( D ) Membrane association of ubiquitinated 3HA - Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation . Cells of the indicated genotype and expressing myc - tagged ubiquitin were grown as in ( C ) . Lysates were subjected to centrifugation on an optiprep density gradient . Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti - HA antibodies . Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively . Input fraction corresponds to 10 % of the total amount used for IP . " Top " , " mid " , " bottom " indicate the fractions from the gradient . Asterisk marks a faster migrating unspecific band in the input bottom fraction .

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

(D) Membrane association of ubiquitinated 3HA-Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation. Cells of the indicated genotype and expressing myc-tagged ubiquitin were grown as in (C). Lysates were subjected to centrifugation on an optiprep density gradient. Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti-HA antibodies. Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively. Input fraction corresponds to 10% of the total amount used for IP. "Top", "mid", "bottom" indicate the fractions from the gradient. Asterisk marks a faster migrating unspecific band in the input bottom fraction.


( A ) Localization of chromosomally expressed GFP - Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells . Arrowheads indicate GFP - Pgc1 labeling at the ER , which is stained by Sec63 - Cherry . LDs were visualized upon staining with the neutral lipid dye MDH . On the bottom , box plot with quantitation of GFP - Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ . GFP - Pgc1 measurements were performed as described in the Materials and Methods section . Scale bar : 5 µm .

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

(A) Localization of chromosomally expressed GFP-Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells. Arrowheads indicate GFP-Pgc1 labeling at the ER, which is stained by Sec63-Cherry. LDs were visualized upon staining with the neutral lipid dye MDH. On the bottom, box plot with quantitation of GFP-Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ. GFP-Pgc1 measurements were performed as described in the Materials and Methods section. Scale bar: 5 µm.


( C ) Crude membranes from wt and doa10Δ cells expressing 3HA - Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet ( P ) and supernatant ( S ) . ( D ) Crude membranes from wt and doa10Δ cells expressing 3HA - GFP - Pgc1275 - 321 were analyzed as in ( C ) .

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

(C) Crude membranes from wt and doa10Δ cells expressing 3HA-Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet (P) and supernatant (S).(D) Crude membranes from wt and doa10Δ cells expressing 3HA-GFP-Pgc1275-321 were analyzed as in (C).


( A ) Localization of GFP - Pgc1 in are1Δ are2Δ lro1Δ dga1Δ cells in presence ( no LDs ) or absence of DOA10 ( no LDs doa10Δ ) . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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

(A) Localization of GFP-Pgc1 in are1Δ are2Δ lro1Δ dga1Δ cells in presence (no LDs) or absence of DOA10 (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( B ) GFP - Pgc1 was expressed under the ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δ . Fluorescence microscopy was used to follow GFP - Pgc1 localization over time . LDs were visualized upon staining with the neutral lipid dye MDH . Scale bar : 5 µm .

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

(B) GFP-Pgc1 was expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δ. Fluorescence microscopy was used to follow GFP-Pgc1 localization over time. LDs were visualized upon staining with the neutral lipid dye MDH. Scale bar: 5 µm.


( C ) Photo - conversion of tdEOS - Pgc1 expressed under the ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells . The red square marks the photo - converted region . The time ( in minutes ) after photoconversion is indicated . Arrowheads point LDs containing photoconverted tdEOS - Pgc1 . Scale bar 5µm .

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

(C) Photo-conversion of tdEOS-Pgc1 expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells. The red square marks the photo-converted region. The time (in minutes) after photoconversion is indicated. Arrowheads point LDs containing photoconverted tdEOS-Pgc1. Scale bar 5µm.


( D ) FRAP experiment of GFP - Pgc1 in wt and doa10Δcells . Representative examples are shown . The bleached areas are marked by red squares and include a LD adjacent to ER . The time ( in seconds ) after photobleaching is indicated . Each graph shows average fluorescence intensities for 10 cells normalized to pre - bleached plotted over time . Error bars indicate standard deviation .

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

(D) FRAP experiment of GFP-Pgc1 in wt and doa10Δcells. Representative examples are shown. The bleached areas are marked by red squares and include a LD adjacent to ER. The time (in seconds) after photobleaching is indicated. Each graph shows average fluorescence intensities for 10 cells normalized to pre-bleached plotted over time. Error bars indicate standard deviation.


( A ) The degradation of 3HA - Pgc1 was analyzed in cells with the indicated genotype as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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

(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) The degradation of the Doa10 substrates 3HA - Pgc1 and 3HA - Vma12 - Ndc10C ' was analyzed in cells of the indicated genotypes treated with oleic acid . 3HA - Pgc1 and 3HA - Vma12 - Ndc10C ' were detected with anti - HA antibodies . Dpm1 was used as a loading control and detected with anti - Dpm1 antibodies . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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

(B) The degradation of the Doa10 substrates 3HA-Pgc1 and 3HA-Vma12-Ndc10C' was analyzed in cells of the indicated genotypes treated with oleic acid. 3HA-Pgc1 and 3HA-Vma12-Ndc10C' were detected with anti-HA antibodies. Dpm1 was used as a loading control and detected with anti-Dpm1 antibodies. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( A ) Localization of GFP - tagged Pgc1 derivatives in which the hydrophobic hairpin ( aa275 - 321 ) was replaced by membrane anchor of Scs2 ( Pgc1Scs2MA ) or Bos1 ( Pgc1Bos1MA ) . ER and LDs were visualized by expression of Sec63 - Cherry and MDH staining , respectively . Scale bar : 5 µm .

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

(A) Localization of GFP-tagged Pgc1 derivatives in which the hydrophobic hairpin (aa275-321) was replaced by membrane anchor of Scs2 (Pgc1Scs2MA) or Bos1 (Pgc1Bos1MA). ER and LDs were visualized by expression of Sec63-Cherry and MDH staining, respectively. Scale bar: 5 µm.


( B ) The degradation of 3HA - Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig . 1A . The blot is representative of at least three independent experiments .

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

(B) The degradation of 3HA-Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig. 1A. The blot is representative of at least three independent experiments.


( C ) The degradation of 3HA - GFP - Pgc1275 - 321 in wt and doa10Δ cells was analyzed as in Fig . 1A . The graph shows the average of four independent experiments ; error bars represent the standard deviation .

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

(C) The degradation of 3HA-GFP-Pgc1275-321 in wt and doa10Δ cells was analyzed as in Fig. 1A. The graph shows the average of four independent experiments; error bars represent the standard deviation.


( D ) Localization of 3HA - GFP - Pgc1275 - 321 in doa10Δ cells . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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

(D) Localization of 3HA-GFP-Pgc1275-321 in doa10Δ cells. The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( A ) The degradation of 3HA - Pgc1 - GPAT160 - 216 , containing GPAT4 hydrophobic hairpin ( aa160 - 216 ) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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

(A) The degradation of 3HA-Pgc1-GPAT160-216, containing GPAT4 hydrophobic hairpin (aa160-216) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) Localization of GFP - Pgc1 - GPAT4160 - 216 in cells with the indicated genotype . Arrowheads indicate GFP - Pgc1 - GPAT4160 - 216 labeling at the ER , marked with Sec63 - Cherry . LDs were visualized upon staining with MDH . Scale bar : 5 µm .

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

(B) Localization of GFP-Pgc1-GPAT4160-216 in cells with the indicated genotype. Arrowheads indicate GFP-Pgc1-GPAT4160-216 labeling at the ER, marked with Sec63-Cherry. LDs were visualized upon staining with MDH. Scale bar: 5 µm.


( C ) The degradation of 3HA - Pgc1 - GPAT160 - 216 in cells with the indicated genotype was analyzed as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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

(C) The degradation of 3HA-Pgc1-GPAT160-216 in cells with the indicated genotype was analyzed as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( D ) Localization of GFP - Pgc1 - GPAT160 - 216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant ( no LDs doa10Δ ) . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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

(D) Localization of GFP-Pgc1-GPAT160-216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young ( 2 months ) and old ( 22 months ) mice ( n = 3 mice per group ) . Scale bar 200 µm .

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

B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young (2 months) and old (22 months) mice (n = 3 mice per group). Scale bar 200 µm.


C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young ( 20 - 35 years old ) and old ( 60 - 80 years old ) humans ( n = 4 - 7 individuals per group ) . Scale bar , 200 µm .

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

C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young (20-35 years old) and old (60-80 years old) humans (n = 4-7 individuals per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs from young and old mice ( n = 3 - 6 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs from young and old mice (n = 3-6 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


E Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1 - positive and CD31 - positive endothelia in lungs from young and old humans ( n = 4 - 7 individuals per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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

E Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1-positive and CD31-positive endothelia in lungs from young and old humans (n = 4-7 individuals per group). Scale bar, 100 µm. a.u. = arbitrary units.


C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 200 µm .

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

C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F / F at the indicated time points following 4OHT treatment and consequent TRF2 knockout . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

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A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown . Scale bar , 25 µm . D RT - qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C ( n = 6 independent experiments ) .

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

C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm. D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments).


E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce TRF2 loss and telomere uncapping ) or vehicle . The animals as been injected also with PBS . Scale bar , 10 µm . F RT - qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E ( n = 5 - 7 mice per group ) .

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

E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm. F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).


B Relative luciferase activity in HeLa shTRF2 following ionizing radiation ( 5 Gy ) or TRF2 knockdown upon doxycycline - induced shTRF2 expression ( n = 3 independent experiments ) . Error bars represent the s . e . m . * P < 0 . 05 . Two - way paired ANOVA .

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

B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. *P<0.05. Two-way paired ANOVA.


A Immunofluorescence showing gammaH2AX foci in Trf2F / F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

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

A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce telomere uncapping ) or vehicle and injected with the indicated ASOs or PBS as control . Scale bar , 10 µm . D RT - qPCR detection of Ace2 mRNA levels in livers of mice treated as in C ( n = 5 - 8 mice per group ) .

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

C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm. D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group).


E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 200 µm .

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

E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm.


F Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive cells in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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

F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


( A ) Profile of transcript levels of autophagy - related genes in iBAT which are significantly modified in cold exposed mice ( 4ºC , 24h ) , compared with mice maintained at thermoneutral temperature ( 29ºC ) . ( Fisher ' s exact test corrected by the Benjamini - Hochberg method , p < 0 . 05 ) . Heatmap represents log - fold change of RPKM values of cold versus thermoneutral ( TN ) sample data obtained from RNAseq analysis . Each square represents replicates ( n = 4 ) . The most repressed genes are shown on the bottom of the heatmap .

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

(A)Profile of transcript levels of autophagy-related genes in iBAT which are significantly modified in cold exposed mice (4ºC, 24h), compared with mice maintained at thermoneutral temperature (29ºC). (Fisher's exact test corrected by the Benjamini-Hochberg method, p< 0.05). Heatmap represents log-fold change of RPKM values of cold versus thermoneutral (TN) sample data obtained from RNAseq analysis. Each square represents replicates (n=4). The most repressed genes are shown on the bottom of the heatmap.


Parkin expression in iBAT from mice exposed to cold ( 4ºC ) for 1 day ( 1d ) or 21 days ( 21d ) compared with control mice ( ctl ) maintained at 22ºC . Left : Relative transcript levels . Right : representative immunoblot and Parkin protein quantification . Ponceau staining ( PS ) was used as loading control . ( n = 6 ; transcript levels ) ( n = 3 ; protein levels ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , cold vs . control and # # p < 0 . 01 , 1d vs . 21d . One - way ANOVA with Tukey ' s post hoc test .

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

Parkin expression in iBAT from mice exposed to cold (4ºC) for 1 day (1d) or 21 days (21d) compared with control mice (ctl) maintained at 22ºC. Left: Relative transcript levels. Right: representative immunoblot and Parkin protein quantification. Ponceau staining (PS) was used as loading control. (n=6; transcript levels) (n=3; protein levels). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ##p<0.01, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.


Relative transcript levels of Park2 in sWAT and eWAT . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , cold vs . control and # # # p < 0 . 001 , 1d vs . 21d . One - way ANOVA with Tukey ' s post hoc test .

O O O O B-GENEPROD O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative transcript levels of Park2 in sWAT and eWAT. (n= 6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ###p<0.001, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.


Relative transcript levels of Parkin in iBAT , sWAT and eWAT of mice injected with the β3 - AR agonist CL316 , 243 , every day for 1 week . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * * p < 0 . 001 , CL316 , 243 treatment vs . saline . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Parkin in iBAT, sWAT and eWAT of mice injected with the β3-AR agonist CL316,243, every day for 1 week. (n=6). Data are presented as means ±s.e.m. *p<0.05, ***p<0.001, CL316,243 treatment vs. saline. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in mature brown adipocytes compared with the stromal vascular fraction ( SVF ) in iBAT . ( n = 3 ) . Data are presented as means ± s . e . m . * * p < 0 . 01 , * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in mature brown adipocytes compared with the stromal vascular fraction (SVF) in iBAT. (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


mRNA expression profiles of Park2 and Ucp1 during differentiation . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to day ( d ) 4 . Two - tailed unpaired Student ' s t - test .

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

mRNA expression profiles of Park2 and Ucp1 during differentiation. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to day (d) 4. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture with fetal bovine serum ( FBS , to trigger in vitro differentiation ) or charcoal - stripped serum ( CSS , non - permissive for differentiation ) . ( n = 3 ) . Data are presented as means ± s . e . m . * * p < 0 . 01 , * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture with fetal bovine serum (FBS, to trigger in vitro differentiation) or charcoal-stripped serum (CSS, non-permissive for differentiation). (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture treated with norepinephrine ( NE ) or cAMP for indicated times . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to non - treated cells . One - way ANOVA with Dunnett ' s post hoc test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture treated with norepinephrine (NE) or cAMP for indicated times. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to non-treated cells. One-way ANOVA with Dunnett's post hoc test.


Parkin protein levels in brown adipocytes treated with NE or cAMP for the indicated times in the presence or absence of 3 - methyladenine ( 3 - MA ) . Top : Representative immunoblots . p62 and LC3BII proteins are shown as positive controls of 3 - MA treatment . β - actin was use as loading control . Bottom : Parkin protein quantification ( performed using 3 immunoblots processed in parallel due to sample size ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 compared to NE / cAMP non - treated cells . One - way ANOVA with Dunnett ' s post hoc test .

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

Parkin protein levels in brown adipocytes treated with NE or cAMP for the indicated times in the presence or absence of 3-methyladenine (3-MA). Top: Representative immunoblots. p62 and LC3BII proteins are shown as positive controls of 3-MA treatment. β-actin was use as loading control. Bottom: Parkin protein quantification (performed using 3 immunoblots processed in parallel due to sample size). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to NE/cAMP non-treated cells. One-way ANOVA with Dunnett's post hoc test.


Relative transcript levels of Park2 in brown adipocytes treated with NE in the presence or absence of cycloheximide ( CHX ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 in brown adipocytes treated with NE in the presence or absence of cycloheximide (CHX). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


Relative mRNA expression levels of Parkin in brown adipocytes treated with NE in the presence or absence of actinomycin D ( n = 3 ) . Data are presented as means ± s . e . m . One - way ANOVA with Tukey ' s post hoc test comparing treatments .

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

Relative mRNA expression levels of Parkin in brown adipocytes treated with NE in the presence or absence of actinomycin D (n=3). Data are presented as means ±s.e.m. One-way ANOVA with Tukey's post hoc test comparing treatments.


Park2 gene promoter activity in HIB - 1B cells transfected with a pGL4 - Park2 - Luc reporter construct treated with or without NE or cAMP . Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that in untreated cells . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 compared to non - treated cells . ANOVA with Dunnett ' s post hoc

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

Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct treated with or without NE or cAMP. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that in untreated cells. (n=6). Data are presented as means ±s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test


Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated with cAMP ( 12 h ) plus H89 ( a PKA inhibitor ) or SB202190 ( a p38 MAPK inhibitor ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 with vs . without inhibitor and # p < 0 . 05 , and # # p < 0 . 01 , # # # p < 0 . 001 with vs . without cAMP . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated with cAMP (12 h) plus H89 (a PKA inhibitor) or SB202190 (a p38 MAPK inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 , and concentration of glycerol in the culture medium of brown adipocytes treated with cAMP ( 24h ) plus Atglistatin ( an adipose triglyceride lipase inhibitor ) or CAY10499 ( a hormone sensitive lipase inhibitor ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 with vs . without inhibitor and # p < 0 . 05 , and # # p < 0 . 01 , # # # p < 0 . 001 with vs . without cAMP . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2, and concentration of glycerol in the culture medium of brown adipocytes treated with cAMP (24h) plus Atglistatin (an adipose triglyceride lipase inhibitor) or CAY10499 (a hormone sensitive lipase inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated for 24h with a PPARα activator ( GW7647 ) , a PPARα inhibitor ( GW6471 ) with or without norepinephrine ( NE ) , or all - trans - retinoic acid ( RA ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated for 24h with a PPARα activator (GW7647), a PPARα inhibitor (GW6471) with or without norepinephrine (NE), or all-trans-retinoic acid (RA). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 in wild - type and PPARα - KO brown adipocytes treated for 24h with NE ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

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

Relative transcript levels of Park2 in wild-type and PPARα-KO brown adipocytes treated for 24h with NE (n=3). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Park2 gene promoter activity in HIB - 1B cells transfected with a pGL4 - Park2 - Luc reporter construct and , when indicated , with a pSG5 - Ppara expression vector . Cells were treated with the PPARα activator GW7647 or NE . Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that only transfected with the pGL4 - Park2 - Luc construct ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 compared to non - treated cells . ANOVA with Dunnett ' s post hoc test .

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

Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct and, when indicated, with a pSG5-Ppara expression vector. Cells were treated with the PPARα activator GW7647 or NE. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that only transfected with the pGL4-Park2-Luc construct (n=6). Data are presented as means ± s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test.


Left : Representative optical microscopic images of H & E - stained iBAT ( scale bars , 110µm ) and electron microscopic images of iBAT ( scale bars , 20µm ) from wild - type ( WT ) and Parkin - KO ( KO ) mice . Right : Quantification of lipid droplet area from electron microscopic images ( 10 images per condition were used , n = 3 WT , n = 3 Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . Two - tailed unpaired Student ' s t - test .

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

Left: Representative optical microscopic images of H&E-stained iBAT (scale bars, 110µm) and electron microscopic images of iBAT (scale bars, 20µm) from wild-type (WT) and Parkin-KO (KO) mice. Right: Quantification of lipid droplet area from electron microscopic images (10 images per condition were used, n =3 WT, n=3 Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. Two-tailed unpaired Student's t-test.


Representative thermal images ( left ) and quantification of surface temperature ( right ) for the interscapular region . ( n = 6 ) . Data are presented as means ± s . e . m . * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

O O B-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative thermal images (left) and quantification of surface temperature (right) for the interscapular region. (n=6). Data are presented as means ±s.e.m. ***p<0.001. Two-tailed unpaired Student's t-test.


Representative immunoblot of UCP1 protein and the loading control ( Ponceau staining , PS ) in iBAT samples .

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

Representative immunoblot of UCP1 protein and the loading control (Ponceau staining, PS) in iBAT samples.


Relative transcript levels of thermogenesis - related genes of iBAT from WT and Parkin - KO mice exposed to acute cold ( 4ºC , 24h ) compared to those maintained at the control temperature ( 22ºC ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , CTL vs . cold ; # p < 0 . 05 , WT vs . Parkin - KO . ANOVA with Tukey ' s post hoc test .

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

Relative transcript levels of thermogenesis-related genes of iBAT from WT and Parkin-KO mice exposed to acute cold (4ºC, 24h) compared to those maintained at the control temperature (22ºC). (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001, CTL vs. cold; #p<0.05, WT vs. Parkin-KO. ANOVA with Tukey's post hoc test.


Weight gain from WT and Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

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

Weight gain from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


, blood glucose levels and plasma insulin levels from WT and Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

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

, blood glucose levels and plasma insulin levels from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


insulin tolerance test ( ITT ) from WT and Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

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

insulin tolerance test (ITT) from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


Relative transcript levels of thermogenesis - related genes in iBAT from WT and Parkin - KO mice given HFD . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . # p < 0 . 05 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of thermogenesis-related genes in iBAT from WT and Parkin-KO mice given HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. #p<0.05. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 in iBAT from WT mice fed CD or HFD . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . Two - tailed unpaired Student ' s t - test .

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Relative transcript levels of Park2 in iBAT from WT mice fed CD or HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. Two-tailed unpaired Student's t-test.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Top : Total protein content in iBAT during cold deaclimation . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 compared to 21d . ANOVA with Dunnett ' s post hoc test . Bottom : Representative optical microscopic images of H & E - stained iBAT ( scale bars , 100µm ) and electron microscopic images of iBAT ( scale bars , 10µm ) .

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

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Top: Total protein content in iBAT during cold deaclimation. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to 21d. ANOVA with Dunnett's post hoc test. Bottom: Representative optical microscopic images of H&E-stained iBAT (scale bars, 100µm) and electron microscopic images of iBAT (scale bars, 10µm).


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Relative transcript levels of thermogenesis - related genes and Park2 in iBAT . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to 21d . ANOVA with Dunnett ' s post hoc test .

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

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Relative transcript levels of thermogenesis-related genes and Park2 in iBAT. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to 21d. ANOVA with Dunnett's post hoc test.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative immunoblot of the indicated thermogenic and autophagic proteins in iBAT during cold acclimation and deacclimation . PS : Ponceau staining .

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WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative immunoblot of the indicated thermogenic and autophagic proteins in iBAT during cold acclimation and deacclimation. PS: Ponceau staining.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative immunoblot of Parkin phosphoSer65 and total Parkin in whole lysates of iBAT and after Parkin immunoprecipitation . Arrowheads indicate Parkin 52kDa band and asterisk indicates a non - specific band .

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

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative immunoblot of Parkin phosphoSer65 and total Parkin in whole lysates of iBAT and after Parkin immunoprecipitation. Arrowheads indicate Parkin 52kDa band and asterisk indicates a non-specific band.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative electron microscopic images of autophagic and mitophagic events in iBAT after 1 day of cold deacclimation . Yellow arrowheads indicate mitochondrial cristae , blue arrowheads indicate double - membrane autophagosomes , and red arrowheads indicate structures compatible with mitochondria - derived vesicles . LD : lipid droplet . Scale bars , 1 µm .

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

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative electron microscopic images of autophagic and mitophagic events in iBAT after 1 day of cold deacclimation. Yellow arrowheads indicate mitochondrial cristae, blue arrowheads indicate double-membrane autophagosomes, and red arrowheads indicate structures compatible with mitochondria-derived vesicles. LD: lipid droplet. Scale bars, 1 µm.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Representative immunoblot of LC3B in iBAT , and the loading control ( Ponceau staining , PS ) .

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Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Representative immunoblot of LC3B in iBAT, and the loading control (Ponceau staining, PS).


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Immunoblot for p62 and OPTN ( left ) and its quantification ( right ) in iBAT . PS was used as the loading control . Arrowhead indicates p62 band and asterisk indicates a non - specific band . ( n = 4 ; cold ) ( n = 3 ; deacclimation ) . Data are presented as means ± s . e . m . * * p < 0 . 01 . ANOVA with Tukey ' s post hoc test .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Immunoblot for p62 and OPTN (left) and its quantification (right) in iBAT. PS was used as the loading control. Arrowhead indicates p62 band and asterisk indicates a non-specific band. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. **p<0.01. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Quantification of the total protein amounts of the indicated OXPHOS protein subunits and UCP1 in iBAT . ( n = 4 ; cold ) ( n = 3 ; deacclimation ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Quantification of the total protein amounts of the indicated OXPHOS protein subunits and UCP1 in iBAT. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Citrate synthase activity in iBAT protein extracts . ( n = 6 ; WT ) ( n = 8 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Citrate synthase activity in iBAT protein extracts. (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Mitochondrial DNA ( mtDNA ) content in iBAT relative to nuclear DNA ( nDNA ) . ( n = 6 ; WT ) ( n = 8 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O B-TISSUE O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Mitochondrial DNA (mtDNA) content in iBAT relative to nuclear DNA (nDNA). (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Representative electron microscopic images of mitochondrial integrity and autophagic / mitophagic events in iBAT after 1 day of cold - deacclimation . Yellow arrowheads indicate mitochondria cristae and blue arrowheads indicate double - membrane autophagosomes . Left panels scale bars , 2 µm . Right panels scale bars , 1 µm .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Representative electron microscopic images of mitochondrial integrity and autophagic/mitophagic events in iBAT after 1 day of cold-deacclimation. Yellow arrowheads indicate mitochondria cristae and blue arrowheads indicate double-membrane autophagosomes. Left panels scale bars, 2 µm. Right panels scale bars, 1 µm.


Sexing the established ESCs by detecting the Y - linked Zfy gene using genomic DNA ( gDNA ) - qPCR , normalized to Gapdh . n . d . : not detected . Average of presumed males was set to 1 .

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Sexing the established ESCs by detecting the Y-linked Zfy gene using genomic DNA (gDNA)-qPCR, normalized to Gapdh. n.d.: not detected. Average of presumed males was set to 1.


DNA - FISH detection of the number of X - linked Atrx gene foci . Representative images of the DNA - FISH are shown . Nuclei are surrounded by white dotted lines . Magenta : Atrx ( white arrowheads ) , cyan : DAPI . N > 100 .

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DNA-FISH detection of the number of X-linked Atrx gene foci. Representative images of the DNA-FISH are shown. Nuclei are surrounded by white dotted lines. Magenta: Atrx (white arrowheads), cyan: DAPI. N>100.


Heatmap analysis from RNA - seq . Each log10 ( FPKM + 1 ) value was used for clustering . Genes with high or low expression are shown in red and blue , respectively . FPKM : fragments per kilobases per million reads .

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

Heatmap analysis from RNA-seq. Each log10(FPKM+1) value was used for clustering. Genes with high or low expression are shown in red and blue, respectively. FPKM: fragments per kilobases per million reads.


X and Y chromosome mapping . Genes with high expression ( up ) are shown in orange and low expression ( down ) are shown in blue . The defined ratio of up or down genes is > 1 . 5 and < two - thirds , respectively . The comparison is ( each female vs the average for males ) or ( each male vs the average for females ) . The location controls for the expressed genes ( exp . genes ) on the X - chromosome and Y - chromosome are plotted ( red squares ) for genes whose FPKM averages were above 1 for females or males .

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

X and Y chromosome mapping. Genes with high expression (up) are shown in orange and low expression (down) are shown in blue. The defined ratio of up or down genes is > 1.5 and < two-thirds, respectively. The comparison is (each female vs the average for males) or (each male vs the average for females). The location controls for the expressed genes (exp. genes) on the X-chromosome and Y-chromosome are plotted (red squares) for genes whose FPKM averages were above 1 for females or males.


Clonogenic assay for ESCs in response to etoposide ( A ) or camptothecin ( B ) . Non - treated cells were set to 1 .

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Clonogenic assay for ESCs in response to etoposide (A) or camptothecin (B). Non-treated cells were set to 1.


Nanog HR assay . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown . The results for each cell line are shown at right .

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Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown. The results for each cell line are shown at right.


Nanog expression confirmed by RNA - seq .

Nanog expression confirmed by RNA-seq.


Detection of the number of X - linked Atrx gene foci by DNA - FISH in GFP - positive subclones of the Nanog - targeting assay ( nine independent clones , N > 100 each ) , performed as described in Figure 1C .

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Detection of the number of X-linked Atrx gene foci by DNA-FISH in GFP-positive subclones of the Nanog-targeting assay (nine independent clones, N> 100 each), performed as described in Figure 1C.


Sexing of established ESCs ( gDNA - qPCR , Zfy / Gapdh ) , performed as described in Figure 1B .

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Sexing of established ESCs (gDNA-qPCR, Zfy/Gapdh), performed as described in Figure 1B.


Detection of the number of X - linked Atrx gene foci by DNA - FISH with or without dox addition for 96 h ( N > 100 ) , performed as described in Figure 1C .

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Detection of the number of X-linked Atrx gene foci by DNA-FISH with or without dox addition for 96 h (N >100), performed as described in Figure 1C.


Detection of the Xist cloud in # B3 by RNA - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image is shown [ magenta : Xist ( white arrowhead ) , cyan : DAPI . Scale bar : 10 μm ] .

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Detection of the Xist cloud in #B3 by RNA-FISH, with or without dox addition for 96 h ( N >200 each). A representative image is shown [magenta: Xist (white arrowhead), cyan: DAPI. Scale bar: 10 μm].


Allele - distinguishable RT - PCR for detecting Hprt , G6pdx , and Rnf12 X - linked genes . Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown .

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Allele-distinguishable RT-PCR for detecting Hprt, G6pdx, and Rnf12 X-linked genes. Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown.


Nanog HR assay . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C . # B2 or # B3 were cultured with or without dox addition for 96 h . The combined number of GFP - positive cells and total colonies from all experiments are listed and summarized at right .

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

Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C. #B2 or #B3 were cultured with or without dox addition for 96 h. The combined number of GFP-positive cells and total colonies from all experiments are listed and summarized at right.


Detection of the Xist foci by RNA - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image with percentage of Xist - positive cells is shown [ magenta : Xist , cyan : DAPI . ] .

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

Detection of the Xist foci by RNA-FISH, with or without dox addition for 96 h (N >200 each). A representative image with percentage of Xist-positive cells is shown [magenta: Xist, cyan: DAPI.].


Detection of the number of X - linked Atrx gene foci by DNA - FISH , with or without dox addition for 96 h ( N > 100 each ) , performed as described in Figure 1C .

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

Detection of the number of X-linked Atrx gene foci by DNA-FISH, with or without dox addition for 96 h (N >100 each), performed as described in Figure 1C.


Estimated repair events . The flow cytometry plots are shown in Figure EV4 . The percentage of GFP - positive cells in TX - DRGFP # 11 and TX - EJ5GFP # 23 was normalized to the transfection efficiency detected by the VENUS expression vector . Dox ( - ) was set to 1 and the ratio of relative GFP - positive cells is shown ( n = 3 ) .

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

Estimated repair events. The flow cytometry plots are shown in Figure EV4. The percentage of GFP-positive cells in TX-DRGFP#11 and TX-EJ5GFP#23 was normalized to the transfection efficiency detected by the VENUS expression vector. Dox (-) was set to 1 and the ratio of relative GFP-positive cells is shown (n=3).


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 .

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

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 cell lines ( male : # A6 , # A13 , # A17 ; female : # A4 , # A10 , # A11 ) ] .

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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)


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Confirmation of Brcc3 expression from knockdown samples in western blotting


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 ) .

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

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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.


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

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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


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

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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 the first and last pulse , n = 27 control and 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 percentage 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 and Estrogen receptor ( ER antagonists Histogram shows relative bead displacement for the first and last pulse , n > 20 for all

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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 - protein estrogen receptor ( GPER ) antagonists Histogram shows relative bead displacement for the first and last pulse , n > 20 for all

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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


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


Histogram shows percentage 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 and tamoxifen n = 145 . Lines and 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 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

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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


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

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


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

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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 and total protein . ( E ) Quantification of YAP and pYAP Ser127 normalised to total protein , 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)


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

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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


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

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


IF images for total ( MLC - 2 ) and 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


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


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

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


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

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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 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 .

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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 and MAPK target genes in organoid single cell 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 and MMP7 in organoid single cell 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 and 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 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 ) .

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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 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 .

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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 / 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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

(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 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 ) .

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

(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 ) 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 .

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

(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.