d ) Plots show the probability for an individual keratinocyte to belong to a clone of a given size . Experimental data are represented in green bars . Red curve represents the 5 - colour simulation and the blue curve the real size simulation . Although the frequency of large clones is overestimated in experimental data due to the limited 5 - color palette , it is apparent that real clone size progresses over time .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plots show the probability for an individual keratinocyte to belong to a clone of a given size. Experimental data are represented in green bars. Red curve represents the 5-colour simulation and the blue curve the real size simulation. Although the frequency of large clones is overestimated in experimental data due to the limited 5-color palette, it is apparent that real clone size progresses over time.


e ) Histogram charts showing the distribution of clones at D1 - 3 following tamoxifen induction according to total number of cells and number of basal cells .

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

e) Histogram charts showing the distribution of clones at D1-3 following tamoxifen induction according to total number of cells and number of basal cells.


f ) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining . The frequency of larger clones increases over time . Chi square test p = 0 . 0013 for P21 group and p = 0 . 0296 for P42 group . All data are represented as mean ± s . e . m .

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

f) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining. The frequency of larger clones increases over time. Chi square test p=0.0013 for P21 group and p=0.0296 for P42 group. All data are represented as mean ± s.e.m.


a ) Clones were categorized as attached or not attached to hair follicles . Average clone size of attached ( circles ) and non - attached ( squares ) clones were plotted over time . T - test , * * * p < 0 . 0001 , * * p < 0 . 001 . ( Number of clones : 1 week n = 271 , 3 weeks n = 1847 , 5 weeks n = 517 , 12 weeks n = 706 , 24 weeks n = 258 ) . Data are represented as mean ± s . d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Clones were categorized as attached or not attached to hair follicles. Average clone size of attached (circles) and non-attached (squares) clones were plotted over time. T-test, *** p<0.0001, ** p<0.001. (Number of clones: 1 week n = 271, 3 weeks n = 1847, 5 weeks n = 517, 12 weeks n = 706, 24 weeks n = 258). Data are represented as mean ± s.d.


b ) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks . The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF . The orange and pink clones are not attached to the HF . Limits of clones ( lines ) and cells ( dots ) within each clone were identified on this optical section . White dashed circles represent the projection of hair follicle openings ( scale bar = 50 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks. The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF. The orange and pink clones are not attached to the HF. Limits of clones (lines) and cells (dots) within each clone were identified on this optical section. White dashed circles represent the projection of hair follicle openings (scale bar= 50 µm).


c ) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination . In the P42 group , clones attached to HF were more likely to be bigger than those not attached at 3 , 5 , 12 and 24 weeks . ǂǂǂǂ Chi square test p < 0 . 0001 . The number of clones analyzed at each time - point is indicated on the graph . Of note , whether attached or not clones continued to grow over time : * * * * Chi square test p < 0 . 0001 . All data are represented as mean ± s . e . m .

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

c) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination. In the P42 group, clones attached to HF were more likely to be bigger than those not attached at 3, 5, 12 and 24 weeks. ǂǂǂǂ Chi square test p<0.0001.The number of clones analyzed at each time-point is indicated on the graph. Of note, whether attached or not clones continued to grow over time: ****Chi square test p<0.0001. All data are represented as mean ± s.e.m.


d ) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size ( dashed line : predicted curve based on theoretical probabilities according to spacing of hair follicles at different time - points ; squares : experimental data ) . Between different time points the distance between hair follicles varies slightly ; therefore each time point ( 3 , 5 , 12 and 24 weeks ) has a distinct theoretical curve . The observed frequency of clone attachment to hair follicles is at every time point above the random prediction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size (dashed line: predicted curve based on theoretical probabilities according to spacing of hair follicles at different time-points; squares: experimental data). Between different time points the distance between hair follicles varies slightly; therefore each time point (3, 5, 12 and 24 weeks) has a distinct theoretical curve. The observed frequency of clone attachment to hair follicles is at every time point above the random prediction.


a ) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at the time of tissue collection . Clones connected to hair follicles were more likely to be larger in anagen compared to telogen ( Chi square test , * * * p = 0 . 0009 at 3 weeks , * * * p < 0 . 0001 at 5 weeks , * p = 0 . 0193 at 12 weeks , * * * p = 0 . 0001 at 24 weeks ) . However no difference between the two phases was observed in clones that were not attached to HFs . Clone numbers are indicated on the graph . All data are represented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at the time of tissue collection. Clones connected to hair follicles were more likely to be larger in anagen compared to telogen (Chi square test, *** p=0.0009 at 3 weeks, *** p<0.0001 at 5 weeks, * p=0.0193 at 12 weeks, *** p=0.0001 at 24 weeks). However no difference between the two phases was observed in clones that were not attached to HFs. Clone numbers are indicated on the graph. All data are represented as mean ± s.e.m.


b ) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post - induction . Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling . Average clone size of attached ( circles ) and non - attached ( squares ) clones were plotted over the number of club hair . T - test , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post-induction. Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling. Average clone size of attached (circles) and non-attached (squares) clones were plotted over the number of club hair. T-test, **** p<0.0001.


c ) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at induction . Even at a rate of one cell division per 24h , 7 cycles of divisions results in 128 cells . Therefore the findings reported are not incompatible with our claims as suggested . Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe . Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones . Chi square test , * * * p = 0 . 001 ; * * p = 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at induction. Even at a rate of one cell division per 24h, 7 cycles of divisions results in 128 cells. Therefore the findings reported are not incompatible with our claims as suggested. Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe. Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones. Chi square test, *** p=0.001; ** p=0.01.


( a - b ) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway ( a ) and corresponding scatter plots quantification . ( Scale bar 100µm ) ( b ) . Analysis focused on the basal layer of the IFE . Each dot represents one image . Proliferation in the IFE is significantly increased during the anagen phase except in mid - anagen . Unpaired t test , * * * * p < 0 . 0001 , * * p = 0 . 0020 . EA n = 32 , EMA n = 13 , MA n = 15 , C n = 21 , T n = 19 from 6 mice . Black arrows : positive Ki67 cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-b) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway (a) and corresponding scatter plots quantification. (Scale bar 100µm) (b). Analysis focused on the basal layer of the IFE. Each dot represents one image. Proliferation in the IFE is significantly increased during the anagen phase except in mid-anagen. Unpaired t test, ****p<0.0001, **p=0.0020. EA n = 32, EMA n = 13, MA n = 15, C n = 21, T n = 19 from 6 mice. Black arrows: positive Ki67 cells.


( c ) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti - Ki67 antibody or upon BrdU incorporation . Ki67 + cells are labelled with Alexa 647 ( red ) . The density of Ki67 + cells increased in the anagen phase ( iii and iv ) compared to telogen ( i and ii ) . White arrows : ki67 + cells . ( Scale bar = 20µm ) . Similarly , short term BrdU pulse ( 2h ) resulted in significant staining of anagen epidermal areas ( vi , red nuclear stain ) reinforcing hair follicle openings as compared to telogen areas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti-Ki67 antibody or upon BrdU incorporation. Ki67+ cells are labelled with Alexa 647 (red). The density of Ki67+ cells increased in the anagen phase (iii and iv) compared to telogen (i and ii). White arrows: ki67+ cells. (Scale bar = 20µm). Similarly, short term BrdU pulse (2h) resulted in significant staining of anagen epidermal areas (vi, red nuclear stain) reinforcing hair follicle openings as compared to telogen areas.


a - b ) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . a ) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti - BrdU antibody ( red ) . White dots represent hair follicles at different chase time points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-b) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. a) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti-BrdU antibody (red). White dots represent hair follicles at different chase time points.


a - b ) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . b ) At 5 weeks of chase , BrdU staining ( green ) in skinbowskin revealed single cells distant from hair follicles . White dashed circles : hair follicles . White line : small clones . White arrows : BrdU + cells . ( scale bar = 20 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-b) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. b) At 5 weeks of chase, BrdU staining (green) in skinbowskin revealed single cells distant from hair follicles. White dashed circles: hair follicles. White line: small clones. White arrows: BrdU+ cells. (scale bar = 20 µm).


c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . c ) Photomicrographs show optical sections from a confocal image stack acquired on a 0 . 5 mm back skin section immunostained with anti - ki67 antibody . Ki67 + cells are labelled with Alexa 647 ( red ) . Back skin from treated and untreated areas was collected 48hrs after TPA topical application .

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

c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application.


c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . d ) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti - BrdU ( green ) . Small clones not attached to HF ( 2 examples figured by dashed lines ) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation .

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

c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation.


e ) ( i to iv ) Representative individual sections from Z stacks , show small clones ( lines ) identified in back skin treated ( i and ii ) or not with TPA ( iii and iv ) . ( scale bar = 100 µm ) . ( v to viii ) Maximal intensity projection from stack acquired on whole mounted skin . A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA ( v to vi ) compared to skin treated with vehicle ( vii and viii ) . ( scale bars = 100 µm ) . f ) Reduction in small clone frequency in the areas treated with TPA . Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse . Mann Whitney test , * * p = 0 . 0079 . Data pooled from 3 mice represented as median with interquartile range . The number of clones analyzed is indicated on the graph . * p = 0 . 0272 . Data are represented as mean ± s . e . m .

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

e) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in back skin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.


a ) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42 . Data are represented as mean ± s . e . m .

O O O O O O O O O O O O O O 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) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42. Data are represented as mean ± s.e.m.


( G ) Sorted human monocytes were incubated with control medium or SARS - CoV - 2 viruses at an MOI of 1 for 1 h , followed by washing away extracellular viruses and further culture for 1 h . Cells were fixed and stained with the indicated antibodies . Otherwise , monocytes were challenged with 1 μg / ml LPS for 3 h and 10 μM nigericin for 30 min , followed by fixing and staining with the indicated antibodies . Treatment with medium from uninfected Vero cells served as a control . Scale bar : 5 μm . Data information : results were repeated for three times with similar results .

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

(G) Sorted human monocytes were incubated with control medium or SARS-CoV-2 viruses at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture for 1 h. Cells were fixed and stained with the indicated antibodies. Otherwise, monocytes were challenged with 1 μg/ml LPS for 3 h and 10 μM nigericin for 30 min, followed by fixing and staining with the indicated antibodies. Treatment with medium from uninfected Vero cells served as a control. Scale bar: 5 μm. Data information: results were repeated for three times with similar results.


Supernatants from control or SARS - CoV - 2 infected CD14 + monocytes with or without LPS and nigericin stimulation were collected and subjected to an ELISA assay for IL - 1β levels ( H ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . results were repeated for three times with similar results .

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

Supernatants from control or SARS-CoV-2 infected CD14+ monocytes with or without LPS and nigericin stimulation were collected and subjected to an ELISA assay for IL-1β levels (H). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: , Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.


Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells ( I ) . The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants ( J ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . results were repeated for three times with similar results .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells (I). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (J). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.


( B , C ) THP - 1 cells stably expressing the indicated SARS - CoV - 2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants were collected for an ELISA assay to determine supernatant IL - 1β levels ( B ) and cell pellets were subjected to a cell viability assay to verify the percentage of viable cells through checking cellular ATP levels ( C ) . For B - C , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . NS , non - significant . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Similar results were observed for at least three times .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C) THP-1 cells stably expressing the indicated SARS-CoV-2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were collected for an ELISA assay to determine supernatant IL-1β levels (B) and cell pellets were subjected to a cell viability assay to verify the percentage of viable cells through checking cellular ATP levels (C). For B-C, Student's t-test was used and data were shown as means±SD of three technical replicates. NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.


( D - F ) THP - 1 cells stably expressing the nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants and cell pellets were collected and immunoblotted with antibodies against the indicated proteins ( D ) . The percentage of viable cells was determined through checking cellular ATP levels in cell pellets ( E ) . The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants ( F ) . For E , F , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Similar results were observed for at least three times .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) THP-1 cells stably expressing the nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants and cell pellets were collected and immunoblotted with antibodies against the indicated proteins (D). The percentage of viable cells was determined through checking cellular ATP levels in cell pellets (E). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.


( D ) GSDMD + / + and GSDMD - / - THP - 1 cells were transfected with plasmids encoding nucleocapsid for 24 h , followed by cell fixation , permeabilization and stain with antibodies against nucleocapsid and GSDMD . The nucleus was counterstained with DAPI . Scale bar : 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GSDMD+/+ and GSDMD-/- THP-1 cells were transfected with plasmids encoding nucleocapsid for 24 h, followed by cell fixation, permeabilization and stain with antibodies against nucleocapsid and GSDMD. The nucleus was counterstained with DAPI. Scale bar: 5 μm.


( E , F ) THP - 1 ( E ) or human CD14 + monocytes ( F ) were transfected with plasmids encoding FLAG - tagged nucleocapsid for 36 h , followed by pretreatment of 1 μg / ml LPS for 3 h and 10 μM nigericin stimulation for 30 min . Cells were lysed and immunoprecipitated with antibody against FLAG or isotype IgG . Input and precipitates were immunoblotted as indicated .

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

(E, F) THP-1 (E) or human CD14+ monocytes (F) were transfected with plasmids encoding FLAG-tagged nucleocapsid for 36 h, followed by pretreatment of 1 μg/ml LPS for 3 h and 10 μM nigericin stimulation for 30 min. Cells were lysed and immunoprecipitated with antibody against FLAG or isotype IgG. Input and precipitates were immunoblotted as indicated.


( I ) GST - GSDMD and control GST were mixed with HEK293T cell lysate containing FLAG - tagged full - length nucleocapsid or truncated nucleocapsid lacking amino acids 70 - 160 and 290 - 360 , followed by a GST - pulldown assay . Input and precipitates were immunoblotted as indicated .

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

(I) GST-GSDMD and control GST were mixed with HEK293T cell lysate containing FLAG-tagged full-length nucleocapsid or truncated nucleocapsid lacking amino acids 70-160 and 290-360, followed by a GST-pulldown assay. Input and precipitates were immunoblotted as indicated.


( A , B ) THP - 1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Cells were either immunoblotted with antibodies against the indicated proteins ( A ) or stained with propidium iodide ( PI ) ( B , left ) . Scale bar : 20 μm . The percentage of PI positive cells was calculated ( B , right ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Similar results were observed for at least three times .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (A) or stained with propidium iodide (PI) (B, left). Scale bar: 20 μm. The percentage of PI positive cells was calculated (B, right). Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.


( H , I ) THP - 1 cells stably expressing full - length nucleocapsid or mutant lacking amino acids 70 - 160 and 290 - 360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Cells were either immunoblotted with antibodies against the indicated proteins ( H ) . Cells were also stained with PI and the percentage of PI positive cells was calculated ( I ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Results were repeated for three times with similar results . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Similar results were observed for at least three times .

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

(H, I) THP-1 cells stably expressing full-length nucleocapsid or mutant lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (H). Cells were also stained with PI and the percentage of PI positive cells was calculated (I). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.


( A ) GST or GST - GSDMD , p20 ( C285A ) - His , p10 - His and increasing amount of His - nucleocapsid were incubated for a GST - pulldown assay , followed by immunoblotting with antibodies against the indicated protein tags . Data information : Experiments were repeated for three times with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GST or GST-GSDMD, p20 (C285A)-His, p10-His and increasing amount of His-nucleocapsid were incubated for a GST-pulldown assay, followed by immunoblotting with antibodies against the indicated protein tags. Data information: Experiments were repeated for three times with similar results.


( G , H ) Wild - type GSDMD and GSDMDsusti . ( G ) or mCherry - GSDMD - C ( H ) were purified from HEK293T cells and incubated with recombinant p20 / p10 complex and His - tagged nucleocapsid in a cleavage buffer , followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C - termimus . Data information : Experiments were repeated for three times with similar results .

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

(G, H) Wild-type GSDMD and GSDMDsusti. (G) or mCherry-GSDMD-C (H) were purified from HEK293T cells and incubated with recombinant p20/p10 complex and His-tagged nucleocapsid in a cleavage buffer, followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C-termimus. Data information: Experiments were repeated for three times with similar results.


GSDMD - / - THP - 1 cells rescued with wild - type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Cells were immunoblotted with antibodies against the indicated proteins ( A )

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

GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were immunoblotted with antibodies against the indicated proteins (A)


( E , F ) GSDMD - / - THP - 1 cells rescued with wild - type or linker substituted GSDMD were infected with SARS - CoV - 2 at an MOI of 1 for 1h , followed by washing away extracellular viruses and further culture for 3 h . Otherwise , cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants were subjected to an ELISA assay to determine IL - 1β levels ( E ) . Otherwise , cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells ( F ) . For E , F , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results .

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

(E, F) GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Otherwise, cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were subjected to an ELISA assay to determine IL-1β levels (E). Otherwise, cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.


( G ) Sorted CD14 + monocytes from donors were infected with SARS - CoV - 2 at an MOI of 1 for 1h , followed by washing away extracellular viruses and further culture for 3 h . Cells were immunoblotted with antibodies against the indicated proteins . CD14 + monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h and served as a positive control . Data information : experiments were repeated for three time with similar results .

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

(G) Sorted CD14+ monocytes from donors were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Cells were immunoblotted with antibodies against the indicated proteins. CD14+ monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h and served as a positive control. Data information: experiments were repeated for three time with similar results.


A . RT - PCR analysis of the expression of β - actin , Trpv1 , Trpv2 , Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 ( upper ) and 35 ( lower ) thermal cycles with ( RT ( + ) ) and without ( RT ( - ) ) reverse transcription ( RT ) . Control ( Ct . ) lanes indicate the results with each plasmid DNA as a template .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.


B . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes . Expression levels of mRNA were normalized to that of the ribosomal protein gene ( 36B4 ) , a housekeeping gene un - affected by adipogenesis . Data are presented as mean ± SEM , n = 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.


C . Results of real - time PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue ( iBAT ) . mRNA expression levels were normalized to that of 36B4 . Data are presented as mean ± SEM , n = 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.


D . Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes . Upper bands in the TRPV2 blots likely indicate glycosylated forms .

O O O 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. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.


E . Averaged traces of [ Ca2 + ] i changes in response to 500 µmol / L 2APB in differentiated brown adipocytes from WT ( black ) and TRPV2KO ( red ) mice . One µmol / L NE was used to confirm differentiation . Five µmol / L ionomycin was used to confirm cell viability . Ratio values correspond to the real [ Ca2 + ] i of differentiated mousebrown adipocytes . Data are presented as mean ± SEM , n = 149 of WT cells , and n = 112 of TRPV2KO brown adipocytes ; * * P < 0 . 01 . Unpaired Student ' s t - test .

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

E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.


F . A representative trace of whole - cell current activated by 3 mmol / L of 2APB in the presence or absence of 10 µmol / L SKF in mouse differentiated brown adipocyte . The left inset indicates a voltage ramp - pulse protocol . The right inset indicates the current - voltage curves of basal current ( a , black ) , current in the presence of 2APB + SKF ( b , blue ) and current in the presence of 2APB alone ( c , red ) at the time points of a , b and c , respectively . G . Comparison of the mean densities of basal currents ( Base , black ) , currents in the presence of 2APB alone ( red ) and currents in the presence of 2APB + SKF ( blue ) at - 60 mV and + 100 mV in mouse differentiated brown adipocytes . Data are presented as mean ± SEM , n = 10 ; * * P < 0 . 01 vs . Base ; # # P < 0 . 01 vs . 2APB alone . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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

F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression in mouse pre - adipocytes and differentiated brown adipocytes . Expression levels of mRNA were normalized to those of 36B4 . Data are presented as mean ± SEM , n = 6 . * P < 0 . 05 vs . pre - adipocytes . Unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test.


B . The number of 6 day - differentiated mousebrown adipocytes treated with TRPV2 agonists , 100 µmol / L 2APB or 10 µmol / L LPC . Mean ± SEM , n = 6 , * P < 0 . 05 and * * P < 0 . 01 vs . dimethyl sulfoxide ( DMSO ) group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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

B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


C and D . Comparison of the numbers of 6 day - differentiated mouse brown adipocytes ( C ) and triglyceride levels ( D ) in the cells from WT and TRPV2KO mice with different differentiation media . 1 / 10 suppl . indicates differentiation medium ten - time - diluted with DMEM . Mean ± SEM , n = 8 , * * P < 0 . 01 vs . control group , # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Basal mRNA expression of Ucp1 , peroxisome proliferator - activated receptor gamma coactivator 1 - alpha ( Pgc1α ) , peroxisome proliferator - activated receptorγ ( Pparγ ) and β3 - adrenergic receptor 3 ( Adrb3 ) in the differentiated brown adipocytes from WT and TRPV2KO mice . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . WT . Unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptorγ(Pparγ) and β3-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student's t-test.


B and C . Changes in Ucp1 ( B ) and Pgc1α ( C ) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol / L isoproterenol ( ISO ) for 4 h . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . DMSO group ; # P < 0 . 05 ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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

B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D and E . Changes in Ucp1 mRNA expression ( D ) and non - esterified fatty acid ( NEFA ) release ( E ) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol / L forskolin for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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

D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


F and G . Changes in Ucp1 mRNA ( F ) and Pgc1α mRNA ( G ) in the differentiated brown adipocytes treated with 10 µmol / L ISO alone or 10 µmol / L ISO plus 10 µmol / L BAPTA - AM for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . ISO group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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

F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Body weight changes from 3 - weeks to 8 - weeks of age between the two genotypes ( n = 11 ) . Data are presented as mean ± SEM ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B and C . Changes in food intake ( B ) and water intake ( C ) from 3 - weeks to 8 - weeks of ages between WT and TRPV2KO mice ( n = 6 ) .

O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).


D . Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice ( n = 15 ) .

O 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. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).


E . The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice . mRNA expression levels of Ucp1 , Pgc1a , Ppary , Adrb3 , cytochrome c oxidase subunit 4 isoform 1 ( Cox4i1 ) , PR domain containing 16 ( Prdm16 ) , Ucp2 , lipoprotein lipase ( Lpl ) and cluster of differentiation 36 ( Cd36 ) were examined and normalized to the levels of 36B4 . Data are presented as mean ± SEM , n = 15 ; * P < 0 . 05 vs . WT group ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Ppary, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


A . Representative morphological results of iBAT by hematoxylin and eosin staining from 8 - week WT and TRPV2KO mice . Scale bars indicate 100 μm . B . Comparison of lipid droplet size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . Mean values are 9 . 5 ± 0 . 2 µm in 8 - week - old WT adipocytes , and 14 . 7 ± 0 . 2 µm in 8 - week - old TRPV2KO adipocytes ( P < 0 . 01 ) . 1000 to 1400 droplets were counted . Data are presented as mean ± SEM , n = 4 . C . Comparison of adipocyte size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . 800 to 1000 cells were counted . Data are presented as mean ± SEM , n = 4 . D . Mean diameters of adipocytes in 8 - week - old WT mice ( black ) and TRPV2KO mice ( red ) . Mean values are 15 . 8 ± 0 . 4 µm in 8 - week - old WT adipocytes , and 19 . 8 ± 0 . 3 µm in 8 - week - old TRPV2KO adipocytes . Data are presented as mean ± SEM , n = 4 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative morphological results of iBAT by hematoxylin and eosin staining from 8-week WT and TRPV2KO mice. Scale bars indicate 100 μm.B. Comparison of lipid droplet size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. Mean values are 9.5 ± 0.2 µm in 8-week-old WT adipocytes, and 14.7 ± 0.2 µm in 8-week-old TRPV2KO adipocytes (P < 0.01). 1000 to 1400 droplets were counted. Data are presented as mean ± SEM, n = 4.C. Comparison of adipocyte size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. 800 to 1000 cells were counted. Data are presented as mean ± SEM, n = 4.D. Mean diameters of adipocytes in 8-week-old WT mice (black) and TRPV2KO mice (red). Mean values are 15.8 ± 0.4 µm in 8-week-old WT adipocytes, and 19.8 ± 0.3 µm in 8-week-old TRPV2KO adipocytes. Data are presented as mean ± SEM, n = 4; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B , C and D . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) , Pparγ ( C ) and Trpv2 ( D ) in iBAT of WT and TRPV2KO mice before ( 25oC ) , after 1 day and 3 days cold ( 4oC ) exposure . Mean ± SEM , n = 5 - 8 ; * P < 0 . 05 ; * * P < 0 . 01 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


E and F . Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before ( 25oC ) and after 3 days cold ( 4oC ) exposure ( E ) . Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice ( F ) . Mean ± SEM , n = 6 ; * P < 0 . 05 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


G and H . Averages of core body temperatures ( G ) and activities ( H ) of WT and TRPV2KO mice with or without cold ( 4oC ) exposure . Data are presented as mean ± SEM , n = 5 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

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

G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B and C . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) and Pparγ ( C ) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration ( i . p . , arrows ) of saline or a selective β3 - adrenergic receptor agonist , BRL37344 ( 600 µg / kg body weight ) . Mean ± SEM , n = 5 - 7 ; * P < 0 . 05 vs . saline group ; # P < 0 . 01 vs . WT BRL37344 administration group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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

A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D and E . Average traces of changes in iBAT temperature ( D ) or rectal body temperature ( E ) from WT and TRPV2KO mice after BRL37344 ( 600 µg / kg body weight ) administration ( i . p . , arrows ) . Mean ± SEM , n = 5 . * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

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

D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test.


F and G . iBAT NE turnover rates ( F ) and rate constants k ( G ) in WT and TRPV2KO mice upon cold exposure ( 4oC ) for 4 h . Data are presented as mean ± SEM , n = 8 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.


A . Body weight changes between WT and TRPV2KO mice treated with high fat diet ( HFD ) for continuous 8 weeks from 13 - week - old .

O O O O O 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. Body weight changes between WT and TRPV2KO mice treated with high fat diet (HFD) for continuous 8 weeks from 13-week-old.


B and C . Blood glucose level ( B ) and plasma insulin level ( C ) of WT and TRPV2KO mice after 8 - week HFD treatment .

O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.


D . Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8 - week HFD treatment .

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

D. Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8-week HFD treatment.


E . mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8 - week HFD treatment . All data are presented as mean ± SEM , WT mice ( n = 6 ) , TRPV2KO mice ( n = 8 ) ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8-week HFD treatment. All data are presented as mean ± SEM, WT mice (n = 6), TRPV2KO mice (n = 8); * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade . Immunohistochemistry analysis of FOXA1 ( top ) and FOXA2 ( bottom ) in human PDAC . Representative G1 , G2 and G3 areas from one patient are shown . Red dotted lines indicate the borders of poorly differentiated ( G3 ) tumor areas .

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

FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade. Immunohistochemistry analysis of FOXA1 (top) and FOXA2 (bottom) in human PDAC. Representative G1, G2 and G3 areas from one patient are shown. Red dotted lines indicate the borders of poorly differentiated (G3) tumor areas.


Percent of FOXA1 - and FOXA2 - positive nuclei in PDAC cells from areas of different grade . Tissue microarrays were automatically acquired and quantified using QuPath . The number ( n ) of analyzed areas of each grade is indicated .

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

Percent of FOXA1- and FOXA2-positive nuclei in PDAC cells from areas of different grade. Tissue microarrays were automatically acquired and quantified using QuPath. The number (n) of analyzed areas of each grade is indicated.


Expression of FOXA1 and FOXA2 in low - grade and high - grade PDAC cell lines . Vinculin is shown as loading control .

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

Expression of FOXA1 and FOXA2 in low-grade and high-grade PDAC cell lines. Vinculin is shown as loading control.


Western blot showing siRNA - mediated depletion of FOXA1 and / or FOXA2 in the low - grade CFPAC1 cell line . The three biological replicates used in the subsequent experiments are shown .

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

Western blot showing siRNA-mediated depletion of FOXA1 and/or FOXA2 in the low-grade CFPAC1 cell line. The three biological replicates used in the subsequent experiments are shown.


Heatmap showing row - normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1 / 2 ( FDR ≤ 0 . 05 and absolute log2 FC ≥ 1 ) . Blue and orange depict down - and up - regulation , respectively .

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

Heatmap showing row-normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1/2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.


FOXA2 and FOXA1 expression in wild type and genome - edited high - grade PANC1 cells . Three different bulk populations were used for RNA - seq experiments .

FOXA2 and FOXA1 expression in wild type and genome-edited high-grade PANC1 cells. Three different bulk populations were used for RNA-seq experiments.


Heatmap showing row - normalized expression of differentially expressed genes in PANC1 cells deleted of FOXA2 ( FDR ≤ 0 . 05 and absolute log2 FC ≥ 1 ) . Blue and orange depict down - and up - regulation , respectively .

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

Heatmap showing row-normalized expression of differentially expressed genes in PANC1 cells deleted of FOXA2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.


Representative Gene Ontology ( GO ) terms enriched in genes differentially expressed upon FOXA1 / 2 depletion in CFPAC1 cells . Terms are ranked by the negative log10 p - value from the hypergeometric test . Panels on the right show the effects of FOXA1 / 2 depletion on cell morphology and adhesion to multiple substrates . Three replicates per condition were used : circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Gene Ontology (GO) terms enriched in genes differentially expressed upon FOXA1/2 depletion in CFPAC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA1/2 depletion on cell morphology and adhesion to multiple substrates. Three replicates per condition were used: circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition.


Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells . Terms are ranked by the negative log10 p - value from the hypergeometric test . Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates . Three different bulk populations were used as biological replicates : circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates. Three different bulk populations were used as biological replicates: circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition.


Scatter plots showing the correlation between FOXA2 ChIP - seq biological duplicates in CFPAC1 ( top panel ) and PANC1 cells ( bottom panel ) . Pearson ' s correlation ( r ) between samples is indicated .

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

Scatter plots showing the correlation between FOXA2 ChIP-seq biological duplicates in CFPAC1 (top panel) and PANC1 cells (bottom panel). Pearson's correlation (r) between samples is indicated.


Scatter plot showing the correlation between FOXA2 ChIP - seq peaks in CFPAC1 and PANC1 cells . Green and blue dots indicate FOXA2 peaks that are significantly ( FDR < 0 . 001 ) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind . All other peaks are indicated as grey dots . The boxplots on the right show the intensity of CFPAC1 - specific , PANC1 - specific and other FOXA2 peaks . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers .

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

Scatter plot showing the correlation between FOXA2 ChIP-seq peaks in CFPAC1 and PANC1 cells. Green and blue dots indicate FOXA2 peaks that are significantly (FDR < 0.001) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind. All other peaks are indicated as grey dots. The boxplots on the right show the intensity of CFPAC1-specific, PANC1-specific and other FOXA2 peaks. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.


Representative FOXA2 ChIP - seq snapshot showing FOXA2 peaks classified as CFPAC1 - specific , shared or PANC1 - specific .

Representative FOXA2 ChIP-seq snapshot showing FOXA2 peaks classified as CFPAC1-specific, shared or PANC1-specific.


The five top transcription factor motifs over - represented in TSS - distal CFPAC1 - specific FOXA2 peaks . TFs were grouped into families or subfamilies and ranked based on the best PWM q - value . The POU domain TF family includes HNF1β . Top scoring motifs are shown on the right .

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

The five top transcription factor motifs over-represented in TSS-distal CFPAC1-specific FOXA2 peaks. TFs were grouped into families or subfamilies and ranked based on the best PWM q-value. The POU domain TF family includes HNF1β. Top scoring motifs are shown on the right.


Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP - seq peaks .

Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP-seq peaks.


Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS ( left ) and JUNB ( right ) ChIP - seq peaks .

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

Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS (left) and JUNB (right) ChIP-seq peaks.


Two representative ChIP - seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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

Two representative ChIP-seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Representative areas from human PDAC tissue showing expression of HNF1β in G1 , G2 and G3 areas . The red dotted line indicates the borders of a G3 area . The percentage of HNF1β - positive cells in areas of different grade from a PDAC tissue microarray is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 areas from human PDAC tissue showing expression of HNF1β in G1, G2 and G3 areas. The red dotted line indicates the borders of a G3 area. The percentage of HNF1β-positive cells in areas of different grade from a PDAC tissue microarray is shown.


Colocalization of HNF1β and FOXA2 in human PDAC areas . PDAC sections from three different patients were stained with the anti - HNF1β antibody , stripped and re - stained with the anti - FOXA2 antibody . The red dotted areas on the left represent poorly differentiated ( G3 ) tumor regions .

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

Colocalization of HNF1β and FOXA2 in human PDAC areas. PDAC sections from three different patients were stained with the anti-HNF1β antibody, stripped and re-stained with the anti-FOXA2 antibody. The red dotted areas on the left represent poorly differentiated (G3) tumor regions.


FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility ( determined by ATAC - seq ) . Clusters 1 - 4 were HNF1β - positive while cluster 5 was HNF1β - negative . The heat - maps on the left show FOXA2 , HNF1β , ATAC - seq and H3K27Ac profiles at FOXA2 - bound genomic regions . The three heat - maps on the right show JunB ChIP - seq data and ATAC - seq data in wild type or Jun / JunB double knockout ( DKO ) CFPAC1 cells . Data are shown in + / - 3 kb regions centered on the summit of FOXA2 peaks .

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

FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility (determined by ATAC-seq). Clusters 1-4 were HNF1β- positive while cluster 5 was HNF1β-negative. The heat-maps on the left show FOXA2, HNF1β, ATAC-seq and H3K27Ac profiles at FOXA2-bound genomic regions. The three heat-maps on the right show JunB ChIP-seq data and ATAC-seq data in wild type or Jun/JunB double knockout (DKO) CFPAC1 cells. Data are shown in +/- 3 kb regions centered on the summit of FOXA2 peaks.


Characterization of clusters 1 - 3 . Left : quantification of the indicated signals at FOXA2 bound regions in wild - type ( WT ) and HNF1β knock - out ( KO ) cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers . Middle : distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks . Right : over - represented motifs in clusters 1 - 3 and inferred binding models .

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

Characterization of clusters 1-3. Left: quantification of the indicated signals at FOXA2 bound regions in wild-type (WT) and HNF1β knock-out (KO) cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Middle: distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks. Right: over-represented motifs in clusters 1-3 and inferred binding models.


Quantification of JunB ChIP - seq signals in clusters 1 to 5 . ( Right ) Quantification of ATAC - seq signals in clusters 1 to 5 in wild type and Jun / JunB double KO ( DKO ) CFPAC1 cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers .

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

Quantification of JunB ChIP-seq signals in clusters 1 to 5. (Right) Quantification of ATAC-seq signals in clusters 1 to 5 in wild type and Jun/JunB double KO (DKO) CFPAC1 cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.


Representative snapshot showing changes in FOXA2 occupancy , chromatin accessibility and histone acetylation in HNF1β knock - out CFPAC1 cells . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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

Representative snapshot showing changes in FOXA2 occupancy, chromatin accessibility and histone acetylation in HNF1β knock-out CFPAC1 cells. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Western blot showing HNF1β over - expression after lentiviral transduction of PANC1 cells . Expression of FOXA2 in wild type and over - expressing ( O . E . ) cells is shown . Vinculin : loading control .

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

Western blot showing HNF1β over-expression after lentiviral transduction of PANC1 cells. Expression of FOXA2 in wild type and over-expressing (O.E.) cells is shown. Vinculin: loading control.


Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β - expressing lentivirus ( HNF1B O . E . ) and in their matched mock - infected control . The fraction of gained , unchanged and lost FOXA2 peaks overlapping HNF1β ChIP - seq peaks in transduced PANC1 cells is shown in the pie - charts on the right .

Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β-expressing lentivirus (HNF1B O.E.) and in their matched mock-infected control. The fraction of gained, unchanged and lost FOXA2 peaks overlapping HNF1β ChIP-seq peaks in transduced PANC1 cells is shown in the pie-charts on the right.


TF motif over - represented in gained or lost FOXA2 - bound genomic regions .

TF motif over-represented in gained or lost FOXA2-bound genomic regions.


Representative snapshot from PANC1 cells transduced with the HNF1β - expressing lentivirus . HNF1B O . E . : HNF1B over - expression .

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 snapshot from PANC1 cells transduced with the HNF1β-expressing lentivirus. HNF1B O.E.: HNF1B over-expression.


Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP - seq peaks .

Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP-seq peaks.


Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP - seq peaks in PANC1 cells . The FOXA2 ChIP - seq track in CFPAC1 cells is also shown for comparison .

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

Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP-seq peaks in PANC1 cells. The FOXA2 ChIP-seq track in CFPAC1 cells is also shown for comparison.


FOXA2 ChIP - seq in CFPAC1 cells transduced with a HOXB8 - expressing lentivirus and in their matched mock - infected control . Gained ( brown ) and lost ( yellow ) FOXA2 peaks are shown . Pie - charts on the right show the fraction of gained , unchanged and lost peaks overlapping HOXB8 ChIP - seq peaks in transduced CFPAC1 cells .

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

FOXA2 ChIP-seq in CFPAC1 cells transduced with a HOXB8-expressing lentivirus and in their matched mock-infected control. Gained (brown) and lost (yellow) FOXA2 peaks are shown. Pie-charts on the right show the fraction of gained, unchanged and lost peaks overlapping HOXB8 ChIP-seq peaks in transduced CFPAC1 cells.


Expression of HNF1β protein in wild - type and HOXB8 - expressing PANC1 cells .

Expression of HNF1β protein in wild-type and HOXB8-expressing PANC1 cells.


Representative snapshots from CFPAC1 cells transduced with the HOXB8 - expressing lentivirus .

Representative snapshots from CFPAC1 cells transduced with the HOXB8-expressing lentivirus.


Figure 1 . CHCHD10 resides in the same complex than Mitofilin , CHCHD3 and CHCHD6 . A . Second dimension of the Blue - Native ( BN ) PAGE showing that CHCHD10 migrates with the MICOS protein subunits Mitofilin , CHCHD3 and CHCHD6 in isolated mitochondria from mouse

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

Figure 1. CHCHD10 resides in the same complex than Mitofilin, CHCHD3 and CHCHD6. A. Second dimension of the Blue-Native (BN) PAGE showing that CHCHD10 migrates with the MICOS protein subunits Mitofilin, CHCHD3 and CHCHD6 in isolated mitochondria from mouse brain


B . BN - PAGE of the MICOS and OXPHOS complexes in control ( C ) and patient ( P1 , P2 ) fibroblasts . Complexes I to IV of OXPHOS were detected with the 39kDa subunit antibody ( CI ) , 70kDa subunit antibody ( CII ) , core I antibody ( CIII ) and cytochrome c oxidase subunit I antibody ( CIV ) . MICOS complex was detected with an anti - Mitofilin antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BN-PAGE of the MICOS and OXPHOS complexes in control (C) and patient (P1, P2) fibroblasts. Complexes I to IV of OXPHOS were detected with the 39kDa subunit antibody (CI), 70kDa subunit antibody (CII), core I antibody (CIII) and cytochrome c oxidase subunit I antibody (CIV). MICOS complex was detected with an anti-Mitofilin antibody.


C . Second dimension of the BN - PAGE showing that the steady - state levels of assembled CHCHD10 in MICOS complex are decreased in patient fibroblasts .

O O O O 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. Second dimension of the BN-PAGE showing that the steady-state levels of assembled CHCHD10 in MICOS complex are decreased in patient fibroblasts.


D . Analysis of OXPHOS supercomplexes in control and patient fibroblasts . BN - PAGE from isolated mitochondria permeabilized with 6g / g ( w / v ) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies . SC , supercomplexes I + III2 + IVn . The dividing lines correspond to gels sections visible in the raw data file .

D. Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. The dividing lines correspond to gels sections visible in the raw data file.


Figure 2 . Interaction of CHCHD10 with components of MICOS complex . A . Co - immunonoprecipitation ( IP ) of endogenous mitofilin , CHCHD10 and CHCHD3 in control and patient fibroblasts ( 1mg of total extracts were used for each IP ( left lower panel ) and 200µg were used for the input ( left upper panel ) ) . The same results were found in P2 fibroblasts ( not shown ) . Reverse co - IP experiment in HeLa cells with an antibody against CHCHD10 ( right lower panel ) . 1mg of total extracts were used for each IP and 200µg were used for the input ( right upper panel ) . The dividing lines correspond to gels sections visible in the raw data file .

Figure 2. Interaction of CHCHD10 with components of MICOS complex. A. Co-immunonoprecipitation (IP) of endogenous mitofilin, CHCHD10 and CHCHD3 in control and patient fibroblasts (1mg of total extracts were used for each IP (left lower panel) and 200µg were used for the input (left upper panel)). The same results were found in P2 fibroblasts (not shown). Reverse co-IP experiment in HeLa cells with an antibody against CHCHD10 (right lower panel). 1mg of total extracts were used for each IP and 200µg were used for the input (right upper panel). The dividing lines correspond to gels sections visible in the raw data file.


B . Duolink proximity ligation assay between Mitofilin and CHCHD10 ( upper panels ) and , CHCHD10 and CHCHD6 ( lower panels ) in control and patient fibroblasts observed by confocal microscopy . Mitochondria were stained with Mitotracker . Duolink spots per cell were quantified for 30 randomly - selected individual cells per each studied fibroblast cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p = 0 . 0001 ) . Scale bar = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Duolink proximity ligation assay between Mitofilin and CHCHD10 (upper panels) and, CHCHD10 and CHCHD6 (lower panels) in control and patient fibroblasts observed by confocal microscopy. Mitochondria were stained with Mitotracker. Duolink spots per cell were quantified for 30 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001). Scale bar = 10 µm.


Figure 3 . Decrease of nucleoid number without mtDNA depletion in patient fibroblasts . A . In control and patient fibroblasts , nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker . Image analysis was performed by confocal microscopy . Scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

Figure 3. Decrease of nucleoid number without mtDNA depletion in patient fibroblasts. A. In control and patient fibroblasts, nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.


B - C . The average number ( B ) and size ( C ) of nucleoids , labeled with antibodies against DNA shown in A , were quantified for 35 randomly - selected individual cells per each studied fibroblast cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p < 0 . 001 ) . Nucleoid number ( B ) : patient 1 versus control * * * : p = 0 . 0001 , patient 2 versus control * * * : p = 0 . 001 . Nucleoid size ( C ) : patient 1 versus control * * * : p = 0 . 0001 . D . Mitochondrial DNA content in control and patientfibroblasts was quantified by real - time PCR . Data were expressed as ratio between mtDNA and nuclear DNA concentration . Results represent the mean of relative PCR ± SD of 3 independent experiments . Statistical analysis were performed by Student ' s t - test ( two - sided ) .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p<0.001). Nucleoid number (B): patient 1 versus control ***: p=0.0001, patient 2 versus control ***: p=0.001. Nucleoid size (C): patient 1 versus control ***: p=0.0001. D. Mitochondrial DNA content in control and patientfibroblasts was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nuclear DNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).


Figure 4 . Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles . A . Transfections were performed with empty vector ( EV ) or vectors encoding wild - type CHCHD10 - FLAG ( WT ) , CHCHD10 - FLAG ( S59L ) or CHCHD10 - FLAG ( P34S ) mutants . Mitochondria were stained with Mitotracker . Cells overexpressing wild - type and mutant CHCHD10 were labeled with FLAG antibodies . Nucleoids were visualized with an antiserum against DNA . Image analysis was performed by confocal microscopy . Scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

Figure 4. Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles. A. Transfections were performed with empty vector (EV) or vectors encoding wild-type CHCHD10-FLAG (WT), CHCHD10-FLAG (S59L) or CHCHD10-FLAG (P34S) mutants. Mitochondria were stained with Mitotracker. Cells overexpressing wild-type and mutant CHCHD10 were labeled with FLAG antibodies. Nucleoids were visualized with an antiserum against DNA. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.


B - C . The average number ( B ) and size ( C ) of nucleoids , labeled with antibodies against DNA shown in A , were quantified for 35 randomly - selected individual cells per each studied cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : very significant ( * * : p = 0 . 0280 ) or extremely significant ( * * * : p = 0 . 0001 ) . D . Mitochondrial DNA content was quantified by real - time PCR . Data were expressed as ratio between mtDNA and nDNA concentration . Results represent the mean of relative PCR ± SD of 3 independent experiments . Statistical analysis were performed by Student ' s t - test ( two - sided ) .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): very significant (**: p=0.0280) or extremely significant (***: p=0.0001). D. Mitochondrial DNA content was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nDNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).


Figure 5 . Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress . A . Total extracts from control ( C ) fibroblasts and intact isolated mitochondria from control and patient fibroblasts ( P1 , P2 ) were analyzed by immunoblotting using antibodies against PCNA ( nuclear protein ) , GAPDH ( cytosolic protein ) or SMAC ( mitochondrial intermembrane space protein ) ( upper panel ) . Mitochondria from patient and control fibroblasts were incubated with NP - 40 and separated into pellets ( P ) and supernatants ( S ) . The fractions of each extraction were subjected to western blot analysis . VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins , respectively ( middle panel ) . Ratio of mtDNA amplified from supernatant / mtDNA amplified from pellet , by qPCR , was quantified in control and patient fibroblasts ( lower panel ) .

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

Figure 5. Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress. A. Total extracts from control (C) fibroblasts and intact isolated mitochondria from control and patient fibroblasts (P1, P2) were analyzed by immunoblotting using antibodies against PCNA (nuclear protein), GAPDH (cytosolic protein) or SMAC (mitochondrial intermembrane space protein) (upper panel). Mitochondria from patient and control fibroblasts were incubated with NP-40 and separated into pellets (P) and supernatants (S). The fractions of each extraction were subjected to western blot analysis. VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins, respectively (middle panel). Ratio of mtDNA amplified from supernatant/mtDNA amplified from pellet, by qPCR, was quantified in control and patient fibroblasts (lower panel).