( C ) In vitro enzymatic assay quantitating the effect of NDST3 on α - tubulin acetylation . Flag - NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti - Flag beads . Flag - EGFP was used as a control . The immunoprecipitated protein was incubated with purified tubulin heterodimers in vitro , with or without 1 mM NAD + , 400 nM TSA , or 5 mM nicotinamide ( NAM ) for 2 h at room temperature . The reaction products were examined by immunoblotting against Ac - α - tubulin or total α - tubulin ( n = 3 independent experiments , * P = 0 . 0185 , * * P < 0 . 01 , * * * * P < 0 . 0001 ) . ( D ) The same in vitro enzymatic assay as in ( C ) , except that the purified tubulin was polymerized in the presence of 20 μM Taxol and 1 mM GTP ( n = 3 independent experiments , * * P < 0 . 01 , * * * P = 0 . 0001 ) . Data information : Error bars represent ± standard deviation . ns means non - significant .

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(C) In vitro enzymatic assay quantitating the effect of NDST3 on α-tubulin acetylation. Flag-NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti-Flag beads. Flag-EGFP was used as a control. The immunoprecipitated protein was incubated with purified tubulin heterodimers in vitro, with or without 1 mM NAD+, 400 nM TSA, or 5 mM nicotinamide (NAM) for 2 h at room temperature. The reaction products were examined by immunoblotting against Ac-α-tubulin or total α-tubulin (n = 3 independent experiments, *P = 0.0185, **P < 0.01, ****P < 0.0001). (D) The same in vitro enzymatic assay as in (C), except that the purified tubulin was polymerized in the presence of 20 μM Taxol and 1 mM GTP (n = 3 independent experiments, **P < 0.01, ***P = 0.0001). Data information: Error bars represent ± standard deviation. ns means non-significant.


( A , B ) NDST3 protein levels relative to β - tubulin in B lymphocytes derived from healthy controls and C9orf72 - linked ALS patients , as examined by immunoblotting ( n = 3 biologically independent samples , * * P = 0 . 0026 ) . Data information : Error bars represent ± standard deviation .

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(A, B) NDST3 protein levels relative to β-tubulin in B lymphocytes derived from healthy controls and C9orf72-linked ALS patients, as examined by immunoblotting (n = 3 biologically independent samples, **P = 0.0026). Data information: Error bars represent ± standard deviation.


( C - F ) The protein levels of NDST3 relative to GAPDH ( D ) ( * * P = 0 . 0013 ) , Ac - α - tubulin relative to total α - tubulin ( E ) ( * P = 0 . 0379 ) , and total α - tubulin relative to GAPDH ( F ) ( P = 0 . 5700 ) , as examined by immunoblotting , in spinal cord tissues from healthy controls and C9orf72 - linked ALS patients ( n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group ) . Data information : Error bars represent ± standard deviation .

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(C-F) The protein levels of NDST3 relative to GAPDH (D) (**P = 0.0013), Ac-α-tubulin relative to total α-tubulin (E) (*P = 0.0379), and total α-tubulin relative to GAPDH (F) (P = 0.5700), as examined by immunoblotting, in spinal cord tissues from healthy controls and C9orf72-linked ALS patients (n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group). Data information: Error bars represent ± standard deviation.


( G - I ) Immunoblotting assays showing down - regulation of NDST3 in C9orf72 shRNA ( shC9orf72 ) - treated RPE1 cells , as compared to control shRNA ( shCTRL ) - treated cells . Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in ( H ) ( n = 4 independent experiments , * * * P = 0 . 0004 ) and ( I ) ( n = 4 independent experiments , * * * P = 0 . 0001 ) , respectively . ( J - L ) Immunoblotting assays showing up - regulation of NDST3 in RPE1 cells over - expressing Flag - C9orf72 compared to those expressing Flag - EGFP as control . Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in ( K ) ( n = 3 independent experiments , * * P = 0 . 0011 ) and ( L ) ( n = 3 independent experiments , * * P = 0 . 0033 ) , respectively . Data information : Error bars represent ± standard deviation .

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(G-I) Immunoblotting assays showing down-regulation of NDST3 in C9orf72 shRNA (shC9orf72)-treated RPE1 cells, as compared to control shRNA (shCTRL)-treated cells. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (H) (n = 4 independent experiments, ***P = 0.0004) and (I) (n = 4 independent experiments, ***P = 0.0001), respectively. (J-L) Immunoblotting assays showing up-regulation of NDST3 in RPE1 cells over-expressing Flag-C9orf72 compared to those expressing Flag-EGFP as control. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (K) (n = 3 independent experiments, **P = 0.0011) and (L) (n = 3 independent experiments, **P = 0.0033), respectively. Data information: Error bars represent ± standard deviation.


( M , N ) Baf A1 sensitivity assay for WT , NDST3 KO , and Noco - treated NDST3 KO RPE1 cells expressing proline - arginine poly - dipeptide ( PR82 ) . 24 h after the transfection of PR82 , WT and NDST3 KO cells were treated with vehicle or 10 μg / mL Noco overnight . The cell survival measured using calcein AM staining at 48 h post - transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells ( n = 3 independent cultures , * P = 0 . 0369 , * * * P = 0 . 0007 ) . Scale bar , 200 μm . Data information : Error bars represent ± standard deviation .

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(M, N) Baf A1 sensitivity assay for WT, NDST3 KO, and Noco-treated NDST3 KO RPE1 cells expressing proline-arginine poly-dipeptide (PR82). 24 h after the transfection of PR82, WT and NDST3 KO cells were treated with vehicle or 10 μg/mL Noco overnight. The cell survival measured using calcein AM staining at 48 h post-transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells (n = 3 independent cultures, *P = 0.0369, ***P = 0.0007). Scale bar, 200 μm. Data information: Error bars represent ± standard deviation.


Representative immunofluorescence ( IF ) staining of Ki67 and TDP - 43 in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Nuclei were stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; upper panel in blue ) or neural marker NeuN ( lower panel in green ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm .

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Representative immunofluorescence (IF) staining of Ki67 and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.


Quantification of cells or neurons with Ki67 immunoreactivity and TDP - 43 mislocalization from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0007 by t - test .

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Quantification of cells or neurons with Ki67 immunoreactivity and TDP-43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0007 by t-test.


Representative data of reverse - transcription PCR ( left panel ) or Western blot ( right panel ) for cell cycle - related genes and semi - quantification of the expression levels in the frontal cortices and hippocampus from the 6 - mon - old WT and FTLD - TDP Tg mice . N = 5 mice per group , data are presented as mean ± SEM ( % ) , statistical analysis by multiple t - test with FDR correction , Q = 1 % . * P < 0 . 05 , * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 and * * * * P ≤ 0 .

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Representative data of reverse-transcription PCR (left panel) or Western blot (right panel) for cell cycle-related genes and semi-quantification of the expression levels in the frontal cortices and hippocampus from the 6-mon-old WT and FTLD-TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t-test with FDR correction, Q = 1%. *P < 0.05, **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001


Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Scale bar : 50 μm . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * p = 0 . 0114 by t - test .

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Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *p = 0.0114 by t-test.


Representative IF staining of γH2AX and TDP - 43 in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Nuclei were stained with DAPI ( upper panel in blue ) or NeuN ( middle panel in green ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Lower panel : quantification of cells or neurons with γH2AX immunoreactivity and TDP - 43 proteinopathies from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0008 by t - test .

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Representative IF staining of γH2AX and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP-43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0008 by t-test.


Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD - TDP Tg mice . Nuclei were stained with DAPI ( upper panel ) or NeuN ( middle panel ) . Scale bar : 50 μm . Subregions , scale bar : 15 μm . Lower panel : quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0004 by t - test .

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Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0004 by t-test.


Top , representative western blot data of HDAC1 and TDP - 43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 - mon - old of FTLD - TDP or WT mice . Bottom , semi - quantification of HDAC1 and TDP - 43 expression levels . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * * * * p < 0 . 0001 by multiple t - tests .

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Top, representative western blot data of HDAC1 and TDP-43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6-mon-old of FTLD-TDP or WT mice. Bottom, semi-quantification of HDAC1 and TDP-43 expression levels. N = 5 mice per group, data are presented as mean ± SEM (%), **** p < 0.0001 by multiple t-tests.


Top , representative western blot data of HDAC1 and TDP - 43 in the cytosolic and nuclear extracts . Bottom , quantification of HDAC1 and TDP - 43 levels . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * * * p = 0 . 0005 ( TDP - 43 ) or 0 . 0004 ( HDAC1 ) ; * * * * p < 0 . 0001 by multiple t - tests .

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Top, representative western blot data of HDAC1 and TDP-43 in the cytosolic and nuclear extracts. Bottom, quantification of HDAC1 and TDP-43 levels. N = 5 mice per group, data are presented as mean ± SEM (%), *** p = 0.0005 (TDP-43) or 0.0004 (HDAC1); **** p < 0.0001 by multiple t-tests.


IF staining of HDAC1 and SMI - 32 in the regions of frontal cortices from WT and FTLD - TDP Tg mice . Scale bar : 150 μm . Blocked area in left bottom picture is showed in a magnified view in the right bottom picture . Scale bar : 50 μm .

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IF staining of HDAC1 and SMI-32 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Scale bar: 150 μm. Blocked area in left bottom picture is showed in a magnified view in the right bottom picture. Scale bar: 50 μm.


Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6 - mon - old of FTLD - TDP and WT mice . TSA : nuclear extracts that were treated with Trichostatin A ( TSA , an HDAC inhibitor ) as a negative control for HDAC1 - transferred fluorescent activity during the HDAC1 activity assay . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0415 by t - test .

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Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6-mon-old of FTLD-TDP and WT mice. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. N = 5 mice per group, data are presented as mean ± SEM (%), *p = 0.0415 by t-test.


Left , representative western blot data of nuclear acetylated - histone H3 ( Lys 9 / 14 ) and total - histone H3 in 6 - mon - old of WT and FTLD - TDP Tg mice . Right , quantification of nuclear acetylated - histone H3 / total - histone H3 ratio . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0147 by t - test .

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Left, representative western blot data of nuclear acetylated-histone H3 (Lys 9/14) and total-histone H3 in 6-mon-old of WT and FTLD-TDP Tg mice. Right, quantification of nuclear acetylated-histone H3/total-histone H3 ratio. N = 5 mice per group, data are presented as mean ± SEM (%), * p = 0.0147 by t-test.


Left graph : IF staining of TDP - 43 and HDAC1 during progression of TDP - 43 proteinopathies in the frontal cortices from the FTLD - TDP Tg and WT mice . Nuclei were stained with DAPI ( in blue ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Right histogram : Quantification of co - localized TDP - 43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1 - , 6 - , and 12 - month - old FTLD - TDP Tg mice . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM . * Nucleus : Tg 1m vs . Tg 6m or 12m ; # Cytosol : Tg 1m vs . Tg 6m or 12m ; @ Tg 6m vs Tg 12m . * * * * / # # # # / @ @ @ @ p < 0 . 0001 by multiple comparison .

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Left graph: IF staining of TDP-43 and HDAC1 during progression of TDP-43 proteinopathies in the frontal cortices from the FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co-localized TDP-43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1m vs. Tg 6m or 12m; #Cytosol: Tg 1m vs. Tg 6m or 12m; @ Tg 6m vs Tg 12m. ****/####/@@@@ p < 0.0001 by multiple comparison.


Left graph : IF staining of γH2AX and HDAC1 in the frontal cortices from the 12 - month - old FTLD - TDP Tg and WT mice . Nuclei were stained with DAPI ( in blue ) . Arrow head : γH2AX + nucleus ; arrow : mislocalized HDAC1 . Scale bar : 50 μm . Right histogram : Quantification of γH2AX + and HDAC1 mislocalized cells in the WT or 1 - , 6 - , and 12 - month - old FTLD - TDP Tg mice . n = 4 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM . * * p = 0 . 0079 , * * * * p < 0 . 0001 by multiple comparison .

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Left graph: IF staining of γH2AX and HDAC1 in the frontal cortices from the 12-month-old FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Arrow head: γH2AX+ nucleus; arrow: mislocalized HDAC1. Scale bar: 50 μm. Right histogram: Quantification of γH2AX+ and HDAC1 mislocalized cells in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 4 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. ** p = 0.0079, **** p < 0.0001 by multiple comparison.


Left panel : flag - tagged full - length HDAC1 was overexpressed with myc - tagged TDP - 43 in HEK - 293T cells ; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP - 43 and flag . Right panel : myc - tagged TDP - 43 was overexpressed with flag - tagged full - length HDAC1 in HEK - 293T cells ; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP - 43 .

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Left panel: flag-tagged full-length HDAC1 was overexpressed with myc-tagged TDP-43 in HEK-293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP-43 and flag. Right panel: myc-tagged TDP-43 was overexpressed with flag-tagged full-length HDAC1 in HEK-293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP-43.


Upper left : flag - tagged full - length HDAC1 ( b . Ⅰ ) or various truncation mutations ( b . Ⅱ - Ⅳ ) were overexpressed with myc - tagged TDP - 43 ; the catalytic domain is indicated in red . Lower panel : the western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP - 43 .

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Upper left: flag-tagged full-length HDAC1 (b.Ⅰ) or various truncation mutations (b.Ⅱ-Ⅳ) were overexpressed with myc-tagged TDP-43; the catalytic domain is indicated in red. Lower panel: the western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP-43.


Upper panel : Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP - 43 in WT and FTLD - TDP Tg mice . Lower histogram : Quantification of immunoprecipitation results of HDAC1 and TDP - 43 in WT and Tg mice . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0149 , * * * p = 0 . 0003 by t - test .

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Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP-43 in WT and FTLD-TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP-43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * p =0.0149, *** p =0.0003 by t-test.


Western - blot of HDAC1 and TDP - 43 in urea - soluble fractions . N = 5 mice per group .

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Western-blot of HDAC1 and TDP-43 in urea-soluble fractions. N = 5 mice per group.


Escape latency of mice in Morris water maze task . * WT + Vehicle mice vs . Tg + Vehicle mice , # Tg + 5104434 mice vs . Tg + Vehicle mice . Statistical analysis by two - way ANOVA with Bonferroni ' s multiple comparisons . # P < 0 . 05 , * * * P ≤ 0 . 001 and * * * * / # # # # P ≤ 0 . 0001 Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison .

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Escape latency of mice in Morris water maze task. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by two-way ANOVA with Bonferroni's multiple comparisons. #P < 0.05, ***P ≤ 0.001 and ****/####P ≤ 0.0001 Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.


The representative searching path of mice in the probe test .

The representative searching path of mice in the probe test.


Scores of mice with respect to the number of times they crossed the hidden platform , time spent searching in the target quadrant , and the velocity of swimming in the probe test at 24 h after escape training . * * p = 0 . 009 , * * * p = 0 . 0002 by multiple comparison . Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison .

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Scores of mice with respect to the number of times they crossed the hidden platform, time spent searching in the target quadrant, and the velocity of swimming in the probe test at 24 h after escape training. ** p = 0.009, *** p = 0.0002 by multiple comparison. Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.


Scores of the discrimination index in the novel object recognition test . Scores of mice in the rotarod test . Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison .

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Scores of the discrimination index in the novel object recognition test.   Scores of mice in the rotarod test.   Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.


Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment . The nuclear extracts of the frontal cortices and hippocampus from 8 - mon - old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1 , and were then conducted to HDAC1 activity assay . N = 6 mice per group . TSA : nuclear extracts that were treated with Trichostatin A ( TSA , an HDAC inhibitor ) as a negative control for HDAC1 - transferred fluorescent activity during the HDAC1 activity assay . * p = 0 . 0203 ( WT vs Tg - Veh ) or 0 . 0408 ( Tg - Veh vs Tg + 5104434 ) by multiple comparison . Data information : All data are presented as mean ± SEM ( %

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Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment. The nuclear extracts of the frontal cortices and hippocampus from 8-mon-old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1, and were then conducted to HDAC1 activity assay. N = 6 mice per group. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. * p = 0.0203 (WT vs Tg-Veh) or 0.0408 (Tg-Veh vs Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%


Representative data of western blot for nuclear acetylated - histone 3 ( Lys 9 / 14 ) , total histone H3 , and quantification of protein levels . N = 6 mice per group . * p = 0 . 0105 or * * * p = 0 . 0008 by multiple comparison . Data information : All data are presented as mean ± SEM ( % )

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Representative data of western blot for nuclear acetylated-histone 3 (Lys 9/14), total histone H3, and quantification of protein levels. N = 6 mice per group. * p = 0.0105 or *** p = 0.0008 by multiple comparison. Data information: All data are presented as mean ± SEM (%)


Representative data of western blot for nuclear HDAC1 levels and semi - quantification of the expression levels . N = 6 mice per group . * * p = 0 . 0022 ( Tg ) / 0 . 0012 ( Tg + 5104434 ) by multiple comparison . Data information : All data are presented as mean ± SEM ( % )

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Representative data of western blot for nuclear HDAC1 levels and semi-quantification of the expression levels. N = 6 mice per group. ** p = 0.0022 (Tg)/ 0.0012 (Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%)


Left panel : representative data of western blot for cell cycle - and DNA damage - related proteins . Right histogram : semi - quantification of protein levels . N = 6 per group . * WT + Vehicle mice vs . Tg + Vehicle mice , # Tg + 5104434 mice vs . Tg + Vehicle mice . Statistical analysis by multiple t - test with FDR correction , Q = 1 % . * * P ≤ 0 . 01 ; # # # P ≤ 0 . 001 and * * * * / # # # # P ≤ 0 . 0001 . Data information : All data are presented as mean ± SEM ( % )

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Left panel: representative data of western blot for cell cycle- and DNA damage-related proteins. Right histogram: semi-quantification of protein levels. N = 6 per group. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by multiple t-test with FDR correction, Q = 1%. **P ≤ 0.01; ###P ≤ 0.001 and ****/####P ≤ 0.0001. Data information: All data are presented as mean ± SEM (%)


IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8 - mon - old of WT + Vehicle , Tg + Vehicle , Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * p = 0 . 0421 or * * * * p < 0 . 0001 by multiple comparison . Data information : number of cells in each view is presented .

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IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8-mon-old of WT+Vehicle, Tg+Vehicle, Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. * p = 0.0421 or **** p < 0.0001 by multiple comparison. Data information: number of cells in each view is presented.


Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice , 8 - mon - old WT + Vehicle , Tg + Vehicle and Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * p = 0 . 0414 or * * * p = 0 . 0006 by multiple comparison . Data information : All data are presented as mean ± SEM ( % )

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Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 8-mon-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm.* p = 0.0414 or *** p = 0.0006 by multiple comparison. Data information: All data are presented as mean ± SEM (%)


IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice , 12 - month - old WT + Vehicle , Tg + Vehicle and Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * * p = 0 . 002 and * * * * p < 0 . 0001 by multiple comparison . Data information : where number of cells in each view is presented .

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IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 12-month-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. ** p = 0.002 and **** p < 0.0001 by multiple comparison. Data information: where number of cells in each view is presented.


Representative IF staining of TDP - 43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD - TDP . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm .

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Representative IF staining of TDP-43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD-TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.


Quantification of cells with co - mislocalized HDAC1 and TDP - 43 from each view of microscope . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Quantification of cells with co-mislocalized HDAC1 and TDP-43 from each view of microscope. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Linear regression analysis of cells with HDAC1 mislocalization and TDP - 43 proteinopathies . Total cell counts : 3000 per samples . P = 0 . 0001 by Pearson correlation analysis . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Linear regression analysis of cells with HDAC1 mislocalization and TDP-43 proteinopathies. Total cell counts: 3000 per samples. P = 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD - TDP from each view of microscope . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm .

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Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD-TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.


Quantification of cells with γH2AX and Ki67 immunoreactivity . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Linear regression analysis of cells with DNA damage and aberrant cell cycle activity . Cell counts : 40 - 113 ( normal ) and 418 - 887 ( FTLD ) per samples . p < 0 . 0001 by Pearson correlation analysis . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Cell counts: 40 -113 (normal) and 418-887 (FTLD) per samples. p < 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


Dot - blot of HDAC1 and TDP - 43 in urea - soluble fractions from the frontal cortex of normal individuals and patients with FTLD - TDP .

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Dot-blot of HDAC1 and TDP-43 in urea-soluble fractions from the frontal cortex of normal individuals and patients with FTLD-TDP.


Quantification of HDAC1 and TDP - 43 expression levels in urea - soluble fractions . N = 5 per group . Data information : All data represent mean ± SEM , * * * * p < 0 . 0001 by t - test .

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Quantification of HDAC1 and TDP-43 expression levels in urea-soluble fractions. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.


B . Changes in the expression of cytokines in THP1 macrophages ( left ) and PBMCs ( right ) upon treatment with full - length S1 subunit at 1 µg / ml for 24 hr . Data information ,

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B. Changes in the expression of cytokines in THP1 macrophages (left) and PBMCs (right) upon treatment with full-length S1 subunit at 1 µg/ml for 24 hr. Data information,


E . Effect of an anti - RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs . Left , a schematic showing the domain structure of S1 spike protein and anti - RBD antibody . Right , changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti - RBD antibody . Gene expression was measured by qPCR . Cytokine release in the conditioned media was measured by Luminex . Full - length S1 and S1 subunits are purified from HEK293 cells . Data information , data are shown as the mean of three technical replicates ; in ( D ) , data are shown as the mean of 20 individual donors . Error bars denote SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Multiple t - test .

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E. Effect of an anti-RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs. Left, a schematic showing the domain structure of S1 spike protein and anti-RBD antibody. Right, changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti-RBD antibody. Gene expression was measured by qPCR. Cytokine release in the conditioned media was measured by Luminex. Full-length S1 and S1 subunits are purified from HEK293 cells. Data information, data are shown as the mean of three technical replicates; in (D), data are shown as the mean of 20 individual donors. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Multiple t-test.


C . Validation of predicted kinases as drivers of cytokine release by siRNAs in THP1 cells . Cytokine release was measured by Luminex and normalized to scrambled siRNA .

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C. Validation of predicted kinases as drivers of cytokine release by siRNAs in THP1 cells. Cytokine release was measured by Luminex and normalized to scrambled siRNA.


C . Comparison of kinase inhibition profile of Ponatinib and Baricitinib .

C. Comparison of kinase inhibition profile of Ponatinib and Baricitinib.


B . Dose - response curves of ponatinib treatment on SARS - Cov2 NTD ( Wuhan ) - mediated cytokine release in PBMCs . Right , a table showing EC50 ( nM ) values of ponatinib - mediated inhibition of cytokine release in response to indicated SARS - Cov2 variant NTD treatments in PBMCs .

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B. Dose-response curves of ponatinib treatment on SARS-Cov2 NTD (Wuhan)-mediated cytokine release in PBMCs. Right, a table showing EC50 (nM) values of ponatinib-mediated inhibition of cytokine release in response to indicated SARS-Cov2 variant NTD treatments in PBMCs.


A . Comparison of LPS and full length S1 spike protein - mediated changes in cytokines in THP1 macrophages . Data information , , data are shown as the mean of three technical replicates . Error bars denote SEM .

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A. Comparison of LPS and full length S1 spike protein-mediated changes in cytokines in THP1 macrophages. Data information, , data are shown as the mean of three technical replicates. Error bars denote SEM.


D . Representative H & E images showing Ponatinib alleviates LPS - induced inflammatory cell infiltration , septal thickening , alveolar edema in mouse lungs . Scale bars 100 µm ( upper ) and 50 µm ( lower ) .

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D. Representative H&E images showing Ponatinib alleviates LPS-induced inflammatory cell infiltration, septal thickening, alveolar edema in mouse lungs. Scale bars 100 µm (upper) and 50 µm (lower).


E . Plots showing pre - treatment ( 60 min ) with Ponatinib ( 35 mg / Kg ) inhibits LPS - induced cytokine release in BALF . Cytokine levels were measured using Luminex . Data information , data are mean of five biological replicates from two independent studies , error bars denote SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Welch ' s t - test .

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E. Plots showing pre-treatment (60 min) with Ponatinib (35 mg/Kg) inhibits LPS-induced cytokine release in BALF. Cytokine levels were measured using Luminex. Data information, data are mean of five biological replicates from two independent studies, error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Welch's t-test.


( A ) CP20 and OVCAR4 cells were transfected with either non - target miRNA control ( miR - CTL ) , miR - 15a , or miR - 195 . 72h following transfection , cells were lysed and immunoblotted for detection of MICU1 , BMI1 , BCL2 , and MFN2 . GAPDH and VDAC were used as loading control .

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(A) CP20 and OVCAR4 cells were transfected with either non-target miRNA control (miR-CTL), miR-15a, or miR-195. 72h following transfection, cells were lysed and immunoblotted for detection of MICU1, BMI1, BCL2, and MFN2. GAPDH and VDAC were used as loading control.


( B ) miR - 195 expression in FTE188 and ovarian cell lines as determined by RT - qPCR normalized with U6 . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

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(B) miR-195 expression in FTE188 and ovarian cell lines as determined by RT-qPCR normalized with U6. Data are mean ± S.D., n=3 (Biological repeats). *P<0.05, Student's t-test.


( C ) Expression of MICU1 in FTE188 and various ovarian cell lines as determined by immunoblotting . Actin is used as the loading control .

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(C) Expression of MICU1 in FTE188 and various ovarian cell lines as determined by immunoblotting. Actin is used as the loading control.


( D ) Anti - miR - 195 was transfected in the FTE188 cell line , level of miR - 195 was measured using RT - qPCR ( left ) and MICU1 levels were measured using immunoblotting ( right ) . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

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(D) Anti-miR-195 was transfected in the FTE188 cell line, level of miR-195 was measured using RT-qPCR (left) and MICU1 levels were measured using immunoblotting (right). Data are mean ± S.D., n=3 (Biological repeats). *P<0.05, Student's t-test.


( A , B ) CP20 , OVCAR4 , and OVSAHO cells were transfected with non - target micro RNA control ( miR - CTL ) or miR - 195 , 24 h post - transfection , cells were recounted and plated as single cells for anchorage - dependent ( A ) or anchorage - independent ( B ) clonal growth . After 10 ( CP20 ) or 14 ( OVCAR4 and OVSAHO ) days , colonies were quantified using an Optronix GelCount colony counter . The left panel shows representative images of the colonies and the right panel depicts a graphical representation of data presented as percent clonal growth relative to the control ( miR - CTL ) .

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(A, B) CP20, OVCAR4, and OVSAHO cells were transfected with non-target micro RNA control (miR-CTL) or miR-195, 24 h post-transfection, cells were recounted and plated as single cells for anchorage-dependent (A) or anchorage-independent (B) clonal growth. After 10 (CP20) or 14 (OVCAR4 and OVSAHO) days, colonies were quantified using an Optronix GelCount colony counter. The left panel shows representative images of the colonies and the right panel depicts a graphical representation of data presented as percent clonal growth relative to the control (miR-CTL).


( C ) Migration of miR - CTL , miR - 195 , and miR - 195 + MICU1 ( pLYS1 - MICU1 - Flag ) transfected cells , towards serum gradient was examined and the number of cells per field was counted . The left panel shows representative images and the right panel depicts a graphical representation of relative migration compared to the control ( miR - CTL ) .

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(C) Migration of miR-CTL, miR-195, and miR-195+ MICU1 (pLYS1-MICU1-Flag) transfected cells, towards serum gradient was examined and the number of cells per field was counted. The left panel shows representative images and the right panel depicts a graphical representation of relative migration compared to the control (miR-CTL).


( D ) Invasion of miR - CTL , miR - 195 , and miR - 195 + MICU1 cells through fibronectin - coated filters was examined using Boyden chamber and number of cells per field was counted . Left panel shows representative images and the right panel depicts a graphical representation of relative invasion compared to the control ( miR - CTL ) .

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(D) Invasion of miR-CTL, miR-195, and miR-195 + MICU1 cells through fibronectin-coated filters was examined using Boyden chamber and number of cells per field was counted. Left panel shows representative images and the right panel depicts a graphical representation of relative invasion compared to the control (miR-CTL).


( B ) MICU1 3 ' UTR in LightSwitch ™ 3 ′ UTR Reporter Vector ( wild type or miR - 195 binding site deleted ) was co - transfected with either non - target miR control ( miR - CTL ) or different concentrations of miR - 195 . Post 24h transfection luciferase ( Renilla ) activity was measured . Relative light units ( RLU ) compared to miR - CTL were plotted . Data are mean ± S . D . n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

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(B) MICU1 3'UTR in LightSwitch™ 3′UTR Reporter Vector (wild type or miR-195 binding site deleted) was co-transfected with either non- target miR control (miR-CTL) or different concentrations of miR-195. Post 24h transfection luciferase (Renilla) activity was measured. Relative light units (RLU) compared to miR-CTL were plotted. Data are mean ± S.D. n=3 (Biological repeats). *P<0.05, Student's t-test.


CP20 cells were transfected as indicated , 48 h post transfection 4x106 cells were permeabilized , and extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in miR - CTL and miR - 195 transfected cells . [ Ca2 + ] out pulses and FCCP were delivered as indicated .

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CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in miR-CTL and miR-195 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.


CP20 cells were transfected as indicated , 48 h post transfection 4x106 cells were permeabilized , and extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in control siRNA ( siCTL ) and siMICU1 transfected cells . [ Ca2 + ] out pulses and FCCP were delivered as indicated .

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CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in control siRNA (siCTL) and siMICU1 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.


( E ) Bar graph illustrating the number of Ca2 + pulses handled by control miR , miR - 195 , siCTL and siMICU1 transfected cells . Mean ± SEM ; n = 3 - 4 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

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(E) Bar graph illustrating the number of Ca2+ pulses handled by control miR, miR-195, siCTL and siMICU1 transfected cells. Mean ±SEM; n=3-4 (Biological repeats). *P<0.05, Student's t-test.


( F ) Representative [ Ca2 + ] ( m ) traces after addition of FCCP ( 10 µM ) in permeabilized CP20 cells transfected with non - target miR - CTL or miR - 195 . ( G ) Quantification of resting matrix [ Ca2 + ] ( m ) after addition of FCCP . n = 5 ( Biological repeats ) * P < 0 . 05 , Student ' s t - test ,

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(F) Representative [Ca2+](m) traces after addition of FCCP (10 µM) in permeabilized CP20 cells transfected with non-target miR-CTL or miR-195. (G) Quantification of resting matrix [Ca2+](m) after addition of FCCP. n=5 (Biological repeats) *P<0.05, Student's t-test,


( H ) Representative traces of mitochondrial membrane potential ( ∆ Ψμ ) in CP20 cells transfected with miR - CTL or miR - 195 . ( I ) Quantification of relative ∆ Ψμ . ( n = 5 , Biological repeats ) .

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(H) Representative traces of mitochondrial membrane potential (∆Ψμ) in CP20 cells transfected with miR-CTL or miR-195. (I) Quantification of relative ∆Ψμ. (n=5, Biological repeats).


( J ) Intracellular lactate was measured in miR - CTL , miR - 195 , siCTL , and siMICU1 expressing OVCAR4 , CP20 , and OVSAHO cells . n = 3 , biological repeats * P < 0 . 05 , Student ' s t - test .

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(J) Intracellular lactate was measured in miR-CTL, miR-195, siCTL, and siMICU1 expressing OVCAR4, CP20, and OVSAHO cells. n=3, biological repeats *P<0.05, Student's t-test.


( K & L ) miR - CTL , miR - 195 , or miR - 195 + pLYS1 - MICU1 - Flag transfected cells were stained with MitoSOX Red , and mitochondrial ROS levels were determined by flow cytometry . The histogram shows representative staining and bar graph ( right ) shows results of three independent experiments . Mean ± SEM ; . n = 3 , biological repeats . * P < 0 . 05 when comparing with miR - CTL , # P < 0 . 05 when comparing with miR - 195 by Student ' s t - test .

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(K&L) miR-CTL, miR-195, or miR-195 + pLYS1-MICU1-Flag transfected cells were stained with MitoSOX Red, and mitochondrial ROS levels were determined by flow cytometry. The histogram shows representative staining and bar graph (right) shows results of three independent experiments. Mean ± SEM; . n=3, biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.


( A & B ) Stable cell lines for CP20 and OVCAR4 expressing GFP - miR - 195 or non - target miR - GFP were generated using lentiviral mediated transduction and were selected using puromycin ; representative images are shown . Scale bars , 50 µm .

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(A&B) Stable cell lines for CP20 and OVCAR4 expressing GFP-miR-195 or non-target miR-GFP were generated using lentiviral mediated transduction and were selected using puromycin; representative images are shown. Scale bars, 50 µm.


( C ) Relative miR - 195 expression was measured using RT - qPCR in stable cell lines , normalized with U6 . Mean ± SEM ; n = 3 , Biological repeats . * P < 0 . 05 , Student ' s t - test .

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(C) Relative miR-195 expression was measured using RT-qPCR in stable cell lines, normalized with U6. Mean ± SEM; n=3, Biological repeats. *P<0.05, Student's t-test.


( D ) CP20 and OVCAR4 stable cell lines were transfected as either empty vector ( EV ) or pLYS1 - MICU1 - Flag ( MICU1 ) and relative MICU1 levels were evaluated using immunoblotting .

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(D) CP20 and OVCAR4 stable cell lines were transfected as either empty vector (EV) or pLYS1-MICU1-Flag (MICU1) and relative MICU1 levels were evaluated using immunoblotting.


( E , F ) CP20 and OVCAR4 stable cell lines transfected as in ( D ) were counted and re - plated for anchorage - dependent ( E ) and independent clonal growth ( F ) . Quantification compared to the miR - CTL stable cells transfected with EV is shown . Mean ± SD ; n = 3 , Biological repeats . * P < 0 . 05 when comparing with miR - CTL , # P < 0 . 05 when comparing with miR - 195 by Student ' s t - test .

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(E, F) CP20 and OVCAR4 stable cell lines transfected as in (D) were counted and re-plated for anchorage-dependent (E) and independent clonal growth (F). Quantification compared to the miR-CTL stable cells transfected with EV is shown. Mean ± SD; n=3, Biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.


( A ) Female athymic mice were injected subcutaneously with either CP20 GFP - miR - CTL or CP20 - GFP - miR - 195 cells . Mice injected with the miR - 195 expressing cells ( miR - 195 ) had significantly smaller tumor volumes than control mice ( miR - CTL ) . Data are mean ± S . D , n = 10 . * P < 0 . 05 , Student ' s t - test .

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(A) Female athymic mice were injected subcutaneously with either CP20 GFP-miR-CTL or CP20- GFP-miR-195 cells. Mice injected with the miR-195 expressing cells (miR-195) had significantly smaller tumor volumes than control mice (miR-CTL). Data are mean ± S.D, n=10. *P<0.05, Student's t-test.


( B ) Tumor doubling time was significantly longer in the mice injected with miR - 195 expressing cells than in control mice . Data are mean ± S . D , n = 10 . * P < 0 . 05 , Student ' s t - test .

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(B) Tumor doubling time was significantly longer in the mice injected with miR-195 expressing cells than in control mice. Data are mean ± S.D, n=10. *P<0.05, Student's t-test.


( C ) Percent survival based on humane endpoint criteria , was calculated by the Kaplan - Meier method and P values determined by Log Rank Test .

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(C) Percent survival based on humane endpoint criteria, was calculated by the Kaplan-Meier method and P values determined by Log Rank Test.


( D ) Four tumor samples from each group were analyzed for the expression of MICU1 , pPDH , PDH , PARP , and BCL2 using immunoblotting . α tubulin was used as the loading control .

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(D) Four tumor samples from each group were analyzed for the expression of MICU1, pPDH, PDH, PARP, and BCL2 using immunoblotting. α tubulin was used as the loading control.


Representative histology images of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) cells with Ki67 expression ( Scale Bar = 50 μm ) ( E ) , CD31 positive vessels ( Scale Bar = 100 μm ) and TUNEL staining ( Scale Bar = 50 μm )

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Representative histology images of tumors from mice xenografts of CP20-GPF-miR-CTL (miR-CTL) or CP20-GPF-miR-195 (miR-195) cells with Ki67 expression (Scale Bar = 50 μm) (E), CD31 positive vessels (Scale Bar = 100 μm) and TUNEL staining (Scale Bar = 50 μm)


Representative histology images of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) cells with Ki67 expression ( Scale Bar = 50 μm ) ( F ) graph showing quantification of each marker in tumors from the experimental mice ; values are mean ± SD , n = 10 , * P < 0 . 05 , Student ' s t - test .

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Representative histology images of tumors from mice xenografts of CP20-GPF-miR-CTL (miR-CTL) or CP20-GPF-miR-195 (miR-195) cells with Ki67 expression (Scale Bar = 50 μm) (F) graph showing quantification of each marker in tumors from the experimental mice; values are mean ±SD, n=10, *P<0.05, Student's t-test.


( G ) Female athymic mice were injected subcutaneously with either CP20 GFP - miR - CTL or CP20 - GFP - miR - 195 or CP20 - GFP - miR - 195 + MICU1 cells and tumor volume was measured , values are mean ± SE , n = 10 . * P < 0 . 05 , Student ' s t - test .

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(G) Female athymic mice were injected subcutaneously with either CP20 GFP-miR-CTL or CP20- GFP-miR-195 or CP20- GFP-miR-195 + MICU1 cells and tumor volume was measured, values are mean ±SE, n=10. *P<0.05, Student's t-test.


Percentage of circulating highly differentiated CD4 T cells within CD4 cells ( upper graph ) in age - matched healthy donors or NSCLC patients before undergoing immunotherapies . G1 and G2 , groups of patients classified according to high THD cells ( G1 , > 40 % CD4 THD cells ) and low THD cells ( G2 , < 40 % CD4 THD cells ) . Relevant statistical comparisons are shown by the test of Mann - Whitney . In green , objective responders ( OR ) . In red , no OR . Below the graph , correlation of objective responses to G1 and G2 groups by the Fisher ´ s exact test .

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Percentage of circulating highly differentiated CD4 T cells within CD4 cells (upper graph) in age-matched healthy donors or NSCLC patients before undergoing immunotherapies. G1 and G2, groups of patients classified according to high THD cells (G1, >40% CD4 THD cells) and low THD cells (G2, <40% CD4 THD cells). Relevant statistical comparisons are shown by the test of Mann-Whitney. In green, objective responders (OR). In red, no OR. Below the graph, correlation of objective responses to G1 and G2 groups by the Fisher´s exact test.


Waterfall plot of change in lesion size in patients with measurable disease classified as having a G1 ( blue ) or G2 ( red ) profile . Dotted lines represents the limit to define significant progression ( upper line ) or significant regression ( lower line ) .

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Waterfall plot of change in lesion size in patients with measurable disease classified as having a G1 (blue) or G2 (red) profile. Dotted lines represents the limit to define significant progression (upper line) or significant regression (lower line).


Kaplan - Meier plot for PFS in patients treated with immunotherapies stratified only by G1 ( green ) and G2 ( red ) CD4 T cell profiles . Patients starting therapy with a G2 profile had an overall response rate ( ORR ) of 0 % and 82 % of them experienced progression or death by week 9 . ORR was 44 . 8 % for G1 patients , and the 12 - week PFS was 50 . 2 % .

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Kaplan-Meier plot for PFS in patients treated with immunotherapies stratified only by G1 (green) and G2 (red) CD4 T cell profiles. Patients starting therapy with a G2 profile had an overall response rate (ORR) of 0% and 82% of them experienced progression or death by week 9. ORR was 44.8% for G1 patients, and the 12-week PFS was 50.2%.


The scatter plot shows PD - 1 expression after co - culture of CD4 T cells from healthy donors or NSCLC patients , as indicated , with A459 - SC3 lung cancer cells . Relevant statistical comparisons with the test of Mann - Whitney are indicated .

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The scatter plot shows PD-1 expression after co-culture of CD4 T cells from healthy donors or NSCLC patients, as indicated, with A459-SC3 lung cancer cells. Relevant statistical comparisons with the test of Mann-Whitney are indicated.


Upper graphs , flow cytometry density plots of PD - 1 and LAG - 3 co - expression in CD4 T cells from healthy donors , a G1 responder ( G1 R ) , a G1 non - responder ( G1 NR ) and a G2 non - responder as indicated , following stimulation with A549 - SC3 cells . Percentage of expressing cells are indicated within each quadrant . Below , same as in the upper graphs but as a scatter plot of the percentage of CD4 T cells that simultaneously co - express PD - 1 and LAG - 3 . Relevant statistical comparisons are shown with the test of Mann - Whitney .

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Upper graphs, flow cytometry density plots of PD-1 and LAG-3 co-expression in CD4 T cells from healthy donors, a G1 responder (G1 R), a G1 non-responder (G1 NR) and a G2 non-responder as indicated, following stimulation with A549-SC3 cells. Percentage of expressing cells are indicated within each quadrant. Below, same as in the upper graphs but as a scatter plot of the percentage of CD4 T cells that simultaneously co-express PD-1 and LAG-3. Relevant statistical comparisons are shown with the test of Mann-Whitney.


Upper flow cytometry histograms of Ki67 expression in CD4 T cells from the representative subjects as indicated on the right , after stimulation with A549 - SC3 cells . Vertical dotted line indicates the cut - off value of positive vs negative Ki67 expression . The percentage of Ki67 - expressing CD4 T cells is shown within the histograms . Below , same data represented as a scatter plot from a sample of G1 and G2 donors as indicated , with relevant statistical comparisons with the test Mann - Whitney .

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Upper flow cytometry histograms of Ki67 expression in CD4 T cells from the representative subjects as indicated on the right, after stimulation with A549-SC3 cells. Vertical dotted line indicates the cut-off value of positive vs negative Ki67 expression. The percentage of Ki67-expressing CD4 T cells is shown within the histograms. Below, same data represented as a scatter plot from a sample of G1 and G2 donors as indicated, with relevant statistical comparisons with the test Mann-Whitney.


Proliferation of CD4 T cells stimulated by A549 - SC3 cells from the indicated patient groups . CD28 expression is shown together with the proliferation marker ki67 . Percentages of cells within each quadrant are shown .

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Proliferation of CD4 T cells stimulated by A549-SC3 cells from the indicated patient groups. CD28 expression is shown together with the proliferation marker ki67. Percentages of cells within each quadrant are shown.


Same as in ( D ) but in the presence of an isotype control antibody or an anti - PD - 1 antibody with the equivalent sequence to pembrolizumab . The effects on CD4 T cells from a G1 and a G2 patient are shown , divided in CD28 high or low / negative subsets as indicated . Relevant statistical comparisons are shown with paired Student ´ s t test .

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Same as in (D) but in the presence of an isotype control antibody or an anti-PD-1 antibody with the equivalent sequence to pembrolizumab. The effects on CD4 T cells from a G1 and a G2 patient are shown, divided in CD28 high or low/negative subsets as indicated. Relevant statistical comparisons are shown with paired Student´s t test.


Top , flow cytometry density plots of Ki67 expression in CD4 T cells from representative G1 or G2 patients after three cycles of therapy , activated by incubation with A549 - SC3 cells . Below , same as above but as a dot - plot graph . A comparison between proliferating CD4 T cells before and after therapy is shown in unpaired patient samples .

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Top, flow cytometry density plots of Ki67 expression in CD4 T cells from representative G1 or G2 patients after three cycles of therapy, activated by incubation with A549-SC3 cells. Below, same as above but as a dot-plot graph. A comparison between proliferating CD4 T cells before and after therapy is shown in unpaired patient samples.


Scatter plot graph with the percentage of lung cancer - specific systemic CD4 T cells quantified by an autologous DC - based antigen presentation assay ( See Materials and Methods ) , in a sample of G1 and G2 patients as indicated . Objective responses ( OR ) are shown in green . In red , patients with no OR .

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Scatter plot graph with the percentage of lung cancer-specific systemic CD4 T cells quantified by an autologous DC-based antigen presentation assay (See Materials and Methods), in a sample of G1 and G2 patients as indicated. Objective responses (OR) are shown in green. In red, patients with no OR.


The scatter plot graph on the left represents the percentage of CD4 THD cells within lung - cancer specific CD4 T cells in a sample of patients from the indicated G1 / G2 groups . On the right , same as left but representing the percentage of CD28 + CD4 T cells within lung - cancer specific CD4 T cells . Objective responders ( OR ) are shown in green .

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The scatter plot graph on the left represents the percentage of CD4 THD cells within lung-cancer specific CD4 T cells in a sample of patients from the indicated G1/G2 groups. On the right, same as left but representing the percentage of CD28+ CD4 T cells within lung-cancer specific CD4 T cells. Objective responders (OR) are shown in green.


Column graphs representing the percentage of CD4 T cells from NSCLC patients or age - matched healthy donors as represented in the graph , expressing the indicated cytokines after T cell stimulation with anti - CD3 / anti - CD28 antibodies . Data on total CD4 ( left graph ) , CD28 + subsets ( center graph ) and CD28negative subsets ( right graph ) are shown . Error bars correspond to standard deviations and bars represent means from 5 independent biological replicates ( patients ) .

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Column graphs representing the percentage of CD4 T cells from NSCLC patients or age-matched healthy donors as represented in the graph, expressing the indicated cytokines after T cell stimulation with anti-CD3/anti-CD28 antibodies. Data on total CD4 (left graph), CD28+ subsets (center graph) and CD28negative subsets (right graph) are shown. Error bars correspond to standard deviations and bars represent means from 5 independent biological replicates (patients).


representing CD4 T cells expressing only one cytokine


representing CD4 T cells expressing only cytokine ,

representing CD4 T cells expressing only cytokine, two


CD4 T cells expressing only three cytokines simultaneously


Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549 - SC3 cells from the indicated patient groups , before the start of immunotherapy . Relevant statistical comparisons are shown with the test of Mann - Whitney .

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Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney.


Dot plots of the percentage of Ki67 + proliferating CD8 T cells after ex vivo activation by A549 - SC3 cells . CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti - PD - 1 therapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . Green , objective responders ( OR ) and red , no ORs .

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Dot plots of the percentage of Ki67+ proliferating CD8 T cells after ex vivo activation by A549-SC3 cells. CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti-PD-1 therapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. Green, objective responders (OR) and red, no ORs.


Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549 - SC3 cells from the indicated patient groups , before the start of immunotherapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . but in the presence of an isotype control antibody or an anti - PD - 1 antibody molecularly equivalent to pembrolizumab . Relevant statistical comparisons are shown with comparisons carried out with paired Student ´ s t test .

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Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. but in the presence of an isotype control antibody or an anti-PD-1 antibody molecularly equivalent to pembrolizumab. Relevant statistical comparisons are shown with comparisons carried out with paired Student´s t test.


Change in CD8 CD28 + T cells from baseline to post - therapy in G1 patients ( left ) or in G2 patients ( right ) . Statistical comparisons were carried out with paired Student ´ s t test .

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Change in CD8 CD28+ T cells from baseline to post-therapy in G1 patients (left) or in G2 patients (right). Statistical comparisons were carried out with paired Student´s t test.


Scatter plots of PD - 1 / LAG - 3 - expressing CD8 T cells after activation by A459 - SC3 cells in a sample of G1 and G2 patients within CD28 + and CD28negative populations as indicated in the figure .

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Scatter plots of PD-1/LAG-3-expressing CD8 T cells after activation by A459-SC3 cells in a sample of G1 and G2 patients within CD28+ and CD28negative populations as indicated in the figure.


Dot - plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy , activated ex vivo by A549 - SC3 cells in the presence of the indicated antibodies . " Dual " represents the addition of both anti - PD - 1 and anti - LAG - 3 antibodies . Appropriate statistical comparisons are shown within the graph with two - way paired ANOVA . Data from CD28 + and CD28negative subsets are represented as indicated .

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Dot-plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated.


Dot - plot representing the percentage of proliferating cells from a sample of G2 patients before starting immunotherapy , activated ex vivo by A549 - SC3 cells in the presence of the indicated antibodies . " Dual " represents the addition of both anti - PD - 1 and anti - LAG - 3 antibodies . Appropriate statistical comparisons are shown within the graph with two - way paired ANOVA . Data from CD28 + and CD28negative subsets are represented as indicated . for CD8 T 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Dot-plot representing the percentage of proliferating cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated. for CD8 T cells.


Kaplan - Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD - L1 + tumors ( blue ) and remaining patients for whom their PD - L1 tumor status is known ( red ) .

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Kaplan-Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD-L1+ tumors (blue) and remaining patients for whom their PD-L1 tumor status is known (red).


Same as ( A ) but including all patients in the study cohort . Remaining patients ( red ) also included G1 patients with PD - L1 low / negative tumors , G1 patients with unknown PD - L1 tumor status and G2 patients with either PD - L1 + or PD - L1 negative tumors .

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Same as (A) but including all patients in the study cohort. Remaining patients (red) also included G1 patients with PD-L1 low/negative tumors, G1 patients with unknown PD-L1 tumor status and G2 patients with either PD-L1+ or PD-L1 negative tumors.


B Equilibrium titration of Hsf1 binding to Alexa Fluor ® 488 - labeled HSE - DNA . Fluorescence polarization plotted versus the Hsf1 trimer concentration . KD = 4 . 7 ± 1 . 9 nM ( n = 4 ) .

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B Equilibrium titration of Hsf1 binding to Alexa Fluor® 488-labeled HSE-DNA. Fluorescence polarization plotted versus the Hsf1 trimer concentration. KD = 4.7 ± 1.9 nM (n = 4).


C Hsf1 rapidly switches between different HSE - containing double stranded DNA oligonucleotides . Trimeric Hsf1 was pre - incubated with Alexa Fluor ® 488 - labeled HSE - DNA in MgCl2 - free buffer . At timepoint 0 ( arrow ) buffer , 4 mM MgCl2 , 2 mM ATP + 4 mM MgCl2 , control DNA ( DNACtrl at 10 - fold molar excess over HSE - DNA ) , and HSE - containing DNA was added as indicated and fluorescence polarization followed over time .

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C Hsf1 rapidly switches between different HSE-containing double stranded DNA oligonucleotides. Trimeric Hsf1 was pre-incubated with Alexa Fluor® 488-labeled HSE-DNA in MgCl2-free buffer. At timepoint 0 (arrow) buffer, 4 mM MgCl2, 2 mM ATP+4 mM MgCl2, control DNA (DNACtrl at 10-fold molar excess over HSE-DNA), and HSE-containing DNA was added as indicated and fluorescence polarization followed over time.


D Trimeric Hsf1 were bound to HSE - DNA and the indicated components added at timepoint 0 .

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D Trimeric Hsf1 were bound to HSE-DNA and the indicated components added at timepoint 0.


E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70 . Rates were

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E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70. Rates were determined


The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on temperature ( for 25 ° C n = 6 , for 37 ° C n = 5 , T - test , p = 0 . 011 ) ( F )

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The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on temperature (for 25°C n = 6, for 37°C n = 5, T-test, p = 0.011) (F)


The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of Hsc70 and DnaJB1 ( n = 3 ) ( G )

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The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of Hsc70 and DnaJB1 (n = 3) (G)


The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of ATP ( n = 3 ) ( H )

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The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of ATP (n = 3) (H)


The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of the nucleotide exchange factor Apg2 ( HSPA4 ) ( n = 3 , ANOVA , * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ) ( I ) . Shown are mean ± SD .

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The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of the nucleotide exchange factor Apg2 (HSPA4) (n = 3, ANOVA, **, p < 0.01; ****, p < 0.0001) (I). Shown are mean ± SD.


A , B Standard Hsc70 / DnaJB1 - mediated dissociation reaction of Hsf1 from Alexa Fluor ® 488 - labeled HSE - DNA monitored by fluorescence polarization ( A ) . The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x - axis , the reaction stopped by addition of blue - native loading buffer and stored on ice until separation by blue - native PAGE , blotted onto a PVDF membrane and detected with an Hsf1 - specific antiserum ( B ) . Lanes 1 , purified Hsf1 trimer ( T ) ; 2 , purified Hsf1 monomer ( M ) ; 3 - 11 , samples from the Hsc70 / DnaJB1 - mediated Hsf1 dissociation reaction ( 0 to 80 min ) ; 12 - 15 , Dissociation reaction incubated for 80 min missing individual components as indicated . HO , higher order oligomers ; T , trimer ; D , dimer ; M , mono mer . C Quantification of the amounts of Hsf1 species of the blot shown in ( B ) and five similar blots . For each lane the intensities of the two areas indicated by the brackets to the right were integrated ; upper bracket , DNA bound timers and higher order oligomers ( Hsf1b ) ; lower bracket , monomers and Hsf1 - species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA ( Hsf1d ) . Shown are means ± SD ( n = 3 ) . Inset , deoligomerization rate as determined by fitting an exponential decay function to the data . Shown are means ± SD ( n = 3 )

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A,B Standard Hsc70/DnaJB1-mediated dissociation reaction of Hsf1 from Alexa Fluor® 488-labeled HSE-DNA monitored by fluorescence polarization (A). The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x-axis, the reaction stopped by addition of blue-native loading buffer and stored on ice until separation by blue-native PAGE, blotted onto a PVDF membrane and detected with an Hsf1-specific antiserum (B). Lanes 1, purified Hsf1 trimer (T); 2, purified Hsf1 monomer (M); 3-11, samples from the Hsc70/DnaJB1-mediated Hsf1 dissociation reaction (0 to 80 min); 12-15, Dissociation reaction incubated for 80 min missing individual components as indicated. HO, higher order oligomers; T, trimer; D, dimer; M, monomer. C Quantification of the amounts of Hsf1 species of the blot shown in (B) and five similar blots. For each lane the intensities of the two areas indicated by the brackets to the right were integrated; upper bracket, DNA bound timers and higher order oligomers (Hsf1b); lower bracket, monomers and Hsf1-species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA (Hsf1d). Shown are means ± SD (n = 3). Inset, deoligomerization rate as determined by fitting an exponential decay function to the data. Shown are means ± SD (n = 3)


C Fit of two Gaussian distributions to the peak intensities for peptic peptides 159 - 168 ( left ) , 169 - 175 ( middle ) , and 176 - 189 ( right ) . Representative plots of 3 independent experiments .

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C Fit of two Gaussian distributions to the peak intensities for peptic peptides 159-168 (left), 169-175 (middle), and 176-189 (right). Representative plots of 3 independent experiments.


E Relative deuteron incorporation of the low ( solid bars ) and high ( open bars ) exchanging subpopulations .

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E Relative deuteron incorporation of the low (solid bars) and high (open bars) exchanging subpopulations.


B KD values for the indicated peptides as determined by fluorescence polarization . Peptides N74 - Q86 - C ( NMYGFRKVVHIEQC ) , P223 - H235 - C ( PKYSRQFSLEHVHC ) and A343 - R352 - C ( ASVTALTDARC ) represent the most hydrophobic part of HX protected segments 78 - 98 , 229 - 252 , and 347 - 375 , respectively ; N200 - K208 - C ( NRILGVKRKC ) , N200 - K208 - C _ SSS ( NRSSGSKRKC ) , G204 - N214 - C ( GVKRKIPLMLNC ) , and G204 - N214 - C _ SSS ( GVKRKSPSMSNC ) represent the two potential Hsc70 binding sites in segment 202 - 213 in original sequence or with hydrophobic residues replaced by serine ; S461 - Q471 - C ( SGKQLVHYTAQC ) and Y468 - S480 - C ( YTAQPLFLLDPGSC ) represent the two potential Hsc70 binding sites in segment 442 - 474 .

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B KD values for the indicated peptides as determined by fluorescence polarization. Peptides N74-Q86-C (NMYGFRKVVHIEQC), P223-H235-C (PKYSRQFSLEHVHC) and A343-R352-C (ASVTALTDARC) represent the most hydrophobic part of HX protected segments 78-98, 229-252, and 347-375, respectively; N200-K208-C (NRILGVKRKC), N200-K208-C_SSS (NRSSGSKRKC), G204-N214-C (GVKRKIPLMLNC), and G204-N214-C_SSS (GVKRKSPSMSNC) represent the two potential Hsc70 binding sites in segment 202-213 in original sequence or with hydrophobic residues replaced by serine; S461-Q471-C (SGKQLVHYTAQC) and Y468-S480-C (YTAQPLFLLDPGSC) represent the two potential Hsc70 binding sites in segment 442-474.


C Hsf1 ( 1 - 408 ) with a deleted TAD is dissociated from HSE - DNA by Hsc70 , DnaJB1 and ATP with similar rates as Hsf1wt . Fluorescence polarization assays containing Alexa Fluor ® 488 - HSE - DNA bound Hsf1 ( 1 - 408 ) trimers and the indicated components .

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C Hsf1(1-408) with a deleted TAD is dissociated from HSE-DNA by Hsc70, DnaJB1 and ATP with similar rates as Hsf1wt. Fluorescence polarization assays containing Alexa Fluor® 488-HSE-DNA bound Hsf1(1-408) trimers and the indicated components.


D The HR - B proximal Hsc70 binding site is essential for Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1 .

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D The HR-B proximal Hsc70 binding site is essential for Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1.


A The rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1 depends on the number of HR - B proximal Hsc70 binding sites in the Hsf1 trimer . Hsf1wt ( wt ) and Hsf1 ∆ ( 202 - 213 ) ( ∆ ) monomers were mixed in the indicated ratios and heat shocked at 42 ° C for 10 min to form mixtures of different homo - and heterotrimers as indicated with the cartoons ( the red line and the arrow head indicate the Hsc70 binding site ) . Numbers indicate the relative fraction of the different species .

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A The rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1 depends on the number of HR-B proximal Hsc70 binding sites in the Hsf1 trimer. Hsf1wt (wt) and Hsf1∆(202-213) (∆) monomers were mixed in the indicated ratios and heat shocked at 42°C for 10 min to form mixtures of different homo- and heterotrimers as indicated with the cartoons (the red line and the arrow head indicate the Hsc70 binding site). Numbers indicate the relative fraction of the different species.


B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA - bound Hsf1 . Hsf1 _ t10 / 20 / 30 , region 202 - 213 moved by 10 , 20 , or 30 residues towards the C - terminus .

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B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA-bound Hsf1. Hsf1_t10/20/30, region 202-213 moved by 10, 20, or 30 residues towards the C-terminus.


C Anti - FLAG antibodies are not able to dissociate HSE - DNA - bound Hsf1 . Red lines in the cartoon indicate the Hsc70 binding site ; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK . Hsf1 _ i201 / 211 / 221 , FLAG - epitope inserted after residue 201 , 211 or 221 . Anti - FLAG antibodies were split in halfmers by incubation with 2 mM

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C Anti-FLAG antibodies are not able to dissociate HSE-DNA-bound Hsf1. Red lines in the cartoon indicate the Hsc70 binding site; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK. Hsf1_i201/211/221, FLAG-epitope inserted after residue 201, 211 or 221. Anti-FLAG antibodies were split in halfmers by incubation with 2 mM DTT


Anti - FLAG antibody halfmers bound to FLAG - epitope containing Hsf1 variants . Hsf1wt and FLAG - insertion variants were analyzed by blue - native gel electrophoresis in the absence or presence of DTT - treated anti - FLAG antibodies as indicated and Hsf1 detected by immunoblotting ; t - bound , FLAG antibody - bound Hsf1 trimers ; m - bound , FLAG antibody bound Hsf1 monomer .

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Anti-FLAG antibody halfmers bound to FLAG-epitope containing Hsf1 variants. Hsf1wt and FLAG-insertion variants were analyzed by blue-native gel electrophoresis in the absence or presence of DTT-treated anti-FLAG antibodies as indicated and Hsf1 detected by immunoblotting; t-bound, FLAG antibody-bound Hsf1 trimers; m-bound, FLAG antibody bound Hsf1 monomer.


F Trimer - to - monomer ratio of freshly purified Hsf1wt and Hsf1 - I190S , I194S in the absence of heat shock , determined by gel filtration and BN -

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F Trimer-to-monomer ratio of freshly purified Hsf1wt and Hsf1-I190S,I194S in the absence of heat shock, determined by gel filtration and BN-Gel


G Rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1wt and Hsf1 - I190S ,

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G Rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1wt and Hsf1-I190S,I194S