with target sequences complementary to the gRNA spacer. NS activator - non specific activator (ssDNA oligonucleotide or dsDNA duplex) with no sequence complementarity to the spacer of the gRNA.


( B ) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes . Background - subtracted traces from fluorescent reporter assays with favored ssDNA ( 5 ' - CCCCCCCC - 3 ' ) or ssRNA ( 5 ' - CCCCCCCC - 3 ' ) probes . RFU - relative fluorescence units . ( C ) Background - subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes . ( D ) Rates of trans - degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets . Collateral ssDNAse activity is about 3 - fold higher than the rate of ssRNA degradation . Fluorescence intensities in B - C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters . Data information : In ( B - D ) , individual data points from n = 3 replicates from independent experiments are plotted . In ( D ) , bars represent the mean .

O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE 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 B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O O O O O O O O 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) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes. Background-subtracted traces from fluorescent reporter assays with favored ssDNA (5'-CCCCCCCC-3') or ssRNA (5'-CCCCCCCC-3') probes. RFU - relative fluorescence units. (C) Background-subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes. (D) Rates of trans-degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets. Collateral ssDNAse activity is about 3-fold higher than the rate of ssRNA degradation. Fluorescence intensities in B-C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters. Data information: In (B-D), individual data points from n = 3 replicates from independent experiments are plotted. In (D), bars represent the mean.


Distribution of 25 - 35 mer reads mapping to the mouse genome . Proportion of 30 - 35 nt genomic reads matching mouse tRNA sequences . The sequencing was done in duplicates . Error bars represent SEM , significance was calculated using unpaired t - test .

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

Distribution of 25-35 mer reads mapping to the mouse genome. Proportion of 30-35 nt genomic reads matching mouse tRNA sequences. The sequencing was done in duplicates. Error bars represent SEM, significance was calculated using unpaired t-test.


Perbase sequence coverage of 30 - 35 nt reads across parental tRNA sequence . Anticodon positions are demarcated by discontinuous lines ( Blue = LIF - treated , Grey = Wnt3 + LIF - treated , Red = RA - treated mESCs ) . Perbase coverage of 30 - 35 nt reads across the 468 individual parental tRNAs plotted as heatmaps .

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

Perbase sequence coverage of 30-35 nt reads across parental tRNA sequence. Anticodon positions are demarcated by discontinuous lines (Blue = LIF-treated, Grey = Wnt3+LIF- treated, Red = RA- treated mESCs). Perbase coverage of 30-35 nt reads across the 468 individual parental tRNAs plotted as heatmaps.


Northern blot of candidate tRNAs and tsRNAs tsGlnCTG , tsGlyGCC , tsGluTTC , tsValCAC and tsLysTTT . The black arrows represent the tRNAs and the red arrows mark the tsRNAs .

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

Northern blot of candidate tRNAs and tsRNAs tsGlnCTG, tsGlyGCC, tsGluTTC, tsValCAC and tsLysTTT. The black arrows represent the tRNAs and the red arrows mark the tsRNAs.


Northern Blot of 5 ' - tsRNAs ; tsGlyGCC , tsGlnCTG , tsGluTTC , tsLysTTT , tsValCAC at various timepoints of RA - induced mESCs differentiation showing dynamic expression of these 5 ' - tsRNAs . Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates . The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs .

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

Northern Blot of 5'-tsRNAs; tsGlyGCC, tsGlnCTG, tsGluTTC, tsLysTTT, tsValCAC at various timepoints of RA-induced mESCs differentiation showing dynamic expression of these 5'-tsRNAs. Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates. The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs.


Effect of ASO - mediated inhibition of 5 ' - tsRNAs on alkaline phosphatase activity in RA - treated mESCs . ( n = 3 ) , Error bars represent SEM , significance was calculated using unpaired t - test .

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

Effect of ASO-mediated inhibition of 5'-tsRNAs on alkaline phosphatase activity in RA-treated mESCs. (n = 3), Error bars represent SEM, significance was calculated using unpaired t-test.


Relative expression of stemness markers in RA - induced differentiation mESCs blocked for 5 ' - tsRNA function with ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE 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

Relative expression of stemness markers in RA-induced differentiation mESCs blocked for 5'-tsRNA function with ASOs. (n = 2) Error bars represent SEM, significance was calculated using unpaired t-test.


Volcano plot of the transcriptome depicting the overexpressed transcripts ( in green ) and downregulated transcripts ( in pink ) in ASO treated - differentiating cells .

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Volcano plot of the transcriptome depicting the overexpressed transcripts (in green) and downregulated transcripts (in pink) in ASO treated-differentiating cells.


QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5 ' - tsRNA knockdowns using ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

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

QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5'-tsRNA knockdowns using ASOs. (n =2) Error bars represent SEM, significance was calculated using unpaired t-test.


Scatter plot showing Log2 fold - change enrichment of protein interactome between LIF - and RA - treated mESCs . All peptides identified by LC - MS / MS had < 1 % FDR ( Fig EV4E ) . LC - MS / MS was conducted twice ( R2 = 0 . 85 between LIF duplicates and R2 = 0 . 82 between RA - treated mESCs duplicates ) .

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

Scatter plot showing Log2 fold-change enrichment of protein interactome between LIF- and RA-treated mESCs. All peptides identified by LC-MS/MS had < 1% FDR (Fig EV4E). LC-MS/MS was conducted twice (R2 = 0.85 between LIF duplicates and R2 = 0.82 between RA-treated mESCs duplicates).


Scatter plot showing Log2 fold - change enrichment of mRNAs associated with tsGlnCTG over Scramble ( Scr1 ) in LIF - versus RA - treated mESCs .

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Scatter plot showing Log2 fold-change enrichment of mRNAs associated with tsGlnCTG over Scramble (Scr1) in LIF- versus RA-treated mESCs.


Bar plots and representative gene lists depicting association of tsGlnCTG with ' pluripotency - associated ' ( D ) and ' differentiation - responsive ' ( D ' ) genes in LIF - or RA - treated mESCs .

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 B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_TYPE O

Bar plots and representative gene lists depicting association of tsGlnCTG with 'pluripotency-associated' (D) and 'differentiation-responsive' (D') genes in LIF- or RA-treated mESCs.


In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo ( ASO ) against tsGlnCTG . ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1 . ( n = 2 ) , Error bars represent S . E . M .

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

In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. (n = 2), Error bars represent S.E.M.


Quantification of the Igf2bp1 bound c - Myc mRNA between LIF - versus RA - treated mESCs ( n = 2 ; see Fig . EV5F for duplicate data ) .

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

Quantification of the Igf2bp1 bound c-Myc mRNA between LIF- versus RA-treated mESCs (n = 2; see Fig. EV5F for duplicate data).


Association of c - Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition . ( n = 2 ) , Error bars represent SD , significance was calculated by one - tailed unpaired t - test .

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

Association of c-Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition. (n = 2), Error bars represent SD, significance was calculated by one-tailed unpaired t-test.


Relative enrichment of c - Myc mRNA in translating ( 80S and Polysome ) and non - translating ( mRNPs ) pools ( fractionated from polysome profiling ) between ASO - treated and Mock - treated RA - induced differentiating mESCs . ( n = 2 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

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

Relative enrichment of c-Myc mRNA in translating (80S and Polysome) and non-translating (mRNPs) pools (fractionated from polysome profiling) between ASO-treated and Mock-treated RA-induced differentiating mESCs. (n =2) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Relative levels of c - Myc mRNA in ASO - treated and Mock - treated RA - induced differentiating mESCs compared to LIF treated mESCs . ( n = 3 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

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

Relative levels of c-Myc mRNA in ASO-treated and Mock-treated RA-induced differentiating mESCs compared to LIF treated mESCs. (n = 3) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Epistatic analysis of 5 ' - tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity . ( n = 3 ) , Error bars represent SD , significance was calculated using unpaired t - test .

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

Epistatic analysis of 5'-tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity. (n = 3), Error bars represent SD, significance was calculated using unpaired t-test.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Time course of antibody response . Serum samples were diluted 40 - fold prior to ELISA analysis . Animals in the high - dose and low - dose group were immunized with 1 × 109 and 1 × 108 pfu virus each time , respectively . Arrows indicate the time point for immunization .

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

Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Time course of antibody response. Serum samples were diluted 40-fold prior to ELISA analysis. Animals in the high-dose and low-dose group were immunized with 1×109 and 1×108 pfu virus each time, respectively. Arrows indicate the time point for immunization.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Determination of virus neutralization antibody titer using 96 - well microneutralization assay . An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples . Statistical significance between different data sets was calculated using t - test .

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

Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Determination of virus neutralization antibody titer using 96-well microneutralization assay. An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples. Statistical significance between different data sets was calculated using t-test.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . ELISPOT analysis of IFN - γ response in PBMC samples . Pool A and B contain peptides corresponding to S1 - orf8 fusion and N , respectively . Data represent two animals from each group .

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

Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. ELISPOT analysis of IFN-γ response in PBMC samples. Pool A and B contain peptides corresponding to S1-orf8 fusion and N, respectively. Data represent two animals from each group.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Average food consumption after live virus challenge .

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

Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Average food consumption after live virus challenge.


Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS - CoV . All animals were euthanized 7 days after viral challenge , and anti - SARS - CoV monoclonal antibody was used to identify SARS antigens in the tissue . Representative data from lung sections were shown . Histology samples include : a . Control group . b . High - dose group . c . Placebo group . The arrowheads point to alveoli space filled with proteinaceous fluid . IHC samples include : d . Control group . e . High - dose group . f . Placebo group . Arrows point to cells that were positive for SARS antigen expression .

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

Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS-CoV. All animals were euthanized 7 days after viral challenge, and anti-SARS-CoV monoclonal antibody was used to identify SARS antigens in the tissue. Representative data from lung sections were shown. Histology samples include: a. Control group. b. High-dose group. c. Placebo group. The arrowheads point to alveoli space filled with proteinaceous fluid. IHC samples include: d. Control group. e. High-dose group. f. Placebo group. Arrows point to cells that were positive for SARS antigen expression.


( B ) Bar graph representing the normalized average value of the neuronal ATP / ATP + ADP ratio on the basal condition , on glutamate stimulation for 5 minutes , on glutamate stimulation with anisomycin ( 25μM ) and on glutamate stimulation with cycloheximide ( 350μM ) . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n = 5 independent platings . One - way ANOVA followed by Bonferroni ' s multiple comparison test .

O O O O O O O O O O O O B-CELL_TYPE B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE 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 O O O O O O O O O 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) Bar graph representing the normalized average value of the neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with anisomycin (25μM) and on glutamate stimulation with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. **p<0.01, ***p<0.001, ****p<0.0001, n=5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.


( C ) Bar graph representing the normalized average value of neuronal ATP / ATP + ADP ratio on the basal condition , on glutamate stimulation for 5 minutes , on glutamate stimulation with MPEP , on glutamate stimulation with CNQX + D - AP5 and on glutamate stimulation with CNQX + D - AP5 + anisomycin . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . . * p < 0 . 05 , * * * p < 0 . 001 , n = 4 - 5 independent platings . One - way ANOVA followed by Bonferroni ' s multiple comparison test .

O O O O O O O O O O O B-CELL_TYPE B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O

(C) Bar graph representing the normalized average value of neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with MPEP, on glutamate stimulation with CNQX + D-AP5 and on glutamate stimulation with CNQX + D-AP5 + anisomycin. Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment.. *p<0.05, ***p<0.001, n=4-5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.


( D ) Representative current - clamp and voltage - clamp traces showing a single neuron firing spontaneous bursts of action potentials . The traces on the right shows a single representative burst at a higher temporal resolution .

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

(D) Representative current-clamp and voltage-clamp traces showing a single neuron firing spontaneous bursts of action potentials. The traces on the right shows a single representative burst at a higher temporal resolution.


( E ) Bar graph representing the burst frequency and burst duration both in the voltage and current - clamp mode . n = 16 neurons from ≥ 3 independent platings . Data presented as mean ± SEM with scattered data points .

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

(E) Bar graph representing the burst frequency and burst duration both in the voltage and current-clamp mode. n=16 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.


( F ) Representative voltage - clamp traces of miniature EPSCs recorded ( Vhold = - 70mV ) from a neuron in presence of TTX .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O

(F) Representative voltage-clamp traces of miniature EPSCs recorded (Vhold= -70mV) from a neuron in presence of TTX.


( G ) Bar graph representing the baseline mEPSC frequency and amplitude in the current - clamp mode . N = 11 neurons from ≥ 3 independent platings . Data presented as mean ± SEM with scattered data points .

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

(G) Bar graph representing the baseline mEPSC frequency and amplitude in the current-clamp mode. N=11 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.


( I ) Immunoblots depicting levels of PSD 95 , GFAP , Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations . Quantification representing the average fold enrichments of PSD 95 , Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate . Data presented as mean ± SEM with scattered data points . Data points for all the proteins were normalized to their respective whole lysate values . * * p < 0 . 05 , * * p < 0 . 01 , n ≥ 7 animals for each group . One - sample t - test .

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

(I) Immunoblots depicting levels of PSD 95, GFAP, Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations. Quantification representing the average fold enrichments of PSD 95, Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate. Data presented as mean ± SEM with scattered data points. Data points for all the proteins were normalized to their respective whole lysate values. **p<0.05, **p<0.01, n ≥ 7 animals for each group. One-sample t-test.


( J ) Bar graph representing the normalized average value of synaptic ATP level on the basal condition , on DHPG treatment for 5 minutes , on DHPG treatment with MPEP , on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide ( 350μM ) . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . * p < 0 . 05 , n = 8 animals .

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

(J) Bar graph representing the normalized average value of synaptic ATP level on the basal condition, on DHPG treatment for 5 minutes, on DHPG treatment with MPEP, on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. *p<0.05, n=8 animals.


( K ) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition , on NMDA treatment for 5 minutes and on NMDA treatment with D - AP5 . Data presented as mean + / - SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . n = 8 independent platings .

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

(K) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition, on NMDA treatment for 5 minutes and on NMDA treatment with D-AP5. Data presented as mean +/- SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. n=8 independent platings.


( B ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in somatic compartments on the bath application of DHPG and NMDA . Scale bar 10μm . ( C ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in dendritic compartments on the bath application of DHPG and NMDA . Scale bar 10μm .

O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

(B) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in somatic compartments on the bath application of DHPG and NMDA. Scale bar 10μm. (C) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in dendritic compartments on the bath application of DHPG and NMDA. Scale bar 10μm.


( D ) Average traces depicting the time course of the normalized somatic ATP / ADP ratio on the basal conditions , on DHPG treatment , and on NMDA treatment . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 5 - 6 cells from ≥ 3 independent platings .

O O O O O O O O O O O O B-CELL_TYPE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE 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 O O O O O O O O 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) Average traces depicting the time course of the normalized somatic ATP/ADP ratio on the basal conditions, on DHPG treatment, and on NMDA treatment. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 5-6 cells from ≥3 independent platings.


( E ) Average traces depicting the time course of normalized dendritic ATP / ADP ratio on the basal condition , on the basal condition in the presence of anisomycin , on DHPG treatment , on NMDA treatment , on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 6 - 9 cells from ≥ 3 independent platings .

O O O O O O O O O O O B-CELL_TYPE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Average traces depicting the time course of normalized dendritic ATP/ADP ratio on the basal condition, on the basal condition in the presence of anisomycin, on DHPG treatment, on NMDA treatment, on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 6-9 cells from ≥3 independent platings.


( G ) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 for comparison with 0 minute . # # p < 0 . 01 and # # # # p < 0 . 0001 for comparison between DHPG + anisomycin . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 5 animals per group .

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

(G) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, *p<0.05, **p<0.01, ****p<0.0001 for comparison with 0 minute. ##p<0.01 and ####p<0.0001 for comparison between DHPG + anisomycin. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 5 animals per group.


( H ) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * * p < 0 . 01 for comparison with 0 minutes . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 4 animals per group .

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

(H) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, **p<0.01 for comparison with 0 minutes. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 4 animals per group.


( B ) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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

(B) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( C ) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons at various time points following DHPG and NMDA treatment . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O

(C) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons at various time points following DHPG and NMDA treatment. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( D ) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments . Data presented as mean + / - SEM . Data points in all groups were normalized to the average of the basal group . * * * p < 0 . 001 n = 21 - 54 neurons per group from 3 independent platings .

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 B-SMALL_MOLECULE 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 B-CELL_TYPE O O O O O O O

(D) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments. Data presented as mean +/- SEM. Data points in all groups were normalized to the average of the basal group. ***p<0.001 n= 21-54 neurons per group from 3 independent platings.


( E ) Representative images showing phospho - eEF2 immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O

(E) Representative images showing phospho-eEF2 immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( F ) Box plot showing phospho - eEF2 normalized intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 31 - 49 cells per group from 3 independent platings .

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

(F) Box plot showing phospho-eEF2 normalized intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=31-49 cells per group from 3 independent platings.


( G ) Representative immunoblots describing changes in the phospho - eEF2 and total - eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes . Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho / total undefined ratio .

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

(G) Representative immunoblots describing changes in the phospho-eEF2 and total-eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes. Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho/total undefined ratio.


( H ) Line graph showing the average value of the synaptic phospho / total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery . Data presented as mean + / - SEM , * * p < 0 . 01 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * * p < 0 . 001 , * * * * p < 0 . 0001 , n ≥ 5 animals per group .

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

(H) Line graph showing the average value of the synaptic phospho/total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery. Data presented as mean +/- SEM, **p<0.01. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. ***p<0.001, ****p<0.0001, n≥ 5 animals per group.


( A ) Representative images showing phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

(A) Representative images showing phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( B ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 30 - 46 cells per group from 3 independent platings .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=30-46 cells per group from 3 independent platings.


( C ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total ratio of AMPK .

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

(C) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total ratio of AMPK.


( D ) Line graph showing the normalized average value of the synaptic phospho / total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 5 animals per group .

O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE 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 O O O O O O 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) Line graph showing the normalized average value of the synaptic phospho/total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. *p<0.05, **p<0.01, ****p<0.0001, n= 5 animals per group.


( E ) Representative immunoblots depicting the changes in the α - SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin .

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

(E) Representative immunoblots depicting the changes in the α-SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin.


( F ) Line graph showing the normalized average value of the synaptic α - SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . * p < 0 . 05 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , n ≥ 4 animals per group .

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

(F) Line graph showing the normalized average value of the synaptic α-SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. *p<0.05. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, n≥ 4 animals per group.


( G ) Representative images showing the α - SNAP ( cyan ) and AMPK ( magenta ) immunolabeling in low - density cortical neurons . Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes . Scale bar 10μm . Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions . Scale bar 5μm .

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

(G) Representative images showing the α-SNAP (cyan) and AMPK (magenta) immunolabeling in low-density cortical neurons. Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes. Scale bar 10μm. Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions. Scale bar 5μm.


( H ) Box plot depicting the quantification of co - localization through Pearson ' s correlation coefficient between α - SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 01 , n ≥ 35 cells per group from 4 independent platings . Unpaired - sample t - test .

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

(H) Box plot depicting the quantification of co-localization through Pearson's correlation coefficient between α-SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.01, n≥35 cells per group from 4 independent platings. Unpaired-sample t-test.


( I ) Representative images showing the phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α - SNAP siRNA . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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

(I) Representative images showing the phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α-SNAP siRNA. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( J ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA , on DHPG treatment for 5 minutes in the presence of scrambled siRNA , on the basal condition in the presence of α - SNAP siRNA , on DHPG treatment for 5 minutes in the presence of α - SNAP siRNA . Data points in all groups were normalized to the average of the scrambled siRNA basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , n = 29 - 35 cells per group from 3 independent platings .

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

(J) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA, on DHPG treatment for 5 minutes in the presence of scrambled siRNA, on the basal condition in the presence of α-SNAP siRNA, on DHPG treatment for 5 minutes in the presence of α-SNAP siRNA. Data points in all groups were normalized to the average of the scrambled siRNA basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, n=29-35 cells per group from 3 independent platings.


( A ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2 + . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total AMPK ratio .

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

(A) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2+. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total AMPK ratio.


( B ) Line graph showing the normalized average value for the synaptic phospho / total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2 + . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 6 animals per group .

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

(B) Line graph showing the normalized average value for the synaptic phospho/total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2+. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, ****p<0.0001, n= 6 animals per group.


( C ) Representative images depicting intracellular Ca2 + levels through Fluo8 fluorescence ( Pseudo - colored ) of a cortical neuron plated at low - density before stimulation , 15 sec ( immediate ) after NMDA treatment , 300 sec ( delayed ) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2 + . Scale bar 20μm .

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

(C) Representative images depicting intracellular Ca2+ levels through Fluo8 fluorescence (Pseudo-colored) of a cortical neuron plated at low-density before stimulation, 15 sec (immediate) after NMDA treatment, 300 sec (delayed) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2+. Scale bar 20μm.


( D ) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2 + and D - AP5 . Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point . Ionomycin treatment in the presence of 10mM Ca2 + was used to calculate fluorescence maximum for a particular cell .

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

(D) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2+ and D-AP5. Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point. Ionomycin treatment in the presence of 10mM Ca2+ was used to calculate fluorescence maximum for a particular cell.


( E ) Box plot showing the distribution of ΔF / F0 across multiple neurons on the basal condition , on NMDA treatment in the presence Ca2 + , on NMDA treatment in the absence of Ca2 + , on NMDA treatment with D - AP5 and on NMDA treatment along with MK - 801 . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n ≥ 30 neurons per group from 3 independent platings .

O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O

(E) Box plot showing the distribution of ΔF/F0 across multiple neurons on the basal condition, on NMDA treatment in the presence Ca2+, on NMDA treatment in the absence of Ca2+, on NMDA treatment with D-AP5 and on NMDA treatment along with MK-801. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n≥30 neurons per group from 3 independent platings.


( B ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( pseudo - colored ) in cortical neurons on the basal condition and on DHPG ( 50μM ) treatment for 5 minutes in the presence or absence of AICAR ( 1mM ) . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O

(B) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (pseudo-colored) in cortical neurons on the basal condition and on DHPG (50μM) treatment for 5 minutes in the presence or absence of AICAR (1mM). MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( C ) Box plot showing the normalized phospho - eEF2 intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 17 - 46 cells per group from 3 independent platings .

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

(C) Box plot showing the normalized phospho-eEF2 intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=17-46 cells per group from 3 independent platings.


( D ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n = 18 - 57 cells per group from 3 independent platings .

O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O O

(D) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n=18-57 cells per group from 3 independent platings.


( E ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( Pseudo - colored ) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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

(E) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (Pseudo-colored) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( F ) Box plot showing phospho - eEF2 intensity distribution across multiple neurons on basal condition ( Vehicle control ) , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 21 - 36 cells per group from 3 independent platings .

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

(F) Box plot showing phospho-eEF2 intensity distribution across multiple neurons on basal condition (Vehicle control), on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=21-36 cells per group from 3 independent platings.


( G ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 24 - 52 cells per group from 3 independent platings .

O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=24-52 cells per group from 3 independent platings.


Freezing phenotype ( A ) of Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA for non - acclimation , − 5 ° C , 30 min ; CA for cold acclimation , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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

Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) , and ion leakage ( C ) of Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA for non - acclimation , − 5 ° C , 30 min ; CA for cold acclimation , − 8 ° C , 40 min ) .

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

survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min).


Expression of CBFs ( D ) in Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were placed at 4 ° C for 3 h ( D ) and total RNA was extracted and subjected to qRT - PCR analysis . Relative transcript levels in untreated Col - 0 plants were set to 1 .

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

Expression of CBFs (D) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 3 h (D) and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.


Expression of CBFs targets ( E ) in Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were placed at 4 ° C for 24 h ( E ) , and total RNA was extracted and subjected to qRT - PCR analysis . Relative transcript levels in untreated Col - 0 plants were set to 1 .

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

Expression of CBFs targets (E) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 24 h (E), and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.


A Time - course analysis of cytosolic free calcium concentration ( [ Ca2 + ] cyt ) dynamics in 10 - d - old wild type ( Col - 0 ) , atann1 , and atann1 AtANN1 complementation line ( # 10 ) after treatment with ice - cold water or ice - cold water containing 10 mM LaCl3 ( arrow shows the time point of treatment ) . Luminescence was recorded at 1 - s intervals . B Quantification of the cold - induced [ Ca2 + ] cyt changes shown in ( A ) . Peak [ Ca2 + ] cyt indicates the highest [ Ca2 + ] cyt after treatment .

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

A Time-course analysis of cytosolic free calcium concentration ([Ca2+]cyt) dynamics in 10-d-old wild type (Col-0), atann1, and atann1 AtANN1 complementation line (#10) after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.


C GST pull - down assay showing that OST1 interacts with AtANN1 in vitro . Purified recombinant GST - OST1 , GST - OST1G33R , or GST proteins from E . coli were immunoprecipitated with GST beads and then incubated with MBP - His - AtANN1 . Precipitated proteins were detected with anti - GST and anti - His antibodies .

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

C GST pull-down assay showing that OST1 interacts with AtANN1 in vitro. Purified recombinant GST-OST1, GST-OST1G33R, or GST proteins from E. coli were immunoprecipitated with GST beads and then incubated with MBP-His-AtANN1. Precipitated proteins were detected with anti-GST and anti-His antibodies.


D Interaction of OST1 and AtANN1 detected by bimolecular fluorescence complementation ( BiFC ) assays . The construct combinations were co - transformed into N . benthamiana leaves and expressed for 2 d . The signal was detected by confocal microscopy . Scale bar , 25 µm . The fluorescence intensity was scanned using the ImageJ plot profile tool . The y - axes indicate relative pixel intensity .

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D Interaction of OST1 and AtANN1 detected by bimolecular fluorescence complementation (BiFC) assays. The construct combinations were co-transformed into N. benthamiana leaves and expressed for 2 d. The signal was detected by confocal microscopy. Scale bar, 25 µm. The fluorescence intensity was scanned using the ImageJ plot profile tool. The y-axes indicate relative pixel intensity.


E , F Interaction of OST1 and AtANN1 detected by Co - IP assays in Arabidopsis . Twelve - day - old OST1 - Myc overexpressing plants grown on MS medium at 22 ° C were placed at 4 ° C for 0 , 0 . 5 , 2 h , and total proteins were extracted and immunoprecipitated with anti - Myc agarose beads . The wild type ( Col - 0 ) treated with 4 ° C for 2 h was used as control . The OST1 - Myc protein was detected with anti - Myc antibody and the AtANN1 protein was detected with anti - AtANN1 antibody . Representative pictures are shown in ( E ) and relative protein level in ( F ) .

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

E,F Interaction of OST1 and AtANN1 detected by Co-IP assays in Arabidopsis. Twelve-day-old OST1-Myc overexpressing plants grown on MS medium at 22°C were placed at 4°C for 0, 0.5, 2 h, and total proteins were extracted and immunoprecipitated with anti-Myc agarose beads. The wild type (Col-0) treated with 4°C for 2 h was used as control. The OST1-Myc protein was detected with anti-Myc antibody and the AtANN1 protein was detected with anti-AtANN1 antibody. Representative pictures are shown in (E) and relative protein level in (F).


G Co - IP assay showing the interaction between OST1 and AtANN1 in vivo . The construct combinations were expressed in N . benthamiana leaves . Total proteins were extracted after the N . benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti - Myc agarose beads . The proteins were detected with anti - Myc and anti - GFP antibodies .

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

G Co-IP assay showing the interaction between OST1 and AtANN1 in vivo. The construct combinations were expressed in N. benthamiana leaves. Total proteins were extracted after the N. benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti-Myc agarose beads. The proteins were detected with anti-Myc and anti-GFP antibodies.


Freezing phenotype ( A ) of Col - 0 , ost1 - 3 , atann1 , and atann1 ost1 - 3 . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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

Freezing phenotype (A) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) and ion leakage ( C ) of Col - 0 , ost1 - 3 , atann1 , and atann1 ost1 - 3 . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

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survival rate (B) and ion leakage (C) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


OST1 gene expression D ) in Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 .

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OST1 gene expression D) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1.


protein levels of OST1 and AtANN1 ( E ) in Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . OST1 and AtANN1 proteins were detected with anti - Myc and anti - AtANN1 antibodies . HSP90 was used as a control .

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protein levels of OST1 and AtANN1 (E) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1. OST1 and AtANN1 proteins were detected with anti-Myc and anti-AtANN1 antibodies. HSP90 was used as a control.


Freezing phenotype ( F ) of Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , - 5 ° C , 30 min ; CA , - 8 ° C , 40 min ) . Representative pictures are shown in ( F ) .

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

Freezing phenotype (F) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min). Representative pictures are shown in (F).


survival rate ( G ) , and ion leakage ( H ) of Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , - 5 ° C , 30 min ; CA , - 8 ° C , 40 min ) .

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survival rate (G), and ion leakage (H) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min).


A , B OST1 phosphorylates AtANN1 ( A ) and AtANN4 ( B ) in vitro . Purified recombinant MBP - His - OST1 was incubated with GST - AtANN1 , GST - AtANN4 or GST in kinase reaction buffer with 1 µCi [ γ - 32P ] ATP for 30 min at 30 ° C , followed by separation with SDS - PAGE . Phosphorylated AtANN1 and AtANN4 were detected by autoradiography . Recombinant OST1 , AtANN1 and AtANN4 were stained by Coomassie brilliant blue ( CBB ) .

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A,B OST1 phosphorylates AtANN1 (A) and AtANN4 (B) in vitro. Purified recombinant MBP-His-OST1 was incubated with GST-AtANN1, GST-AtANN4 or GST in kinase reaction buffer with 1 µCi [γ-32P] ATP for 30 min at 30°C, followed by separation with SDS-PAGE. Phosphorylated AtANN1 and AtANN4 were detected by autoradiography. Recombinant OST1, AtANN1 and AtANN4 were stained by Coomassie brilliant blue (CBB).


C In - gel kinase assays of OST1 in Col - 0 and ost1 - 3 mutant under cold stress . Twelve - day - old Col - 0 and ost1 - 3 mutant were treated at 4 ° C for 2 h . Total protein extracts were prepared and separated on a SDS - PAGE gel containing 0 . 2 mg / mL GST - AtANN1 as a substrate , and incubated with 70 µCi [ γ - 32P ] ATP . Top , autoradiograph ; bottom , CBB staining .

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

C In-gel kinase assays of OST1 in Col-0 and ost1-3 mutant under cold stress. Twelve-day-old Col-0 and ost1-3 mutant were treated at 4°C for 2 h. Total protein extracts were prepared and separated on a SDS-PAGE gel containing 0.2 mg/mL GST-AtANN1 as a substrate, and incubated with 70 µCi [γ-32P] ATP. Top, autoradiograph; bottom, CBB staining.


D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1 . After phosphorylation , the proteins were separated by SDS - PAGE and subjected to autoradiography . Top , autoradiograph ; bottom , CBB staining .

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D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1. After phosphorylation, the proteins were separated by SDS-PAGE and subjected to autoradiography. Top, autoradiograph; bottom, CBB staining.


E OST1 phosphorylates AtANN1 at S289 in vivo in LC / MS analysis . Total proteins were extracted from 12 - d - old AtANN1 - Myc overexpressing plants treated at 4 ° C for 0 , 10 , 30 and 120 min , followed by trypsin digestion . Phosphopeptides were enriched for mass spectrometry analysis . Representative picture for 10 min was shown .

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E OST1 phosphorylates AtANN1 at S289 in vivo in LC/MS analysis. Total proteins were extracted from 12-d-old AtANN1-Myc overexpressing plants treated at 4°C for 0, 10, 30 and 120 min, followed by trypsin digestion. Phosphopeptides were enriched for mass spectrometry analysis. Representative picture for 10 min was shown.


Freezing phenotype ( A ) of Col - 0 , atann1 , and atann1 AtANN1S289E ( # 5 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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

Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1S289E (#5) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) , and ion leakage ( C ) of Col - 0 , atann1 , and atann1 AtANN1S289E ( # 5 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

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survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1S289E (#5) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


Freezing phenotype ( D ) of Col - 0 , Atann1 , and atann1 AtANN1S289A ( # 16 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( D ) .

Freezing phenotype (D) of Col-0, Atann1, and atann1 AtANN1S289A (#16) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (D).


survival rate ( E ) , and ion leakage ( F ) of Col - 0 , Atann1 , and atann1 AtANN1S289A ( # 16 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

survival rate (E), and ion leakage (F) of Col-0, Atann1, and atann1 AtANN1S289A (#16) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


A AtANN1 current recordings in Xenopus oocytes . Whole - cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1 , OST1 , or AtANN1 + OST1 cRNA . Bath solution was described in Materials and Methods . The voltage protocol as well as time and current scale bars for the recordings are shown . B Current - voltage ( I - V ) relationship of the steady - state whole - cell currents in Xenopus oocytes described in ( A ) . Negative current is influx of cations into the oocytes .

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

A AtANN1 current recordings in Xenopus oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, OST1, or AtANN1 + OST1 cRNA. Bath solution was described in Materials and Methods. The voltage protocol as well as time and current scale bars for the recordings are shown. B Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (A). Negative current is influx of cations into the oocytes.


C Percentage of total Xenophus oocytes in different nA range according to ( A ) .

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C Percentage of total Xenophus oocytes in different nA range according to (A).


D AtANN1 current recordings in oocytes . Whole - cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1 , AtANN1S289A , OST1 , AtANN1 + OST1 , and AtANN1S289A + OST1 cRNA . E Current - voltage ( I - V ) relationship of the steady - state whole - cell currents in Xenopus oocytes described in ( D ) .

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

D AtANN1 current recordings in oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, AtANN1S289A, OST1, AtANN1 + OST1, and AtANN1S289A + OST1 cRNA. E Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (D).


F Percentage of total Xenophus oocytes in different nA range according to ( D ) .

F Percentage of total Xenophus oocytes in different nA range according to (D).


A Time - course analysis of [ Ca2 + ] cyt dynamics between 10 - day - old wild type ( Col - 0 ) and ost1 - 3 mutant after treatment with ice - cold water or ice - cold water containing 10 mM LaCl3 ( arrow shows the time point of treatment ) . Luminescence was recorded at 1 - s intervals . B Quantification of the cold - induced [ Ca2 + ] cyt changes shown in ( A ) . Peak [ Ca2 + ] cyt indicates the highest [ Ca2 + ] cyt after treatment .

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

A Time-course analysis of [Ca2+]cyt dynamics between 10-day-old wild type (Col-0) and ost1-3 mutant after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.


C MST assays of the calcium - binding affinity of GST - AtANN1 , GST - AtANN1S289A , and GST - AtANN1S289E . GST was used as a control .

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

C MST assays of the calcium-binding affinity of GST-AtANN1, GST-AtANN1S289A, and GST-AtANN1S289E. GST was used as a control.


A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours ( n = 4 , 632 tumours in each group ) across TCGA . - log10 ( padj ) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y - axis . Signatures more highly represented in TERThigh tumours are shown on the right , while those in TERTlow tumours are shown on the left .

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A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours (n = 4,632 tumours in each group) across TCGA. -log10(padj) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y-axis. Signatures more highly represented in TERThigh tumours are shown on the right, while those in TERTlow tumours are shown on the left.


D - F RT - qPCR analysis of interferon - related genes ( D ) , western blot analysis of TERT , TBK1 , pTBK1 , IRF3 , pIRF3 , and GAPDH levels ( E ) , and ELISA of IFNβ and CXCL10 in culture supernatant ( F ) in U2OS and HeLa cells ectopically expressing TERT - WT or TERT - K626A .

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D-F RT-qPCR analysis of interferon-related genes (D), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (E), and ELISA of IFNβ and CXCL10 in culture supernatant (F) in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A.


D RT - qPCR analysis of TA - ERVs in U2OS and HeLa cells ectopically expressing TERT - WT or TERT - K626A . E RT - qPCR analysis of TA - ERVs in HeLa and HCT116 cells with TERT knockdown .

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D RT-qPCR analysis of TA-ERVs in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A. E RT-qPCR analysis of TA-ERVs in HeLa and HCT116 cells with TERT knockdown. F


F - H RT - qPCR analysis of interferon - related genes and TA - ERVs ( F ) , western blot analysis of TERT , TBK1 , pTBK1 , IRF3 , pIRF3 , and GAPDH levels ( G ) , and ELISA of IFNβ and CXCL10 in culture supernatant ( H ) in U2OS cells ectopically expressing TERT - WT or TERT - K626A using Dox - inducible system . GFP was used as negative control . Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH , as indicated at the bottom of the blot ( G ) .

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

F-H RT-qPCR analysis of interferon-related genes and TA-ERVs (F), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (G), and ELISA of IFNβ and CXCL10 in culture supernatant (H) in U2OS cells ectopically expressing TERT-WT or TERT-K626A using Dox-inducible system. GFP was used as negative control. Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH, as indicated at the bottom of the blot (G).