Volcano plot of DEGs in ESCs ( # A4 , # A10 , # A11 ) versus ( # A6 , # A13 , # A17 ) . definition of up ( red dots ) or down ( blue dots ) genes is as follows : of up or down genes is > 1 . 5 < two - thirds , respectively , with q - value < 0 . 05 .

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Volcano plot of DEGs in female ESCs (#A4, #A10, #A11) versus males (#A6, #A13, #A17). The definition of up (red dots) or down (blue dots) genes is as follows: the ratio of up or down genes is > 1.5 and < two-thirds, respectively, with q-value< 0.05.


Brcc3 expression confirmed by - seq [ biological replicates ( n = 3 ) with three different lines ( : # A6 , # A13 , # A17 ; : # A4 , # A10 , # A11 ) ] .

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Brcc3 expression confirmed by RNA-seq [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Brcc3 expression confirmed by RT - qPCR [ biological replicates ( n = 3 ) with three different lines ( : # A6 , # A13 , # A17 ; : # A4 , # A10 , # A11 ) ] .

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Brcc3 expression confirmed by RT-qPCR [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Brcc3 expression confirmed by western blotting [ biological replicates ( n = 3 ) with three different lines ( : # A6 , # A13 , # A17 ; : # A4 , # A10 , # A11 ) ] .

Brcc3 expression confirmed by western blotting [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Confirmation of Brcc3 expression from knockdown samples in by RT - qPCR ( technical duplicates )

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Confirmation of Brcc3 expression from knockdown samples in by RT-qPCR (technical duplicates)


Confirmation of Brcc3 expression from knockdown samples in western blotting


Nanog HR with Brcc3 knockdown ( three biological replicates ) . of GFP - positive colonies after gene targeting to Nanog locus is shown , as shown in Figure 2C . combined number of GFP - positive total number of colonies are shown ( please note that total number of # A13 for drug screening was 3 . 67 × 107 because of contamination of 1 out of 12 dishes , compared with other colonies for which 4 × 107 were used ) .

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Nanog HR assay with Brcc3 knockdown (three biological replicates). The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown, as shown in Figure 2C. The combined number of GFP-positive cells and total number of colonies are shown (please note that total cell number of #A13 for drug screening was 3.67×107 because of contamination of 1 out of 12 dishes, compared with the other colonies for which 4×107 cells were used).


Kaplan - Meier curves for overall survival of breast cancer on BRCC3 ( A ) , XIST ( B ) , BRCC3 / XIST ( C ) expression . HR : hazard .

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Kaplan-Meier curves for the overall survival of breast cancer patients based on BRCC3 (A), XIST (B), and BRCC3/XIST(C) expression levels. HR: hazard ratio.


for collagen inserts in panel B represent hematoxylin ( nucleus ) & eosin ( cytoplasm ) for same tissue of collagen In cases n = 4 control mice , ≥ 3 mice for 2mg 5 mg ) for each is shown in histogram in right

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IHC images for collagen The inserts in panel B represent the hematoxylin (nucleus) & eosin (cytoplasm) staining for the same tissue area of the collagen staining In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) The quantification for each staining is shown in the histogram in the right side


fo aSMA , CD68 , CD204 For aSMA , CD68 , CD204 of positive is relative to control condition . In cases n = 4 control mice , ≥ 3 mice for 2mg 5 mg ) Scale bar : 100 mm . for each is shown in histogram in right

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IHC images fo aSMA, CD68, and CD204 For aSMA, CD68, and CD204 the percentage of positive cells is relative to the control condition. In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) Scale bar: 100 mm. The quantification for each staining is shown in the histogram in the right side


Representative traces tracking bead displacements Histogram shows relative bead displacement for first last , n = 27 control n = 37

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Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse, n=27 control and n=37 tamoxifen


Histogram shows of gel contraction , n > 10 gels per

Histogram shows percentage of gel contraction, n>10 gels per condition


Representative mechanosensing trace of PSC treated with tamoxifen Estrogen receptor ( ER antagonists Histogram shows relative bead displacement for first last , n > 20 for

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Representative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor (ER antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions


Representative mechanosensing trace of PSC treated with tamoxifen an G coupled - estrogen receptor ( GPER ) antagonists Histogram shows relative bead displacement for first last , n > 20 for

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Representative mechanosensing trace of PSC treated with tamoxifen an G coupled-protein estrogen receptor (GPER) antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions


Histogram shows of gel contraction by PSCs treated with tamoxifen in presence of indicated

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Histogram shows percentage of gel contraction by PSCs treated with tamoxifen in the presence of indicated antagonist


Histogram shows of gel contraction after treatment with indicated agonist , n > 10 gels per

Histogram shows percentage of gel contraction after treatment with indicated agonist, n>10 gels per conditio


Young " s modulus for matrices previously remodeled by PSCs ; control n = 126 tamoxifen n = 145 . Lines error bars indicate mean ± s . e . m . Four experimental

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Young"s modulus for matrices previously remodeled by PSCs; control n=126 and tamoxifen n=145. Lines and error bars indicate mean±s.e.m. Four experimental repl


Representative of H & E showing AsPC1 cancer invasions in matrices previously remodeled by control PSCs ( top ) or tamoxifen treated PSCs ( bottom ) , scale bar 50 μm AsPC1 invasion , central box represents values from lower to upper quartile . middle represents mean . vertical extends from minimum to maximum value , n = 30 control n = 40

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Representative images of H&E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs (top) or tamoxifen treated PSCs (bottom), scale bar 50 μm AsPC1 invasion quantification, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value, n=30 control and n=40 tamoxifen


Immunofluorescence images of PSCs stained for YAP


of / cytoplasm YAP in PSCs ( four experimental replicates )

Quantification of the nuclear/cytoplasm YAP in PSCs (four experimental replicates)


qPCR mRNA for YAP target genes connective tissue grow factor ( CTGF ) Ankyrin Repeat Domain 1 ( ANRKD1 ) , ( three experimental replicates )

qPCR mRNA levels for YAP target genes connective tissue grow factor (CTGF) and Ankyrin Repeat Domain 1 (ANRKD1), (three experimental replicates)


Western blot bands for YAP , pS127 YAP total . ( E ) of YAP pYAP Ser127 normalised to total , expressed relative to unstimulated control ( n = 8 )

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Western blot bands for YAP, pS127 YAP and total protein. (E) Quantification of YAP and pYAP Ser127 normalised to total protein, expressed relative to unstimulated control (n=8)


for YAP in KPC mice pancreatic tissues ( control treated with 2 5 mg tamoxifen ) , YAP is brown . Scale bar : 100

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IHC images for YAP staining in KPC mice pancreatic tissues (control and treated with 2 and 5 mg tamoxifen), YAP staining is brown. Scale bar: 100 μm


of YAP positive nucleus . n = 5 control mice , ≥ 3 mice for 2mg 5

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Quantification of YAP positive nucleus. n=5 control mice, and ≥3 mice for 2mg and 5 mg


IF for total ( MLC - 2 ) phosphorylated ( pMLC - 2 ) in PSCs , scale bar is 20

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IF images for total (MLC-2) and phosphorylated (pMLC-2) in PSCs, scale bar is 20 μm


of in A . Each data point represents a Western blot for total phosphorylated mlc -

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Quantification of staining in A. Each data point represents a cell Western blot for total and phosphorylated mlc-2


of total active RhoA , expressed as of total RhoA in control

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Quantification of total and active RhoA, expressed as percentage of the total RhoA in the control condition


for phosphorylated mlc - 2 in mice pancreatic tissues ( n = 5 control , n = 5 2mg , n = 3 5mg ) . Scale bar : 100 μm of in

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IHC images for phosphorylated mlc-2 in mice pancreatic tissues (n=5 control, n=5 2mg, and n=3 5mg). Scale bar: 100 μm Quantification of staining in E


D Immunofluorescence of - damage - associated PARP . show adjacent normal crypts of tissue P009T , marked by N T , respectively , tissue of P008 P009 , as indicated . Scale bar 100µm . Significance was assessed by an unpaired t - , after blinded of 10 random per .

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D Immunofluorescence of DNA-damage-associated nuclear protein PARP. Images show adjacent normal and tumor crypts of tissue P009T, marked by N and T, respectively, and tumor tissue of patients P008 and P009, as indicated. Scale bar 100µm. Significance was assessed by an unpaired t-test, after blinded analysis of 10 random images per tumor.


E Activities of Wnt / β - catenin MAPK target genes in organoid single transcriptomes , ordered along latent time .

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E Activities of Wnt/β-catenin and MAPK target genes in organoid single cell transcriptomes, ordered along latent time.


F Activities of TFF3 , FABP1 , MKI67 MMP7 in organoid single transcriptomes , ordered along latent time .

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F Activities of TFF3, FABP1, MKI67 and MMP7 in organoid single cell transcriptomes, ordered along latent time.


A Gene expression of LGR5 EPHB2 , along activity gradients of LGR5 - ISC or MAPK target gene signatures .

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A Gene expression of LGR5 and EPHB2, along activity gradients of LGR5-ISC or MAPK target gene signatures.


C of key CyTOF data . Average activities of selected analytes are given as log fold - change after normalization to DMSO control condition . Range of color scale was adjusted for each analyte . For relative changes between analytes , see Appendix Fig . S7 ) .

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C Heatmap of key CyTOF data. Average activities of selected analytes are given as log fold-change after normalization to DMSO control condition. Range of color scale was adjusted for each analyte. For relative changes between all analytes, see Appendix Fig. S7).


F Scaled signature expression single gene expression moments per , under control ( grey ) preferred treatment ( color ) conditions , as indicated . 10 % of populating end of latent time are displayed in bold . It is of note that latent time is linear thus cannot capture multiple populations located at developmental end points . Densities at side lines display expression in .

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F Scaled signature expression and single gene expression moments per cell, under control (grey) and preferred treatment (color) conditions, as indicated. 10% of cells populating the end of latent time are displayed in bold. It is of note that latent time is linear and thus cannot capture multiple populations located at developmental end points. Densities at the side lines display expression in all cells.


( C ) Graph showing log10 CFU / at 4 days post - inoculation with indicated strains ( n ≥ 5 ) . Each circle represents values obtained in , whereas mean ± s . d . is represented with lines inside each group . No statistical differences were found between infection groups using a one - sided ANOVA followed by post - hoc Fisher ' s PLSD .

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(C) Graph showing log10 CFU/gland at 4 days post-inoculation with the indicated strains (n≥5). Each circle represents the values obtained in individual animals, whereas mean ± s.d. is represented with lines inside each group. No statistical differences were found between infection groups using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test.


Plots showing RT - qPCR conducted to quantify relative expression of indicated interleukins coding genes in mammary . Results are expressed as mean ± s . d ( n = 10 for PBS , n = 5 for WT n = 4 for CV2 ) of 2 - ΔΔCt relative expression values of indicated interleukins ; value from each was calculated from three technical replicates . values obtained in PBS group were used as control for normalization of gene expression ( = 1 ) . Statistical was performed using a one - sided ANOVA followed by post - hoc Fisher ' s PLSD : ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 005 ) .

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Plots showing the RT-qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands. Results are expressed as the mean ± s.d (n=10 for PBS, n=5 for WT and n=4 for CV2) of the 2-ΔΔCt relative expression values of the indicated interleukins; the value from each individual animal was calculated from three technical replicates. The values obtained in the PBS group were used as control for normalization of gene expression (=1). Statistical analysis was performed using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test: (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005).


( B ) Comparison of turbidimetric results obtained with mpn142 mpn142Opt secretion signals . Culture supernatants ( n = 1 ) were collected at 0 h , 5 h or 24 h post - inoculation as indicated . WT medium ( Hayflick ) were added as controls .

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(B) Comparison of the turbidimetric assay results obtained with mpn142 and mpn142Opt secretion signals. Culture supernatants (n=1) were collected at 0 h, 5 h or 24 h post-inoculation as indicated. WT strain and medium (Hayflick) were added as controls.


( A , B ) Mature S . aureus biofilms were allowed to develop for 24 h in polystyrene plates then treated for indicated time intervals with suspensions , or culture supernatants of CV2 or CV2 - DispB strains ( A ) or WT or WT - DispB ( B ) . Biofilm presence was assessed by crystal violet included a negative control ( i . e . , crystal violet without biofilm ) . results are expressed as mean ± s . d . of OD 595 nm absorbance values obtained from three different biological replicates ( n = 3 ) statistically compared by one - sided ANOVA followed by post - hoc Fisher ' s PLSD : * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 ; * * * * P ≤ 0 .

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(A, B) Mature S. aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions, or culture supernatants of the CV2 or the CV2-DispB strains (A) or the WT or the WT-DispB strain (B). Biofilm presence was assessed by crystal violet staining and included a negative staining control (i.e., crystal violet without biofilm). The results are expressed as mean ± s.d. of OD 595 nm absorbance values obtained from three different biological replicates (n=3) and statistically compared by one-sided ANOVA followed by the post-hoc Fisher's PLSD test: *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005; ****P ≤ 0.00005


( C , D ) Plots showing results obtained from in vitro or ex vivo dispersion assays with indicated strains from control . Each circle represents OD 595 nm values obtained for catheters ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Results from one - sided ANOVA followed by Fisher ' s PLSD are shown for treatments statistically different from those on strains not secreting dispersin B ( i . e . , WT or CV2 strains ) . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

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(C, D) Plots showing results obtained from the in vitro or ex vivo dispersion assays with the indicated strains and from the staining control. Each circle represents the OD 595 nm values obtained for individual catheters (n≥4), whereas mean ± s.d. is represented with lines inside each group. Results from one-sided ANOVA followed by Fisher's PLSD test are shown for treatments statistically different from those based on strains not secreting dispersin B (i.e., the WT or CV2 strains). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


( C , D ) Plots showing SUV60 variation ( % ) between day 1 day 4 of different treatments . Each circle represents SUV60 variation obtained for ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Data below dotted lines indicate that SUV 60 values decreased at D4 post - treatment . Results from one - sided ANOVA followed by Fisher ' s PLSD are shown . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

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(C, D) Plots showing the SUV60 variation (%) between day 1 and day 4 of the different treatments. Each circle represents the SUV60 variation obtained for individual animals (n≥4), whereas mean ± s.d. is represented with lines inside each group. Data below the dotted lines indicate that the SUV 60 values decreased at D4 post-treatment. Results from one-sided ANOVA followed by Fisher's PLSD test are shown. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


( G ) Baf A1 sensitivity for NDST3 KO after NDST3 restoration . NDST3 KO with stable expression of Flag - NDST3 or Flag - EGFP ( control ) were treated with 25 nM Baf A1 for 72 h , survival was measured using calcein AM . survival was calculated as of treated relative to untreated ( n = 4 independent cultures , * * * * P < 0 . 0001 ) . Data information : Error bars represent ± standard deviation . Scale bar , 200 μm .

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(G) Baf A1 sensitivity assay for NDST3 KO cells after the NDST3 restoration. The NDST3 KO cells with stable expression of Flag-NDST3 or Flag-EGFP (control) were treated with 25 nM Baf A1 for 72 h, and cell survival was measured using calcein AM staining. The cell survival was calculated as the fluorescence intensity of the treated cells relative to the untreated cells (n = 4 independent cultures, ****P < 0.0001). Data information: Error bars represent ± standard deviation. Scale bar, 200 μm.


( C ) Lysosomal pH values in WT NDST3 KO RPE1 calculated from of LysoSensor Yellow / Blue dextran against pH calibration curve in ( B ) ( n = 3 independent cultures , * P = 0 . 0242 ) . ( D ) Lysosomal pH values measured for WT NDST3 KO RPE1 after treatment with Baf A1 ( 100 nM , 1h ) ( n = 3 independent cultures , * P = 0 . 0305 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm .

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(C) Lysosomal pH values in WT and NDST3 KO RPE1 cells calculated from the fluorescence ratio of LysoSensor Yellow/Blue dextran staining against the pH calibration curve in (B) (n = 3 independent cultures, *P = 0.0242). (D) Lysosomal pH values measured for WT and NDST3 KO RPE1 cells after the treatment with Baf A1 (100 nM, 1h) (n = 3 independent cultures, *P = 0.0305). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.


( F ) Magic Red for Cat B proteolytic activity . WT NDST3 KO RPE1 were treated with vehicle ( VEH ) or Baf A1 ( 100 nM , 1 h ) then stained with MR - ( RR ) 2 to measure Cathepsin B activity ( n = 48 in VEH - treated WT group , n = 37 in VEH - treated NDST3 KO group , n = 64 in Baf A1 - treated WT group , n = 42 in Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) .

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(F) Magic Red assay for Cat B proteolytic activity. WT and NDST3 KO RPE1 cells were treated with vehicle (VEH) or Baf A1 (100 nM, 1 h) and then stained with MR-(RR)2 to measure the Cathepsin B activity (n = 48 cells in the VEH-treated WT group, n = 37 cells in the VEH-treated NDST3 KO group, n = 64 cells in the Baf A1-treated WT group, n = 42 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).


( G ) Alexa Fluor ( AF ) 488 - dextran degradation . WT NDST3 KO RPE1 were loaded with AF488 - dextran in culture medium observed for . AF488 signals without a chase period , with a 4 - h chase period , with 4 - h chase period in presence of Baf A1 ( 100 nM , 1h ) were recorded as of uptake , chase , chase with Baf A1 , respectively . AF488 per was quantified calculated into degradation according to equation in Materials Methods ( n = 62 in VEH - treated WT group , n = 36 in VEH - treated NDST3 KO group , n = 65 in Baf A1 - treated WT group , n = 78 in Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) .

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(G) Alexa Fluor (AF) 488-dextran degradation assay. WT and NDST3 KO RPE1 cells were loaded with AF488-dextran in culture medium and observed for fluorescence. The AF488 fluorescence signals without a chase period, with a 4-h chase period, and with the 4-h chase period in the presence of Baf A1 (100 nM, 1h) were recorded as fluorescence of uptake, chase, and chase with Baf A1, respectively. The AF488 intensity per cell was quantified and calculated into degradation percentage according to the equation in Materials and Methods (n = 62 cells in the VEH-treated WT group, n = 36 cells in the VEH-treated NDST3 KO group, n = 65 cells in the Baf A1-treated WT group, n = 78 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).


( J ) LC3 turnover after a 48 - h Baf A1 treatment . WT NDST3 KO RPE1 were cultured with 25 nM Baf A1 for 48 h analyzed for LC3B - II fold change as in ( I ) ( n = 6 independent experiments , * * P = 0 . 0014 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm .

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(J) The LC3 turnover assay after a 48-h Baf A1 treatment. WT and NDST3 KO RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B-II fold change as in (I) (n = 6 independent experiments, **P = 0.0014). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.


( A ) To examine of V - ATPase V1 - V0 holoenzyme in WT NDST3 KO RPE1 , fractions were analyzed by immunoblotting using antibodies against V - ATPase subunits V1A ( ATP6V1A ) , V1C1 ( ATP6V1C1 ) , V0D ( ATP6V0D ) . LAMP1 vinculin were used as loading controls for , respectively . ( B ) of of ATP6V0D relative to LAMP1 in fraction from ( A ) ( n = 3 independent experiments , P = 0 . 7786 ) . Data information : Error bars represent ± standard deviation .

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(A) To examine the levels of the V-ATPase V1-V0 holoenzyme in WT and NDST3 KO RPE1 cells, cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V-ATPase subunits V1A (ATP6V1A), V1C1 (ATP6V1C1), and V0D (ATP6V0D). LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins, respectively. (B) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from (A) (n = 3 independent experiments, P = 0.7786). Data information: Error bars represent ± standard deviation.


( A , B ) Perinuclear enrichment colocalization of acetylated microtubules , lysosomes , V - ATPase V1 subunits . RPE1 stably expressing LAMP1 - GFP were treated with vehicle ( VEH ) or 10 μg / mL nocodazole ( Noco ) for 6 h before being fixed subjected to immunofluorescence with antibodies against ATP6V1C1 acetylated - α - tubulin ( Ac - α - tubulin ) . nucleus was stained with DAPI . Representative of LAMP1 - GFP , ATP6V1C1 , Ac - α - tubulin immunostaining are shown in ( A ) . Representative distribution profiles of Ac - α - tubulin , LAMP1 - GFP ATP6V1C1 were plotted by Fiji software in ( B ) .

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(A, B) Perinuclear enrichment and colocalization of acetylated microtubules, lysosomes, and V-ATPase V1 subunits. RPE1 cells stably expressing LAMP1-GFP were treated with vehicle (VEH) or 10 μg/mL nocodazole (Noco) for 6 h before being fixed and subjected to immunofluorescence staining with antibodies against ATP6V1C1 and acetylated-α-tubulin (Ac-α-tubulin). The nucleus was stained with DAPI. Representative images of LAMP1-GFP, ATP6V1C1, and Ac-α-tubulin immunostaining are shown in (A). Representative distribution profiles of Ac-α-tubulin, LAMP1-GFP and ATP6V1C1 were plotted by Fiji software in (B).


assembly of V - ATPase V1 - V0 holoenzymes in WT RPE1 over - expressing α - tubulin deacetylase HDAC6 or EGFP as a control . Representative immunoblots for ATP6V0D , ATP6V1A , ATP6V1C1 , LAMP1 GAPDH are shown in ( H ) .

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The assembly of V-ATPase V1-V0 holoenzymes in WT RPE1 cells over-expressing the α-tubulin deacetylase HDAC6 or EGFP as a control. Representative immunoblots for ATP6V0D, ATP6V1A, ATP6V1C1, LAMP1 and GAPDH are shown in (H).


( M - O ) Loss of NDST3 enhances microtubule acetylation colocalization of lysosomes with V - ATPase V1 subunits . WT NDST3 KO PRE1 stably expressing LAMP1 - GFP were fixed analyzed by immunofluorescence with antibodies against ATP6V1C1 Ac - α - tubulin . relative of Ac - α - tubulin ( N ) ( n = 75 in WT group , n = 79 in NDST3 KO group , * * * * P < 0 . 0001 ) colocalization of LAMP1 - GFP with ATP6V1C1 ( O ) ( n = 75 in WT group , n = 79 in NDST3 KO group , * * * * P < 0 . 0001 ) are quantified .

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(M-O) Loss of NDST3 enhances microtubule acetylation and the colocalization of the lysosomes with V-ATPase V1 subunits. WT and NDST3 KO PRE1 cells stably expressing LAMP1-GFP were fixed and analyzed by immunofluorescence staining with antibodies against ATP6V1C1 and Ac-α-tubulin. The relative levels of Ac-α-tubulin (N) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) and the colocalization of LAMP1-GFP with ATP6V1C1 (O) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) are quantified.


( C ) In vitro enzymatic quantitating effect of NDST3 on α - tubulin acetylation . Flag - NDST3 was expressed in HEK 293 immunoprecipitated by anti - Flag beads . Flag - EGFP was used as a control . immunoprecipitated 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 . 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 ) same in vitro enzymatic as in ( C ) , except that purified tubulin was polymerized in presence of 20 μM Taxol 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 relative to β - tubulin in B lymphocytes derived from healthy controls C9orf72 - linked ALS , 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 ) of NDST3 relative to GAPDH ( D ) ( * * P = 0 . 0013 ) , Ac - α - tubulin relative to total α - tubulin ( E ) ( * P = 0 . 0379 ) , total α - tubulin relative to GAPDH ( F ) ( P = 0 . 5700 ) , as examined by immunoblotting , in spinal cord tissues from healthy controls C9orf72 - linked ALS ( n = 4 biologically independent samples in control group n = 5 biologically independent samples in ALS 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 , as compared to control shRNA ( shCTRL ) - treated . of C9orf72 NDST3 expression relative to GAPDH is shown in ( H ) ( n = 4 independent experiments , * * * P = 0 . 0004 ) ( I ) ( n = 4 independent experiments , * * * P = 0 . 0001 ) , respectively . ( J - L ) Immunoblotting assays showing up - regulation of NDST3 in RPE1 over - expressing Flag - C9orf72 compared to those expressing Flag - EGFP as control . of C9orf72 NDST3 expression relative to GAPDH is shown in ( K ) ( n = 3 independent experiments , * * P = 0 . 0011 ) ( L ) ( n = 3 independent experiments , * * P = 0 . 0033 ) , respectively . Data information : Error bars represent ± standard deviation .

O O O O O O O O O O O O B-MEASURED_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 B-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_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

(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 for WT , NDST3 KO , Noco - treated NDST3 KO RPE1 expressing proline - arginine poly - dipeptide ( PR82 ) . 24 h after transfection of PR82 , WT NDST3 KO were treated with vehicle or 10 μg / mL Noco overnight . survival measured using calcein AM at 48 h post - transfection was quantified as of for treated relative to that of untreated ( 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 ) of Ki67 TDP - 43 in regions of frontal cortices from 6 - mon - old WT 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 . circled is emphasized for showing distribution of immunoreactivity in 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.


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

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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 cycle - related genes semi - of expression in frontal cortices hippocampus from 6 - mon - old WT FTLD - TDP Tg mice . N = 5 mice per group , data are presented as mean ± ( % ) , statistical by multiple t - with FDR correction , Q = 1 % . * P < 0 . 05 , * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 * * * * 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 for fragmentation of with comet tails in regions of frontal cortices from 6 - mon - old WT FTLD - TDP Tg mice . Scale bar : 50 μm . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± , * p = 0 . 0114 by t - .

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

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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 of γH2AX Ki67 in regions of frontal cortices from WT 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 : of or neurons with γH2AX Ki67 immunoreactivity from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± , * * * p = 0 . 0004 by t - .

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

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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 TDP - 43 in extracts . Bottom , of HDAC1 TDP - 43 . N = 5 mice per group , data are presented as mean ± ( % ) , * * * p = 0 . 0005 ( TDP - 43 ) or 0 . 0004 ( HDAC1 ) ; * * * * p < 0 . 0001 by multiple t - .

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


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

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

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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 of TDP - 43 HDAC1 during progression of TDP - 43 proteinopathies in frontal cortices from FTLD - TDP Tg WT mice . Nuclei were stained with DAPI ( in blue ) . Scale bar : 50 μm . circled is emphasized for showing distribution of immunoreactivity in subregions . Scale bar : 15 μm . Right histogram : of co - localized TDP - 43 HDAC1 immunoreactivity in cytosol or nucleus in WT or 1 - , 6 - , 12 - month - old FTLD - TDP Tg mice . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± . * 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 of γH2AX HDAC1 in frontal cortices from 12 - month - old FTLD - TDP Tg WT mice . Nuclei were stained with DAPI ( in blue ) . Arrow head : γH2AX + nucleus ; arrow : mislocalized HDAC1 . Scale bar : 50 μm . Right histogram : of γH2AX + HDAC1 mislocalized in WT or 1 - , 6 - , 12 - month - old FTLD - TDP Tg mice . n = 4 sections per mouse , N = 5 mice per group , data are presented as mean ± . * * 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 - HDAC1 was overexpressed with myc - tagged TDP - 43 in HEK - 293T ; lysates were immunoprecipitated for flag immunoblotted for TDP - 43 flag . Right panel : myc - tagged TDP - 43 was overexpressed with flag - tagged full - HDAC1 in HEK - 293T ; lysates were immunoprecipitated for myc immunoblotted for flag 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 - HDAC1 ( b . Ⅰ ) or various truncation mutations ( b . Ⅱ - Ⅳ ) were overexpressed with myc - tagged TDP - 43 ; catalytic domain is indicated in red . Lower panel : western blotting of lysates immunoprecipitated for flag 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 HDAC1 immunoblotting of HDAC1 TDP - 43 in WT FTLD - TDP Tg mice . Lower histogram : of immunoprecipitation results of HDAC1 TDP - 43 in WT Tg mice . N = 5 mice per group , data are presented as mean ± ( % ) , * p = 0 . 0149 , * * * p = 0 . 0003 by t - .

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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 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 by two - way ANOVA with Bonferroni ' s multiple comparisons . # P < 0 . 05 , * * * P ≤ 0 . 001 * * * * / # # # # P ≤ 0 . 0001 Data information : N = 12 mice per group data are presented as mean ± , * * * * 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.


representative searching path of mice in probe .

The representative searching path of mice in the probe test.


Scores of mice with respect to number of times they crossed hidden platform , time spent searching in target quadrant , velocity of swimming in probe at 24 h after escape training . * * p = 0 . 009 , * * * p = 0 . 0002 by multiple comparison . Data information : N = 12 mice per group data are presented as mean ± , * * * * 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 discrimination index in novel object recognition . Scores of mice in rotarod . Data information : N = 12 mice per group data are presented as mean ± , * * * * 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.


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

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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 acetylated - histone 3 ( Lys 9 / 14 ) , total histone H3 , of . N = 6 mice per group . * p = 0 . 0105 or * * * p = 0 . 0008 by multiple comparison . Data information : data are presented as mean ± ( % )

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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 HDAC1 semi - of expression . N = 6 mice per group . * * p = 0 . 0022 ( Tg ) / 0 . 0012 ( Tg + 5104434 ) by multiple comparison . Data information : data are presented as mean ± ( % )

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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 cycle - damage - related . Right histogram : semi - of . N = 6 per group . * WT + Vehicle mice vs . Tg + Vehicle mice , # Tg + 5104434 mice vs . Tg + Vehicle mice . Statistical by multiple t - with FDR correction , Q = 1 % . * * P ≤ 0 . 01 ; # # # P ≤ 0 . 001 * * * * / # # # # P ≤ 0 . 0001 . Data information : data are presented as mean ± ( % )

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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 of γH2AX in frontal cortices of immunoreactive 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 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 of immunoreactive in hippocampus of three groups of mice , 8 - mon - old WT + Vehicle , Tg + Vehicle 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 : data are presented as mean ± ( % )

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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 of GFAP of immunoreactive in hippocampus of three groups of mice , 12 - month - old WT + Vehicle , Tg + Vehicle Tg + 5104434 . n = 9 sections per mouse , N = 6 mice per group . Scale bar : 50 μm . * * p = 0 . 002 * * * * p < 0 . 0001 by multiple comparison . Data information : where number of 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 of TDP - 43 HDAC1 in frontal cortices from normal individuals with FTLD - TDP . Scale bar : 50 μm . circled is emphasized for showing distribution of immunoreactivity in 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.


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

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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 of with HDAC1 mislocalization TDP - 43 proteinopathies . Total : 3000 per samples . P = 0 . 0001 by Pearson correlation . Data information : data represent mean ± , * * * * p < 0 . 0001 by t - .

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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 of γH2AX Ki67 in frontal cortices from normal individuals with FTLD - TDP from each view of microscope . Scale bar : 50 μm . circled is emphasized for showing distribution of immunoreactivity in 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.


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

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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 of with damage aberrant cycle activity . : 40 - 113 ( normal ) 418 - 887 ( FTLD ) per samples . p < 0 . 0001 by Pearson correlation . Data information : data represent mean ± , * * * * p < 0 . 0001 by t - .

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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 TDP - 43 in urea - soluble fractions from frontal cortex of normal individuals 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.


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

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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 expression of cytokines in THP1 macrophages ( left ) PBMCs ( right ) upon treatment with full - 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 expression of cytokines in PBMCs . Left , a schematic showing domain structure of S1 spike anti - RBD antibody . Right , changes in cytokine gene expression in response to S1 spike in presence or abscene of anti - RBD antibody . Gene expression was measured by qPCR . Cytokine release in conditioned media was measured by Luminex . Full - S1 S1 subunits are purified from HEK293 . Data information , data are shown as mean of three technical replicates ; in ( D ) , data are shown as mean of 20 donors . Error bars denote . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Multiple t - .

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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 . Cytokine release was measured by Luminex 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 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 full S1 spike - mediated changes in cytokines in THP1 macrophages . Data information , , data are shown as mean of three technical replicates . Error bars denote .

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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 showing Ponatinib alleviates LPS - induced inflammatory infiltration , septal thickening , alveolar edema in mouse lungs . Scale bars 100 µm ( upper ) 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 were measured using Luminex . Data information , data are mean of five biological replicates from two independent studies , error bars denote . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Welch ' s t - .

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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 OVCAR4 were transfected with either non - target miRNA control ( miR - CTL ) , miR - 15a , or miR - 195 . 72h following transfection , were lysed immunoblotted for detection of MICU1 , BMI1 , BCL2 , MFN2 . GAPDH 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 ovarian lines as determined by RT - qPCR normalized with U6 . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - .

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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 various ovarian lines as determined by immunoblotting . Actin is used as 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 FTE188 , level of miR - 195 was measured using RT - qPCR ( left ) MICU1 were measured using immunoblotting ( right ) . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - .

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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 , OVSAHO were transfected with non - target micro control ( miR - CTL ) or miR - 195 , 24 h post - transfection , were recounted plated as single for anchorage - dependent ( A ) or anchorage - independent ( B ) clonal growth . After 10 ( CP20 ) or 14 ( OVCAR4 OVSAHO ) days , colonies were quantified using an Optronix GelCount colony counter . left panel shows representative of colonies right panel depicts a graphical representation of data presented as clonal growth relative to 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 , miR - 195 + MICU1 ( pLYS1 - MICU1 - Flag ) transfected , towards serum gradient was examined number of per field was counted . left panel shows representative right panel depicts a graphical representation of relative migration compared to 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 , miR - 195 + MICU1 through fibronectin - coated filters was examined using Boyden chamber number of per field was counted . Left panel shows representative right panel depicts a graphical representation of relative invasion compared to 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 units ( RLU ) compared to miR - CTL were plotted . Data are mean ± S . D . n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - .

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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 were transfected as indicated , 48 h post transfection 4x106 were permeabilized , extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in miR - CTL miR - 195 transfected . [ Ca2 + ] out pulses 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 were transfected as indicated , 48 h post transfection 4x106 were permeabilized , extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in control siRNA ( siCTL ) siMICU1 transfected . [ Ca2 + ] out pulses 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 number of Ca2 + pulses handled by control miR , miR - 195 , siCTL siMICU1 transfected . Mean ± ; n = 3 - 4 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - .

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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 transfected with non - target miR - CTL or miR - 195 . ( G ) of resting matrix [ Ca2 + ] ( m ) after addition of FCCP . n = 5 ( Biological repeats ) * P < 0 . 05 , Student ' s t - ,

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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 potential ( ∆ Ψμ ) in CP20 transfected with miR - CTL or miR - 195 . ( I ) 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 , siMICU1 expressing OVCAR4 , CP20 , OVSAHO . n = 3 , biological repeats * P < 0 . 05 , Student ' s t - .

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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 were stained with MitoSOX Red , mitochondrial ROS were determined by flow cytometry . histogram shows representative bar graph ( right ) shows results of three independent experiments . Mean ± ; . n = 3 , biological repeats . * P < 0 . 05 when comparing with miR - CTL , # P < 0 . 05 when comparing with miR - 195 by Student ' s t - .

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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 lines for CP20 OVCAR4 expressing GFP - miR - 195 or non - target miR - GFP were generated using lentiviral mediated transduction were selected using puromycin ; representative 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 lines , normalized with U6 . Mean ± ; n = 3 , Biological repeats . * P < 0 . 05 , Student ' s t - .

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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 OVCAR4 stable lines were transfected as either empty vector ( EV ) or pLYS1 - MICU1 - Flag ( MICU1 ) relative MICU1 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 OVCAR4 stable lines transfected as in ( D ) were counted re - plated for anchorage - dependent ( E ) independent clonal growth ( F ) . compared to miR - CTL stable 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 - .

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

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

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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 ) survival on humane endpoint criteria , was calculated by Kaplan - Meier method P values determined by Log Rank .

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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 samples from each group were analyzed for expression of MICU1 , pPDH , PDH , PARP , BCL2 using immunoblotting . α tubulin was used as 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 of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) with Ki67 expression ( Scale Bar = 50 μm ) ( E ) , CD31 positive vessels ( Scale Bar = 100 μm ) TUNEL ( 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 of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) with Ki67 expression ( Scale Bar = 50 μm ) ( F ) graph showing of each marker in tumors from experimental mice ; values are mean ± SD , n = 10 , * P < 0 . 05 , Student ' s t - .

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

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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.


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

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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 in with measurable disease classified as having a G1 ( blue ) or G2 ( red ) profile . Dotted lines represents limit to define significant progression ( upper ) or significant regression ( lower ) .

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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 treated with immunotherapies stratified only by G1 ( green ) G2 ( red ) CD4 T profiles . starting therapy with a G2 profile had an overall response rate ( ORR ) of 0 % 82 % of them experienced progression or by week 9 . ORR was 44 . 8 % for G1 , 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%.


scatter plot shows PD - 1 expression after co - culture of CD4 T from healthy donors or NSCLC , as indicated , with A459 - SC3 lung cancer . Relevant statistical comparisons with 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 LAG - 3 co - expression in CD4 T from healthy donors , a G1 responder ( G1 R ) , a G1 non - responder ( G1 NR ) a G2 non - responder as indicated , following stimulation with A549 - SC3 . of expressing are indicated within each quadrant . Below , same as in upper graphs but as a scatter plot of of CD4 T that simultaneously co - express PD - 1 LAG - 3 . Relevant statistical comparisons are shown with 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 from representative subjects as indicated on right , after stimulation with A549 - SC3 . Vertical dotted indicates cut - off value of positive vs negative Ki67 expression . of Ki67 - expressing CD4 T is shown within histograms . Below , same data represented as a scatter plot from a sample of G1 G2 donors as indicated , with relevant statistical comparisons with 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 stimulated by A549 - SC3 from indicated groups . CD28 expression is shown together with proliferation marker ki67 . Percentages of 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.