ChIP - seq ( red ) and RIP - seq ( green ) data show enrichment of DNA and RNA , respectively , from var genes in the promoter - targeted and non - targeted " Control " dCas9 immunoprecipitation from non - clonal bulk population of ring stage parasites . Genome location is indicated at the top of each panel . The x - axis is DNA sequence , with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction . The y - axis is input - subtracted dCas9 ChIP enrichment in the var gene promoter - targeted dCas9 strain ( red ) , dCas9 RIP enrichment normalized to IgG control in the var gene promoter - targeted dCas9 strain or non - targeted " Control " dCas9 strain ( green ) , or var gene transcript levels in the promoter - targeted dCas9 strain ( RPKM , grey ) . One replicate was performed for ChIP - seq and RNA - seq and one replicate was performed for HA and IgG control RIP - seq .

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

ChIP-seq (red) and RIP-seq (green) data show enrichment of DNA and RNA, respectively, from var genes in the promoter-targeted and non-targeted "Control" dCas9 immunoprecipitation from non-clonal bulk population of ring stage parasites. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted dCas9 ChIP enrichment in the var gene promoter-targeted dCas9 strain (red), dCas9 RIP enrichment normalized to IgG control in the var gene promoter-targeted dCas9 strain or non-targeted "Control" dCas9 strain (green), or var gene transcript levels in the promoter-targeted dCas9 strain (RPKM, grey). One replicate was performed for ChIP-seq and RNA-seq and one replicate was performed for HA and IgG control RIP-seq.


Volcano plot representation of label - free quantitative proteomic analysis of P . falciparum proteins present in intron - ( left ) and promoter - targeted ( right ) dCas9 immunoprecipitations . Each dot represents a protein , and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment . For the main volcano plot , x - axis = log2 ( fold - change ) , y - axis = - log10 ( p - value ) , horizontal yellow line indicates adjusted p - value = 0 . 05 , and vertical yellow lines indicate absolute fold - change = 1 . 5 . Side panels indicate proteins uniquely identified in either sample ( y - axis = number of peptides per 100 amino acids ) . dCas9 protein is highlighted in green for reference . Two replicates were used for the promoter - targeted dCas9 and four replicates were used for the intron - targeted dCas9 . Fold enrichment and adjusted p - values for each protein highlighted in red ( promoter ) and blue ( intron ) can be found in Tables EV5 and EV6 , respectively .

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

Volcano plot representation of label-free quantitative proteomic analysis of P. falciparum proteins present in intron- (left) and promoter-targeted (right) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. For the main volcano plot, x-axis = log2(fold-change), y-axis = -log10(p-value), horizontal yellow line indicates adjusted p-value = 0.05, and vertical yellow lines indicate absolute fold-change = 1.5. Side panels indicate proteins uniquely identified in either sample (y-axis = number of peptides per 100 amino acids). dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted dCas9. Fold enrichment and adjusted p-values for each protein highlighted in red (promoter) and blue (intron) can be found in Tables EV5 and EV6, respectively.


Correlation plot of label - free quantitative proteomic analysis of P . falciparum proteins enriched in promoter - targeted ( top right quadrant in red ) versus non - targeted control ( top left quadrant in grey ) or intron - ( bottom right quadrant in blue ) dCas9 immunoprecipitations . Each dot represents a protein , and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment . The x - axis = log2 [ fold - change ( promoter / non - targeted control ) ] and y - axis = log2 [ fold - change ( promoter / intron ) ] . dCas9 protein is highlighted in green for reference . Two replicates were used for the promoter - targeted dCas9 and four replicates were used for the intron - targeted and non - targeted dCas9 . Fold enrichment and adjusted p - values can be found for each protein highlighted in red ( promoter ) in Tables EV5 ( intron comparison ) and EV7 ( control comparison ) and for each protein highlighted in blue ( intron ) in Tables EV6 ( promoter comparison ) and EV8 ( control comparison ) .

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

Correlation plot of label-free quantitative proteomic analysis of P. falciparum proteins enriched in promoter-targeted (top right quadrant in red) versus non-targeted control (top left quadrant in grey) or intron- (bottom right quadrant in blue) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. The x-axis = log2[fold-change(promoter/non-targeted control)] and y-axis = log2[fold-change(promoter/intron)]. dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted and non-targeted dCas9. Fold enrichment and adjusted p-values can be found for each protein highlighted in red (promoter) in Tables EV5 (intron comparison) and EV7 (control comparison) and for each protein highlighted in blue (intron) in Tables EV6 (promoter comparison) and EV8 (control comparison).


Western blot analysis of cytoplasmic ( Cty ) and nuclear extracts from a bulk population of ISWI - 3HA - ribo parasites in the absence ( - ) or presence ( + ) of glucosamine ( GlcN ) . ISWI - 3HA is detected with an anti - HA antibody . Antibodies against aldolase ( Ald . ) and histone H3 are controls for the cytoplasmic and nuclear extracts , respectively . Molecular weights are shown to the right .

O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of cytoplasmic (Cty) and nuclear extracts from a bulk population of ISWI-3HA-ribo parasites in the absence (-) or presence (+) of glucosamine (GlcN). ISWI-3HA is detected with an anti-HA antibody. Antibodies against aldolase (Ald.) and histone H3 are controls for the cytoplasmic and nuclear extracts, respectively. Molecular weights are shown to the right.


MA plot of log2 [ glucosamine - treated / untreated , M ] plotted over the mean abundance of each gene ( A ) at 12 hpi . Transcripts with a significantly higher ( above x - axis ) or lower ( below x - axis ) abundance in the presence of glucosamine are highlighted in red ( q ≤ 0 . 05 ) . iswi is highlighted in blue ( q = 6 . 71x10 - 10 ) and the active var gene is highlighted in green ( q = 2 . 53x10 - 5 ) . Two and three replicates were used for untreated and glucosamine - treated parasites , respectively . p - values were calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 ( Love et al , 2014 ) . q = Bonferroni corrected p - value .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MA plot of log2[glucosamine-treated/untreated, M] plotted over the mean abundance of each gene (A) at 12 hpi. Transcripts with a significantly higher (above x-axis) or lower (below x-axis) abundance in the presence of glucosamine are highlighted in red (q ≤ 0.05). iswi is highlighted in blue (q = 6.71x10-10) and the active var gene is highlighted in green (q = 2.53x10-5). Two and three replicates were used for untreated and glucosamine-treated parasites, respectively. p-values were calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected p-value.


Frequency plot showing the time in the IDC of peak transcript level [ comparison to microarray time course in ( Bozdech et al , 2003 ; Data ref : Bozdech et al , 2003 ) ] for genes that are significantly down - regulated ( blue ) or up - regulated ( yellow ) following ISWI knockdown .

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

Frequency plot showing the time in the IDC of peak transcript level [comparison to microarray time course in (Bozdech et al, 2003; Data ref: Bozdech et al, 2003)] for genes that are significantly down-regulated (blue) or up-regulated (yellow) following ISWI knockdown.


Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA parasites at 12 hpi from 1 . 5 kb upstream of the translation start site ( " Start " ) to 1 . 5 kb downstream of the translation stop site ( " Stop " ) for genes that are down - ( blue ) or up - regulated ( yellow ) upon ISWI knockdown . One replicate was used for the ISWI ChIP - seq .

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

Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for genes that are down- (blue) or up-regulated (yellow) upon ISWI knockdown. One replicate was used for the ISWI ChIP-seq.


ChIP - seq data show enrichment of ISWI ( blue ) in clonal ISWI - 3HA parasites at 12 hpi relative to regions of accessible chromatin ( " ATAC , " red ) . Chromatin accessibility ( ATAC - seq ) data were taken from the 15 hpi time point in ( Toenhake et al , 2018 ; Data ref : Toenhake et al , 2018 ) . Genome location is indicated at the top of the panel . The x - axis is DNA sequence , with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction . The y - axis is enrichment ( ChIP / Input or ATAC - seq / genomic DNA ) . One replicate was used for the ISWI ChIP - seq .

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

ChIP-seq data show enrichment of ISWI (blue) in clonal ISWI-3HA parasites at 12 hpi relative to regions of accessible chromatin ("ATAC," red). Chromatin accessibility (ATAC-seq) data were taken from the 15 hpi time point in (Toenhake et al, 2018; Data ref: Toenhake et al, 2018). Genome location is indicated at the top of the panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is enrichment (ChIP/Input or ATAC-seq/genomic DNA). One replicate was used for the ISWI ChIP-seq.


Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA parasites at 12 hpi from 1 . 5 kb upstream of the translation start site ( " Start " ) to 1 . 5 kb downstream of the translation stop site ( " Stop " ) for all genes , which are grouped into quartiles based on their transcript levels ( RPKM ) at 12 hpi . Dark red represents genes with the highest transcript levels ( " 1st " ) , light red represents genes with the second highest transcript levels ( " 2nd " ) , orange represents genes with the third highest transcript levels ( " 3rd " ) , and yellow represents genes with the lowest transcript levels ( " 4th " ) . One replicate was used for the ISWI ChIP - seq and the transcription data are an average from the two replicates of the untreated ISWI - 3HA clone used for the differential expression analysis .

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

Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for all genes, which are grouped into quartiles based on their transcript levels (RPKM) at 12 hpi. Dark red represents genes with the highest transcript levels ("1st"), light red represents genes with the second highest transcript levels ("2nd"), orange represents genes with the third highest transcript levels ("3rd"), and yellow represents genes with the lowest transcript levels ("4th"). One replicate was used for the ISWI ChIP-seq and the transcription data are an average from the two replicates of the untreated ISWI-3HA clone used for the differential expression analysis.


ChIP - seq data show enrichment of ISWI ( blue ) and H3K9ac ( green ) in clonal ISWI - 3HA parasites at 12 hpi at the active var gene ( PF3D7 _ 1240600 ) . RNA - seq data from this clone at 12 hpi show transcript levels for this gene ( grey ) . Genome location is indicated at the top of the panel . The x - axis is DNA sequence , with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction . The y - axis is ChIP / Input for ChIP data and RPKM for RNA - seq data . One replicate was used for each ChIP - seq and the RNA - seq data is from a single replicate from the untreated ISWI - 3HA clone used for the differential expression analysis .

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

ChIP-seq data show enrichment of ISWI (blue) and H3K9ac (green) in clonal ISWI-3HA parasites at 12 hpi at the active var gene (PF3D7_1240600). RNA-seq data from this clone at 12 hpi show transcript levels for this gene (grey). Genome location is indicated at the top of the panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is ChIP/Input for ChIP data and RPKM for RNA-seq data. One replicate was used for each ChIP-seq and the RNA-seq data is from a single replicate from the untreated ISWI-3HA clone used for the differential expression analysis.


Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA parasites at 12 hpi from 1 . 5 kb upstream of the translation start site ( " Start " ) to 1 . 5 kb downstream of the translation stop site ( " Stop " ) for the active var gene ( PF3D7 _ 1240600 , blue ) or silent var genes ( yellow ) . One replicate was used for the ISWI ChIP - seq .

Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for the active var gene (PF3D7_1240600, blue) or silent var genes (yellow). One replicate was used for the ISWI ChIP-seq.


Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL / 6 mouse lines . Tissues were isolated from indicated C57BL / 6 or iNKT TN mouse lines and stained with anti - CD3 and CD1d ( PBS57 ) tetramer .

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

Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d (PBS57) tetramer.


Spleen , mLN , sdLN , liver , adipose , and lung lymphocytes from Jα18 - / - or Vα14 mice were stimulated in vitro with RAW - CD1d cells and 1μg α - GalCer . An additional sample of Vα14 lymphocytes from each organ was plated with RAW - CD1d cells but no α - GalCer . Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array . Error bars are SD of mean values from three different mice per group . Results shown are representative of two independent experiments where n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18-/- or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n=3 biological replicates.


Lymphocytes from the indicated tissues of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer , before they were fixed , permeabilized , and stained with antibodies to T - bet , RORγt , and PLZF . Results shown are gated on CD3 + CD1d - tetramer + cells .

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

Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells.


The percentage of CD3 + CD1d - tetramer + iNKT cells in each organ that stained positively for PLZF , T - bet , and RORγt are shown . * * p < 0 . 01 , Mann - Whitney test . Error bars are SD .

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

The percentage of CD3+ CD1d-tetramer+ iNKT cells in each organ that stained positively for PLZF, T-bet, and RORγt are shown. **p<0.01, Mann-Whitney test. Error bars are SD.


Lymphocytes from the Peyer ' s patches of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer . Percentage of lymphocytes that were CD3 + CD1d - tetramer + iNKT cells among the Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 22 ; Vα14 n = 16 ; Jα18 - / - n = 8 .

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

Lymphocytes from the Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer. Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among the Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=22; Vα14 n=16; Jα18-/- n=8.


( C - E ) Mice were analyzed for total fecal IgA by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 .

(C-E)Mice were analyzed for total fecal IgA by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.


( C - E ) Mice were analyzed for total fecal IgG1 ( D ) or total serum IgG1 ( E ) by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E)Mice were analyzed for total fecal IgG1 (D) or total serum IgG1 (E) by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.


Percentages of total B cells that were IgG1 + in the spleen , mLN , and Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 15 ; Vα14 n = 15 ; Jα18 - / - n =

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

Percentages of total B cells that were IgG1+ in the spleen, mLN, and Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=15; Vα14 n=15; Jα18-/- n=5


CD1d ( PBS57 ) tetramer + CD3 + cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4 + CD3 + CD1d tetramer - cells from PP ( PP CD4 ) . RNAseq was performed . Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample .

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

CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample.


Spleen , mLN , and PP lymphocytes from Vα14 and C57BL / 6 mice were stimulated with PMA and ionomycin . Lymphocytes were stained with anti - CD3 , CD1d ( PBS57 ) tetramer , anti - IL - 4 , and anti - IFNγ . Percentages of iNKT cells and non - iNKT T cells within the population of CD3 + IL - 4 + cells and CD3 + IFNγ + cells , n = 5 mice for C57BL / 6 group and n = 4 mice for Vα14 group . Error bars are SD .

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

Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD.


Vα14 TN mice were administered α - GalCer either 2μg intravenously or 5μg by oral gavage . Mice were given Brefeldin A intraperitoneally after 30 min , and tissues were harvested 3 hours later . Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with antibodies to IL - 4 , IFNγ , and IL - 17 . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.


Quantification of data from C , n = 2 mice per group . Mann - Whitney test . Error bars are SEM .

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

Quantification of data from C, n=2 mice per group. Mann-Whitney test. Error bars are SEM.


Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α - GalCer and with or without agnostic anti - CD40 . IgM was measured by ELISA of culture supernatants 4 days later .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.


Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti - CD40 activated wild type B cells with or without 1μg α - GalCer and with blocking antibody to CD1d ( 1μg / mL , clone 1B1 ) or isotype control as indicated . IgM , IgG1 , and IgA were measured by ELISA of 4 day culture supernatants .

O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants.


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . IL - 4 were measured by ELISA of culture supernatants .

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Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. IL-4 were measured by ELISA of culture supernatants.


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 ( D - E ) were measured by ELISA of culture supernatants .

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Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1 (D-E) were measured by ELISA of culture supernatants.


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + and IgA + class switched B cells were enumerated by flow cytometry after 4 days of

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Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + class switched B cells were enumerated by flow cytometry after 4 days of

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Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


OVA - specific antibody titers from feces were determined by ELISA from the mice in ( A ) . Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD .

OVA-specific antibody titers from feces were determined by ELISA from the mice in (A). Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.


OVA - specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD .

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OVA-specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.


Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with the indicated antibodies . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells . Quantification of data from E , n = 3 mice per group . Mann - Whitney test . Error bars are SEM . * p < 0 . 05 .

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Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with the indicated antibodies. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. Quantification of data from E, n=3 mice per group. Mann-Whitney test. Error bars are SEM. *p<0.05.


Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP - iNKT cells .

Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP-iNKT cells.


Heatmap of FPKM values from genes uniquely upregulated in PP - iNKT cells as compared to both spleen iNKT and PP CD4 cells . Red indicates higher expression and blue indicates lower expression .

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Heatmap of FPKM values from genes uniquely upregulated in PP-iNKT cells as compared to both spleen iNKT and PP CD4 cells. Red indicates higher expression and blue indicates lower expression.


Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies , analyzed by flow cytometry and gated on CD1d ( PBS57 ) tetramer + CD3 + cells .

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Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies, analyzed by flow cytometry and gated on CD1d (PBS57) tetramer+CD3+ cells.


Quantification of data from D , n = 11 mice per group . Mann - Whitney test . Error bars are SEM .

Quantification of data from D, n=11 mice per group. Mann-Whitney test. Error bars are SEM.


Determination of maximum growth rate ( Vmax ) using a label - free cell count - based proliferation assay . Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi - log scale displaying total numbers of cells over time . Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints .

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Determination of maximum growth rate (Vmax) using a label-free cell count-based proliferation assay. Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi-log scale displaying total numbers of cells over time. Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints.


Western blot for NUP58 protein expression in mutant clones ( B - E ) and control cells ( clone A and WT ) . Whole - cell lysate hybridized with customized antibody 1 ( Ab1 , top panel ) and antibody 2 ( Ab2 , bottom panel ) . Actin was used as loading control .

Western blot for NUP58 protein expression in mutant clones (B-E) and control cells (clone A and WT). Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Actin was used as loading control.


Representation of the highest growth rates ( Vmax ) for each technical replicate for each indicated cell line . Each dot represents a technical replicate ( WT n = 35 , C1 n = 27 , C2 n = 28 , C3 n = 34 , C4 n = 32 , C5 n = 35 , C6 n = 38 ) of one independent experimental replicate performed .

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Representation of the highest growth rates (Vmax) for each technical replicate for each indicated cell line. Each dot represents a technical replicate (WT n=35, C1 n=27, C2 n=28, C3 n=34, C4 n=32, C5 n=35, C6 n=38) of one independent experimental replicate performed.


Western blot for NUP58 protein expression in mutant clones and control cells . Whole - cell lysate hybridized with customized antibody 1 ( Ab1 , top panel ) and antibody 2 ( Ab2 , bottom panel ) . Blots were loaded with wild - type HAP1 cell line ( WT ) , empty vector ( EV ) , non - targeting siRNA ( siNT ) and NUP58 targeting siRNA ( siNUP58 ) , followed by NUP58 mutant clones ( C1 / C2 / C3 / C4 in left panels , C5 / C6 in right panels ) . Actin was used as loading control . Asterisks indicate possible NUP58 alternative isoforms .

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Western blot for NUP58 protein expression in mutant clones and control cells. Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Blots were loaded with wild-type HAP1 cell line (WT), empty vector (EV), non-targeting siRNA (siNT) and NUP58 targeting siRNA (siNUP58), followed by NUP58 mutant clones (C1 / C2 / C3 / C4 in left panels, C5 / C6 in right panels). Actin was used as loading control. Asterisks indicate possible NUP58 alternative isoforms.


Cellular viability expressed as number of viable colonies following Cas9 - induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1 - KPNA4 - eGFP ( KAPs ) .

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Cellular viability expressed as number of viable colonies following Cas9-induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1-KPNA4-eGFP (KAPs).


Improvement in active nuclear import upon cell passaging . Dots represent analysed cells ( WT P3 n = 16 , WT P19 n = 25 , C5 P1 n = 24 , C5 P3 n = 27 , C5 P19 n = 23 ) of one independent experimental replicate performed .

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Improvement in active nuclear import upon cell passaging. Dots represent analysed cells (WT P3 n=16, WT P19 n=25, C5 P1 n=24, C5 P3 n=27, C5 P19 n=23) of one independent experimental replicate performed.


NUP58 protein expression in C5 at P1 and P19 . Blots were performed using Antibody 1 ( Ab1 , top ) or Antibody 2 ( Ab2 , bottom ) . Actin was used as loading control . The band corresponding to the full - length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk .

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NUP58 protein expression in C5 at P1 and P19. Blots were performed using Antibody 1 (Ab1, top) or Antibody 2 (Ab2, bottom). Actin was used as loading control. The band corresponding to the full-length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk.


Cellular viability following Cas9 - induced gene editing of native NUP58 ( left ) or NUP85 ( right ) in WT cell line ( black circles ) and in a cell line overexpressing NUP58 mutant isoform ( grey circles ) .

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Cellular viability following Cas9-induced gene editing of native NUP58 (left) or NUP85 (right) in WT cell line (black circles) and in a cell line overexpressing NUP58 mutant isoform (grey circles).


Diagram at the top represents G - banding pattern of chromosome 13 and highlights chromosome locations of tested genes ( NUP58 , GRK1 , ADPRHL1 ) . Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number .

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Diagram at the top represents G-banding pattern of chromosome 13 and highlights chromosome locations of tested genes (NUP58, GRK1, ADPRHL1). Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number.


Representative images of NUP58 gene - specific FISH hybridization for control HAP1 cell lines ( WT P3 - WT P19 ) and mutant C5 at P19 . Each dot represents a NUP58 locus of cluster thereof . Scale bar , 31 . 3

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Representative images of NUP58 gene-specific FISH hybridization for control HAP1 cell lines (WT P3-WT P19) and mutant C5 at P19. Each dot represents a NUP58 locus of cluster thereof. Scale bar, 31.3 μm


A . qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected ( CA : Control , n = 6 ; cKO , n = 6 . DG : Control , n = 6 ; cKO , n = 6 . Cortex : Control , n = 7 ; cKO , n = 7 . Cerebellum : Control , n = 4 ; cKO , n = 4 ) . * p - value < 0 . 05 ( Student t - test ) .

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A. qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected (CA: Control, n = 6; cKO, n = 6. DG: Control, n = 6; cKO, n = 6. Cortex: Control, n = 7; cKO, n = 7. Cerebellum: Control, n = 4; cKO, n = 4). * p-value < 0.05 (Student t-test).


B . Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice ( Control , n = 4 ; cKO , n = 4 ) . * * p - value < 0 . 01 ( Student t - test ) .

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B. Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice (Control, n = 4; cKO, n = 4). ** p-value < 0.01 (Student t-test).


C . Immunohistochemical staining ( upper level ) for marker proteins of neuronal integrity and quantification ( lower panel ) show no difference in control and Setd1b cKO mice ( NeuN : Control , n = 6 ; cKO , n = 6 ; Student t - test p - value = 0 . 57 . MAP2 : Control , n = 4 ; cKO , n = 4 ; Student t - test p - value = 0 . 72 . Iba1 : Control , n = 5 ; cKO , n = 5 ; Student t - test p - value = 0 . 8 . Gfap : Control , n = 5 ; cKO , n = 5 ; Student t - test p - value = 0 . 09 . ) . Scale bar : 100 µm .

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C. Immunohistochemical staining (upper level) for marker proteins of neuronal integrity and quantification (lower panel) show no difference in control and Setd1b cKO mice (NeuN: Control, n = 6; cKO, n = 6; Student t-test p-value = 0.57. MAP2: Control, n = 4; cKO, n = 4; Student t-test p-value = 0.72. Iba1: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.8. Gfap: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.09.). Scale bar: 100 µm.


D . Average speed ( left panel ) and percent time spent in the center ( right panel ) during exposure to the open field test was similar in control and Setd1b cKO mice ( Average speed : Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 075 . Time spent in center : Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 96 ) .

D. Average speed (left panel) and percent time spent in the center (right panel) during exposure to the open field test was similar in control and Setd1b cKO mice (Average speed: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.075. Time spent in center: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.96).


E . Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y - maze test ( Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 3 ) .

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E. Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y-maze test (Control, n = 15; cKO, n = 15; Student t-test p-value = 0.3).


F . Depressive - like behavior was measured in the Porsolt forced swim test . There was no significant difference in the % time spent immobile between groups ( Control , n = 8 ; cKO , n = 8 ; Student t - test p - value = 0 . 5 ) .

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F. Depressive-like behavior was measured in the Porsolt forced swim test. There was no significant difference in the % time spent immobile between groups (Control, n = 8; cKO, n = 8; Student t-test p-value = 0.5).


G . Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice ( Control : n = 15 , cKO : n = 15 . Repeated measures ANOVA , genotype effect : F ( 1 , 28 ) = 82 . 34 , * * * * p - value < 0 . 0001 ) .

G. Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice (Control: n = 15, cKO: n = 15. Repeated measures ANOVA, genotype effect: F (1,28) = 82.34, **** p-value < 0.0001).


H . Plots showing the specific search strategies during water maze training . Note the failure of Setd1b cKO mice to adopt hippocampus - dependent search strategies . I . The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice ( Student t - test : * * * p - value < 0 . 001 ) .

H. Plots showing the specific search strategies during water maze training. Note the failure of Setd1b cKO mice to adopt hippocampus-dependent search strategies. I. The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice (Student t-test: *** p-value < 0.001).


F . Left panel shows representative images of RNAscope performed for Kmt2a , Kmt2b and Setd1b . Right panel shows a bar chart quantifying the dots / cell ( n = 1500 cells from 2 mice ) indicative of the corresponding expression level . Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b ( * * * p - value < 0 , 0001 , Student t - test ) .

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F. Left panel shows representative images of RNAscope performed for Kmt2a, Kmt2b and Setd1b. Right panel shows a bar chart quantifying the dots/cell (n = 1500 cells from 2 mice) indicative of the corresponding expression level. Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b (*** p-value < 0,0001, Student t-test).


B Following treatment with 10 mM metformin overnight , NIH3T3 reporter cells were heat - shocked at 43 ° C for 30 min and recovered at 37 ° C for 5 h . GFP induction was quantitated by flow cytometry . HS : heat shock .

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B Following treatment with 10 mM metformin overnight, NIH3T3 reporter cells were heat-shocked at 43°C for 30 min and recovered at 37°C for 5 h. GFP induction was quantitated by flow cytometry. HS: heat shock.


C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting .

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C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting.


D , E Following heat shock and recovery for 4 h , mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . β - actin was used as the internal control .

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D, E Following heat shock and recovery for 4 h, mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). β-actin was used as the internal control.


F Reporter cells were treated with 10 mM metformin overnight , and viability was measured using Guava ViaCount ® reagents ( mean ± SD , n = 5 , n . s . , not significant , Student ' s t - test ) .

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F Reporter cells were treated with 10 mM metformin overnight, and viability was measured using Guava ViaCount® reagents (mean ± SD, n = 5, n.s., not significant, Student's t-test).


G Left panel , graphic depiction of the ELISA - based DNA binding assay . HSE : heat shock element . Right panel : Immediately following heat shock at 43 ° C for 30 min , nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . Experimental details are described in Materials and Methods .

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G Left panel, graphic depiction of the ELISA-based DNA binding assay. HSE: heat shock element. Right panel: Immediately following heat shock at 43°C for 30 min, nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). Experimental details are described in Materials and Methods.


I HEK293T cells stably expressing a scramble or HSF1 - targeting shRNA were treated with 10 mM metformin overnight and heat - shocked at 43 ° C for 30 min . Cells were fixed for PLA to detect HSF1 - DNA interactions . Experimental details are described in Materials and Methods . Scale bars : 50 μm for low magnification and 10 μm for high magnification .

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I HEK293T cells stably expressing a scramble or HSF1-targeting shRNA were treated with 10 mM metformin overnight and heat-shocked at 43°C for 30 min. Cells were fixed for PLA to detect HSF1-DNA interactions. Experimental details are described in Materials and Methods. Scale bars: 50 μm for low magnification and 10 μm for high magnification.


J Immediately following heat shock , cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting . LDH and Lamin A / C were used as cytoplasmic and nuclear markers , respectively . C : cytoplasm ; N : nucleus .

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J Immediately following heat shock, cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting. LDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. C: cytoplasm; N: nucleus.


K Reporter mice ( ventral side shown ) were pre - treated with metformin for 3 days , 2 mg / mouse / day , via i . p . injection . Following a single i . p . injection of velcade ( 5 mg / kg ) and recovery for 6 h , whole - body luciferase activities were quantitated by bioluminescence imaging . The photon flux of each mouse was expressed as photon / s / cm2 / steradian ( right panel , mean ± SD , n = 3 , one - way ANOVA , * P < 0 . 05 ; * * P < 0 . 01 ) .

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K Reporter mice (ventral side shown) were pre-treated with metformin for 3 days, 2 mg/mouse/day, via i.p. injection. Following a single i.p. injection of velcade (5 mg/kg) and recovery for 6 h, whole-body luciferase activities were quantitated by bioluminescence imaging. The photon flux of each mouse was expressed as photon/s/cm2/steradian (right panel, mean ± SD, n = 3, one-way ANOVA, *P < 0.05; **P < 0.01).


A - C Reporter cells were treated with and without 1 μM A - 769662 for 3 h followed by heat shock at 43 ° C for 30 min and recovery at 37 ° C for 5 h . Hspa1a and Hspb1 mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * * P < 0 . 001 ) . ACC phosphorylation was detected by immunoblotting ( C ) .

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A-C Reporter cells were treated with and without 1 μM A-769662 for 3 h followed by heat shock at 43°C for 30 min and recovery at 37°C for 5 h. Hspa1a and Hspb1 mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001). ACC phosphorylation was detected by immunoblotting (C).


D - F Primary MEFs were derived from Ampkα1fl / fl ; Ampkα2fl / fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1 / 2 in vitro . AMPKα levels were detected 4 days after transductions ( D )

D-F Primary MEFs were derived from Ampkα1fl/fl; Ampkα2fl/fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1/2 in vitro. AMPKα levels were detected 4 days after transductions (D)


Following viral transductions , Hsp mRNA levels were quantitated by qRT - PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43 ° C heat shock for 30 min and recovery for 4 h ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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Following viral transductions, Hsp mRNA levels were quantitated by qRT-PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43°C heat shock for 30 min and recovery for 4 h (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids : the HSF1 - dependent reporter pHSE - SEAP , in which ideal HSEs drive the expression of secreted alkaline phosphatase ( SEAP ) , and the control reporter pCMV - Gaussia Luc , in which a CMV promoter drives the expression of secreted Gaussia luciferase . In HEK293T cells , either LacZ or GST - AMPKα1CA plasmid was co - transfected with the two reporter plasmids . After 24 h , the transfected cells were heat - shocked at 44 ° C for 45 min . The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock . SEAP signals were normalized to Gaussia luciferase signals ( mean ± SD , n = 5 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids: the HSF1-dependent reporter pHSE-SEAP, in which ideal HSEs drive the expression of secreted alkaline phosphatase (SEAP), and the control reporter pCMV-Gaussia Luc, in which a CMV promoter drives the expression of secreted Gaussia luciferase. In HEK293T cells, either LacZ or GST-AMPKα1CA plasmid was co-transfected with the two reporter plasmids. After 24 h, the transfected cells were heat-shocked at 44°C for 45 min. The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock. SEAP signals were normalized to Gaussia luciferase signals (mean ± SD, n = 5, Student's t-test, **P < 0.01, ***P < 0.001).


H , I Following treatment with 10 mM metformin overnight , endogenous AMPKα and HSF1 proteins were co - precipitated using anti - AMPKα agarose conjugates from lysates of immortalized Hsf1 + / + MEFs ( H ) .

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H, I Following treatment with 10 mM metformin overnight, endogenous AMPKα and HSF1 proteins were co-precipitated using anti-AMPKα agarose conjugates from lysates of immortalized Hsf1+/+MEFs (H).


HC : heavy chain . Endogenous HSF1 - AMPKα interactions in immortalized Hsf1 + / + and Hsf1 − / − MEFs were visualized in situ by PLA ( I ) . Experimental details are described in Materials and Methods . Scale bars : 50 μm .

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HC: heavy chain. Endogenous HSF1-AMPKα interactions in immortalized Hsf1+/+ and Hsf1−/− MEFs were visualized in situ by PLA (I). Experimental details are described in Materials and Methods. Scale bars: 50 μm.


A In HEK293T cells that stably express an shRNA targeting the 3 ′ UTR of human HSF1 , HSF1WT or HSF1S121A was expressed . Following treatment with 10 mM metformin overnight , the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho - HSF1 Ser121 antibody ( A8041 , Assay Biotechnology ) .

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A In HEK293T cells that stably express an shRNA targeting the 3′ UTR of human HSF1, HSF1WT or HSF1S121A was expressed. Following treatment with 10 mM metformin overnight, the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho-HSF1 Ser121 antibody (A8041, Assay Biotechnology).


B Primary Ampkα1fl / fl ; Ampkα2fl / fl MEFs were transduced with adenoviral GFP or Cre . Following treatment with 10 mM metformin overnight or treatment with 10 μM A - 769662 for 3 h , HSF1 Ser121 phosphorylation was examined by immunoblotting .

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B Primary Ampkα1fl/fl; Ampkα2fl/fl MEFs were transduced with adenoviral GFP or Cre. Following treatment with 10 mM metformin overnight or treatment with 10 μM A-769662 for 3 h, HSF1 Ser121 phosphorylation was examined by immunoblotting.


C HSF1WT or HSF1S121A was co - expressed with either GFP or GST - AMPKαCA in HSF1 - deficient HEK293T cells . HSF1 Ser121 phosphorylation was examined by immunoblotting .

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C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.


D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight . Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His - tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C ( CC ) . HSF1 Ser121 phosphorylation was detected by immunoblotting .

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D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight. Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His-tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C (CC). HSF1 Ser121 phosphorylation was detected by immunoblotting.


E , F HSF1 activities were measured by the dual reporter system in HSF1 - deficient HEK293T cells . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ orAMPKα1CA ( E ) .

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E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ orAMPKα1CA (E).


E , F HSF1 activities were measured by the dual reporter system in HSF1 - deficient HEK293T cells . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ or AMPKα1CA ( E ) . Following expression of HSF1WT or HSF1S121A , cells were treated with and without 10 mM metformin overnight ( F ) ( mean ± SD , n = 5 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ or AMPKα1CA (E). Following expression of HSF1WT or HSF1S121A, cells were treated with and without 10 mM metformin overnight (F) (mean ± SD, n = 5, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


G , H FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with LacZ or AMPKα1CA plasmids into HSF1 - deficient HEK293T cells for 3 days ( G ) . Following transfection with FLAG - HSF1WT or FLAG - HSF1S121A plasmids for 3 days , HSF1 - deficient HEK293T cells were treated with and without 10 mM metformin overnight ( H ) . Nuclear proteins were extracted to measure HSF1 - DNA binding by the ELISA - based DNA binding assay using anti - FLAG antibodies ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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G, H FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with LacZ or AMPKα1CA plasmids into HSF1-deficient HEK293T cells for 3 days (G). Following transfection with FLAG-HSF1WT or FLAG-HSF1S121A plasmids for 3 days, HSF1-deficient HEK293T cells were treated with and without 10 mM metformin overnight (H). Nuclear proteins were extracted to measure HSF1-DNA binding by the ELISA-based DNA binding assay using anti-FLAG antibodies (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


I , J Following transfections and metformin treatment as described in ( G ) and ( H ) , cytoplasmic and nuclear proteins were extracted for immunoblotting using anti - FLAG antibodies .

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I, J Following transfections and metformin treatment as described in (G) and (H), cytoplasmic and nuclear proteins were extracted for immunoblotting using anti-FLAG antibodies.


A , B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine ( A ) or 4 . 5 g / l glucose ( B ) . Following heat shock at 43 ° C for 30 min and recovery for 5 h , GFP induction was quantitated by flow cytometry .

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A, B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine (A) or 4.5 g/l glucose (B). Following heat shock at 43°C for 30 min and recovery for 5 h, GFP induction was quantitated by flow cytometry.


C , D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA - based DNA binding assay ( mean ± SD , n = 3 , Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C, D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA-based DNA binding assay (mean ± SD, n = 3, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001).


E , F Following nutrient deprivations overnight , the reporter cells were heat - shocked at 43 ° C for 30 min and recovered overnight . Individual proteins were detected by immunoblotting .

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E, F Following nutrient deprivations overnight, the reporter cells were heat-shocked at 43°C for 30 min and recovered overnight. Individual proteins were detected by immunoblotting.


G , H Reporter cells were subjected to nutrient starvation for the indicated time . AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting .

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G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.


I , J HEK293T cells were sequentially transfected with control or AMPKα1 / 2 - targeting siRNAs and dual HSF1 reporter plasmids . 24 h later , transfected cells were starved overnight before reporter activities were measured ( mean ± SD , n = 6 , Student ' s t - test , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).


K , L FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with the dual HSF1 reporter plasmids into HSF1 - deficient HEK293T cells for 2 days , followed by overnight starvation ( mean ± SD , n = 5 , Student ' s t - test , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) .

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K, L FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with the dual HSF1 reporter plasmids into HSF1-deficient HEK293T cells for 2 days, followed by overnight starvation (mean ± SD, n = 5, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).


A Reporter cells were heat - shocked at 43 ° C for the indicated time . AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting .

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A Reporter cells were heat-shocked at 43°C for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.


B Immediately following heat shock at 43 ° C for 40 min , AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates .

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B Immediately following heat shock at 43°C for 40 min, AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates.


C Following heat shock at 43 ° C for 40 min , both detergent - soluble and detergent - insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting .

C Following heat shock at 43°C for 40 min, both detergent-soluble and detergent-insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting.


D , E Immediately following heat shock at 43 ° C for 40 min , HEK293 cells were subjected to nutrient deprivations for 4 h . AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting .

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D, E Immediately following heat shock at 43°C for 40 min, HEK293 cells were subjected to nutrient deprivations for 4 h. AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting.


F - H HSF1WT or HSF1S121A plasmids were transfected into HSF1 - deficient HEK293T cells . Transfected cells were heat - shocked at 43 ° C for 40 min . HSP mRNAs were quantitated by qRT - PCR following overnight recovery . HSF1 - DNA binding was measured immediately after heat shock ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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F-H HSF1WT or HSF1S121A plasmids were transfected into HSF1-deficient HEK293T cells. Transfected cells were heat-shocked at 43°C for 40 min. HSP mRNAs were quantitated by qRT-PCR following overnight recovery. HSF1-DNA binding was measured immediately after heat shock (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001).


A , B Following treatment with 10 μM metformin for 3 days , the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP ( mean ± SD , n = 3 , one - way ANOVA , * * P < 0 . 01 , * * * P < 0 . 001 ) . Normal rabbit IgG served as the negative control ( A ) . Following the same treatment , HSP mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * * P < 0 . 001 ) ( B ) .

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A, B Following treatment with 10 μM metformin for 3 days, the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP (mean ± SD, n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001). Normal rabbit IgG served as the negative control (A). Following the same treatment, HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001) (B).


C , D Following transfection with AMPKα1 / 2 - targeting siRNAs for 2 days , WM115 cells were treated with 10 μM metformin for 3 days . HSP mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 6 , Student ' s t - test , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) .

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C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).


E Following treatment with 10 μM metformin for 7 days , protein levels in WM115 and S462 cells were measured by immunoblotting .

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E Following treatment with 10 μM metformin for 7 days, protein levels in WM115 and S462 cells were measured by immunoblotting.


F , G Following treatment with 10 μM metformin for 7 days , levels of polyubiquitinated proteins were detected in both detergent - soluble and detergent - insoluble fractions in WM115 and A2058 cells using a Lys48 - specific polyubiquitin antibody .

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F, G Following treatment with 10 μM metformin for 7 days, levels of polyubiquitinated proteins were detected in both detergent-soluble and detergent-insoluble fractions in WM115 and A2058 cells using a Lys48-specific polyubiquitin antibody.


H A2058 cells were treated with 10 μM metformin alone or co - treated with 2 μM compound C for 7 days . Individual proteins were detected by immunoblotting .

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H A2058 cells were treated with 10 μM metformin alone or co-treated with 2 μM compound C for 7 days. Individual proteins were detected by immunoblotting.


I HEK293T cells stably expressing either scramble or HSF1 - targeting shRNAs were treated with 10 μM metformin for 7 days . Individual proteins were detected by immunoblotting .

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I HEK293T cells stably expressing either scramble or HSF1-targeting shRNAs were treated with 10 μM metformin for 7 days. Individual proteins were detected by immunoblotting.


J In HEK293T cells , a plasmid encoding HA - polyQ79 - GFP was co - transfected with the indicated plasmids . Following treatment with and without 10 μM metformin for 5 days , the sizes of aggregates in detergent - insoluble fractions were quantitated by a Multisizer ™ 3 Coulter Counter . Experimental details are described in Materials and Methods .

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J In HEK293T cells, a plasmid encoding HA-polyQ79-GFP was co-transfected with the indicated plasmids. Following treatment with and without 10 μM metformin for 5 days, the sizes of aggregates in detergent-insoluble fractions were quantitated by a Multisizer™ 3 Coulter Counter. Experimental details are described in Materials and Methods.


K , L Immortalized and RAS - transformed MEFs were treated with either 10 μM metformin ( K ) or different concentrations of glucose ( L ) for 7 days . Individual proteins were detected by immunoblotting .

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K, L Immortalized and RAS-transformed MEFs were treated with either 10 μM metformin (K) or different concentrations of glucose (L) for 7 days. Individual proteins were detected by immunoblotting.


M Following transfection with LacZ or polyQ79 , HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size .

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M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.


A , B A2058 cells were transduced with lentiviral scramble or HSF1 - targeting ( hA6 and hA9 ) shRNAs . ( A ) Cell numbers were quantitated by Hoechst 33342 DNA staining ( mean ± SD , n = 4 , two - way ANOVA , * * * P < 0 . 001 ) . ( B ) Protein levels were measured by immunoblotting .

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A, B A2058 cells were transduced with lentiviral scramble or HSF1-targeting (hA6 and hA9) shRNAs. (A) Cell numbers were quantitated by Hoechst 33342 DNA staining (mean ± SD, n = 4, two-way ANOVA, ***P < 0.001). (B) Protein levels were measured by immunoblotting.


C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4 . 5 , 1 . 0 , or 0 . 45 g / l glucose . Following treatment with 10 μM metformin , cell proliferation was measured by Hoechst 33342 DNA staining ( mean ± SD , n = 5 , two - way ANOVA , * * * P < 0 . 001 ) .

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C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4.5, 1.0, or 0.45 g/l glucose. Following treatment with 10 μM metformin, cell proliferation was measured by Hoechst 33342 DNA staining (mean ± SD, n = 5, two-way ANOVA, ***P < 0.001).


D , E 1 × 106 LacZ - or HSF1 - expressing A2058 cells were transplanted into female NOD / SCID mice . One day after transplantation , metformin was administered via drinking water at 1 mg / ml . Tumor incidence ( log - rank test ; D ) and volumes ( E ) were measured ( mean ± SEM , two - way ANOVA n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time ( DT ) .

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D, E 1 × 106 LacZ- or HSF1-expressing A2058 cells were transplanted into female NOD/SCID mice. One day after transplantation, metformin was administered via drinking water at 1 mg/ml. Tumor incidence (log-rank test; D) and volumes (E) were measured (mean ± SEM, two-way ANOVA n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time (DT).


F Individual proteins were measured by immunoblotting using lysates of three tumors from each treatment group .

F Individual proteins were measured by immunoblotting using lysates of three tumors from each treatment group.


G Protein levels were measured by immunoblotting using lysates of mouse liver tissues .

G Protein levels were measured by immunoblotting using lysates of mouse liver tissues.


H A2058 cells stably expressing FLAG - HSF1WT or FLAG - HSF1S121A were treated with 10 μM metformin for 7 days . Protein levels were measured by immunoblotting .

H A2058 cells stably expressing FLAG-HSF1WT or FLAG-HSF1S121A were treated with 10 μM metformin for 7 days. Protein levels were measured by immunoblotting.


I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning ® ultra - low attachment 96 - well culture plates with 10 μM metformin and different concentrations of glucose for 7 days , 10 , 000 cells per well . Viable cells were counted by Guava flow cytometer using ViaCount ® reagent ( mean ± SEM , n = 8 , Student ' s t - test , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning®ultra-low attachment 96-well culture plates with 10 μM metformin and different concentrations of glucose for 7 days, 10,000 cells per well. Viable cells were counted by Guava flow cytometer using ViaCount® reagent (mean ± SEM, n = 8, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).


J Tukey box plots showing the inverse correlation between AMPKα Thr172 phosphorylation and HSP mRNA expression in ccRCC . Patients were stratified on the median value of AMPKα Thr172 phosphorylation reverse - phase protein array ( RPPA ) scores ( Student ' s t - test ) .

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J Tukey box plots showing the inverse correlation between AMPKα Thr172 phosphorylation and HSP mRNA expression in ccRCC. Patients were stratified on the median value of AMPKα Thr172 phosphorylation reverse-phase protein array (RPPA) scores (Student's t-test).


( D ) Violin plots showing the distribution of sheath polymerization speed for both modes of T6SS assemblies . 300 assemblies ( 100 from each biological replicate ; 3 biological replicates ) were analyzed over at least 3 min at 5 s intervals . The central dash line indicates the median while the upper and lower dash lines indicate the quartiles . Unpaired t - test was performed ( ns : not significant ) .

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(D) Violin plots showing the distribution of sheath polymerization speed for both modes of T6SS assemblies. 300 assemblies (100 from each biological replicate; 3 biological replicates) were analyzed over at least 3 min at 5 s intervals. The central dash line indicates the median while the upper and lower dash lines indicate the quartiles. Unpaired t-test was performed (ns: not significant).


( D ) The frequency of contact - dependent assembly ( left panel ) and number of assemblies per cell per min ( right panel ) in cells from different strains as indicated on x - axis . Three biological replicates were performed for each strain . At least 500 cells were analyzed per replicate on 3D - SIM time - lapse imaging of sheath dynamics ( TssB - sfGFP ) in 3 min period . Bar graph ( left panel ) and scatter dot plot ( right panel ) show mean ± SD . Unpaired t - test was performed .

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(D) The frequency of contact-dependent assembly (left panel) and number of assemblies per cell per min (right panel) in cells from different strains as indicated on x-axis. Three biological replicates were performed for each strain. At least 500 cells were analyzed per replicate on 3D-SIM time-lapse imaging of sheath dynamics (TssB-sfGFP) in 3 min period. Bar graph (left panel) and scatter dot plot (right panel) show mean ± SD. Unpaired t-test was performed.


Kaplan - Meier analysis of the correlation between expression level of LOC113230 and overall survival of colon adenocarcinoma ( COAD ) patients from TCGA database . P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank test .

Kaplan-Meier analysis of the correlation between expression level of LOC113230 and overall survival of colon adenocarcinoma (COAD) patients from TCGA database. P<0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


qRT - PCR analysis of LOC113230 expression level in 65 paired CRC samples .

qRT-PCR analysis of LOC113230 expression level in 65 paired CRC samples.


Representative RNA - Scope immunostaining images of LOC113230 levels in CRC tissue and corresponding normal tissue from TMA chips . Scale bar represents 100 μM .

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Representative RNA-Scope immunostaining images of LOC113230 levels in CRC tissue and corresponding normal tissue from TMA chips. Scale bar represents 100 μM.


Kaplan - Meier analysis for overall survival of CRC patients with high and low expression of LOC113230 from TMA chips . * P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank test .

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Kaplan-Meier analysis for overall survival of CRC patients with high and low expression of LOC113230 from TMA chips. *P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


Overexpression of LOC113230 in SW - 1116 cells inhibited lung metastasis . Left : Exponentially growing SW - 1116 - LOC113230 - Luc and SW - 1116 - Vector - Luc cells were injected into the tail vein of 8 - week - old nude mice and intensities of lung metastatic tumor cells at the 8th week were analyzed by in vivo photon flux imaging equipment . The red circles in images are to help to see the tumors . Right : Box plot ( central band : median ; box limits : first and third quartile ; whiskers : minimum and maximum ) of lung photon flux at the 8th weeks .

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Overexpression of LOC113230 in SW-1116 cells inhibited lung metastasis. Left: Exponentially growing SW-1116 - LOC113230-Luc and SW-1116-Vector-Luc cells were injected into the tail vein of 8-week-old nude mice and intensities of lung metastatic tumor cells at the 8th week were analyzed by in vivo photon flux imaging equipment. The red circles in images are to help to see the tumors. Right: Box plot (central band: median; box limits: first and third quartile; whiskers: minimum and maximum) of lung photon flux at the 8th weeks.


5 ng / ml TGF - β and DMSO were applied to stimulate SW - 1116 - Vector or SW - 1116 - LOC113230 cells for 24 and 48 hrs , respectively . Cell migration was detected by transwell assays . Scale bar represents 100 μM .

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5 ng/ml TGF-β and DMSO were applied to stimulate SW-1116 -Vector or SW-1116 -LOC113230 cells for 24 and 48 hrs, respectively. Cell migration was detected by transwell assays. Scale bar represents 100 μM.


I ) Stable expression of LOC113230 300 - 800 inhibited cell migration , while LOC113230 antisense showed no apparent effect on cell migration . Scale bar represents 100 μM .

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I)Stable expression of LOC113230 300-800 inhibited cell migration, while LOC113230 antisense showed no apparent effect on cell migration. Scale bar represents 100 μM.


( A ) Top : Images taken by confocal microscopy showed the localization of LOC113230 detected by RNA scope assays in SW - 1116 . Scale bar represents 50 μM . Bottom : qRT - PCR assays showed LOC113230 expression level in the nuclear and cytoplasmic fractions extracted from SW - 1116 cells .

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(A)Top: Images taken by confocal microscopy showed the localization of LOC113230 detected by RNA scope assays in SW-1116. Scale bar represents 50 μM. Bottom: qRT-PCR assays showed LOC113230 expression level in the nuclear and cytoplasmic fractions extracted from SW-1116 cells.


SW - 1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 ( 5 μM ) for 12 hrs . Cell lysates were immunoprecipitated ( IP ) assays were performed with α - Flag antibody . The co - eluted proteins were detected by western blot assays with ubiquitin ( Ub ) , ASS1 and α - Flag antibodies , respectively .

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SW-1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub), ASS1 and α-Flag antibodies, respectively.


SW - 1116 cells stably expressing ASS1WT , or ASS1 K234R and LOC113230 or not were treated with MG132 ( 5 μM ) for 12 hrs . Cell lysates were immunoprecipitated ( IP ) assays were performed with α - Flag antibody . The co - eluted proteins were detected by western blot assays with ubiquitin ( Ub ) and α - Flag antibodies , respectively .

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SW-1116 cells stably expressing ASS1WT, or ASS1 K234R and LOC113230 or not were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub) and α-Flag antibodies, respectively.


Arginine production from each group was determined by a commercially available Arginine assay kit . The cells were stimulated with 5 ng / ml TGF - β or DMSO for 24 hrs . Urea production for each group was determined by a commercially available urea assay kit . The cells were stimulated with 5 ng / ml TGF - β or DMSO for 24 hrs .

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Arginine production from each group was determined by a commercially available Arginine assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs. Urea production for each group was determined by a commercially available urea assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs.


Depletion of ASS1 decreased cell migration in SW - 1116 cells . Scale bar represents 100 μM .

Depletion of ASS1 decreased cell migration in SW-1116 cells. Scale bar represents 100 μM.


Transwell assays showed the effect of ASS1WT and ASS1K234R on Caco - 2 - Vector or Caco - 2 - LOC113323 cell migration . Scale bar represents 100 μM . .

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Transwell assays showed the effect of ASS1WT and ASS1K234R on Caco-2-Vector or Caco-2-LOC113323 cell migration. Scale bar represents 100 μM..


( N ) Kaplan - Meier analysis for overall survival of patients with high or low expression of ASS1 . P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank test .

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(N) Kaplan-Meier analysis for overall survival of patients with high or low expression of ASS1. P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


The interaction of endogenous TRAF2 , ASS1 and LRPPRC was detected in SW - 1116 cells by co - IP assays . GST - TRAF2 , HA - LRPPRC , His - ASS1 and Biotin - LOC113230 were expressed in E . coli BL21 cells , and were used for in vitro binding assays .

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The interaction of endogenous TRAF2, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays. GST-TRAF2, HA-LRPPRC, His-ASS1 and Biotin-LOC113230 were expressed in E. coli BL21 cells, and were used for in vitro binding assays.


SW - 1116 - LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control ( SiRNA - NC ) were treated with MG132 ( 5 µM ) for 12 hrs . Cell lysates were immunoprecipitated with α - Flag antibody . The precipitate proteins and input proteins were analyzed by immunoblotting . NC : Negative control .

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SW-1116-LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control (SiRNA-NC) were treated with MG132 (5 µM) for 12 hrs. Cell lysates were immunoprecipitated with α-Flag antibody. The precipitate proteins and input proteins were analyzed by immunoblotting. NC: Negative control.


A . Left : Telomerase activity measured using ddTRAP assay . The horizontal pink lines represent the threshold used in these samples for background correction . Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal . Right : Quantification of TRAP activity after normalization to SW39 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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A. Left: Telomerase activity measured using ddTRAP assay. The horizontal pink lines represent the threshold used in these samples for background correction. Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal. Right: Quantification of TRAP activity after normalization to SW39/TEL+ cells (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


B . hTR and hTERT expression , normalized to ACTB mRNA and to SW39 / TEL + . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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B. hTR and hTERT expression, normalized to ACTB mRNA and to SW39/TEL+. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


C . RNA Pol II binding at hTR promoter in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines , normalized to H3 ChIP . Control loci with , respectively , high or low transcriptional activity : c - fos promoter and subtelomeric 4qHox locus ( n = 3 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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C. RNA Pol II binding at hTR promoter in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines, normalized to H3 ChIP. Control loci with, respectively, high or low transcriptional activity: c-fos promoter and subtelomeric 4qHox locus (n=3). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


D . H3K9me3 and H3K27me3 ( left ) or H3K4me3 and H3Ac ( right ) densities at hTR promoter in SI14 / TEL + and SI24 / ALT + , normalized to H3 ( n = 3 ) . Control loci as in C . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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D. H3K9me3 and H3K27me3 (left) or H3K4me3 and H3Ac (right) densities at hTR promoter in SI14/TEL+ and SI24/ALT+, normalized to H3 (n=3). Control loci as in C. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


E . NFYB binding at hTR promoter in SI14 / TEL + , SI24 / ALT + , SW39 / TEL + and IMRB / ALT + cells normalized to H3 . Control loci with , respectively , high and low NFYB binding : hnRNPA1 promoter and subtelomeric 4qHox ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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E. NFYB binding at hTR promoter in SI14/TEL+, SI24/ALT+, SW39/TEL+ and IMRB/ALT+ cells normalized to H3. Control loci with, respectively, high and low NFYB binding: hnRNPA1 promoter and subtelomeric 4qHox (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


A . Western blot for hTR chaperone proteins in TEL + and ALT + hybrids . β - Actin is used as loading control . Quantification of western blots is shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 2 - 4 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD .

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A. Western blot for hTR chaperone proteins in TEL+ and ALT+ hybrids. β-Actin is used as loading control. Quantification of western blots is shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=2-4). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD.


B . NHP2 mRNA expression levels , normalized to ACTB mRNA and to SI14 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD .

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B. NHP2 mRNA expression levels, normalized to ACTB mRNA and to SI14/TEL+ cells (n=3). Data information: Data are presented as mean ± SD.


C . Western blot for NHP2 in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines treated with DMSO or 10 μM MG132 for 6 h . β - Actin served as loading control . Quantifications are shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 5 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - test . Data information : Data are presented as mean ± SD .

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C. Western blot for NHP2 in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines treated with DMSO or 10 μM MG132 for 6 h. β-Actin served as loading control. Quantifications are shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=5). Statistical analyses were performed with Student's two-tailed unpaired t-test. Data information: Data are presented as mean ± SD.


D . hTR RNA and NHP2 mRNA expression , normalized to ACTB mRNA and to SI14 / TEL + . Data information : Data are presented as mean ± SD .

D. hTR RNA and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+. Data information: Data are presented as mean ± SD.