IN - DCMRL coronal slice of the chest at the level of the carina demonstrating extensive mediastinal and pulmonary interstitial perfusion ( arrows ) at baseline ( G ) , and after 6 months of treatment with trametinib ( H ) .

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IN-DCMRL coronal slice of the chest at the level of the carina demonstrating extensive mediastinal and pulmonary interstitial perfusion (arrows) at baseline (G), and after 6 months of treatment with trametinib (H).


A . Deep coverage exome sequencing Integrative Genomics Viewer ( IGV ) view of CBL c . 2322T > G in DNA sample that was derived from CD31 selected cells from pleural effusion fluid . The alternate allele fraction was 4 % ( 20 / 493 ) .

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A. Deep coverage exome sequencing Integrative Genomics Viewer (IGV) view of CBL c.2322T>G in DNA sample that was derived from CD31 selected cells from pleural effusion fluid. The alternate allele fraction was 4% (20/493).


B . Droplet digital polymerase chain reaction ( ddPCR ) result with 5 mutant positive droplets ( circle ) from a low concentration left over sample which resulted in ~ 0 . 005 % fractional abundance .

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B. Droplet digital polymerase chain reaction (ddPCR) result with 5 mutant positive droplets (circle) from a low concentration left over sample which resulted in ~0.005% fractional abundance.


A Western blot analysis and quantification of GFUS . Expression of GFUS was analyzed by Western blotting on a 15 % SDS PAGE with cytosolic fractions derived from control and patient fibroblasts . Quantification revealed a significant decrease of GFUS protein . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 4 times , for statistics an unpaired t test was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars and error bars represent mean ± SD .

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A Western blot analysis and quantification of GFUS. Expression of GFUS was analyzed by Western blotting on a 15% SDS PAGE with cytosolic fractions derived from control and patient fibroblasts. Quantification revealed a significant decrease of GFUS protein. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


B mRNA expression studies . qRT - PCR studies showed a significant decrease in mRNA level of GFUS by 40 . 0 % ( ± 9 . 3 % , * * p = 0 . 0010 ) normalized to a control . Also , expression of SLC35C1 and SLC35C2 were significantly decreased in patient - derived fibroblasts . Data were obtained from fibroblasts ; n = 10 ; experiment was independently repeated 3 times , for statistics an one - way ANOVA was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars and error bars represent mean ± SD .

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B mRNA expression studies. qRT-PCR studies showed a significant decrease in mRNA level of GFUS by 40.0% (± 9.3%, **p = 0.0010) normalized to a control. Also, expression of SLC35C1 and SLC35C2 were significantly decreased in patient-derived fibroblasts. Data were obtained from fibroblasts; n = 10; experiment was independently repeated 3 times, for statistics an one-way ANOVA was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


C Conversion of GDP - D - mannose into GDP - L - fucose . The conversion GDP - D - [ 14C ] mannose to GDP - L - [ 14C ] fucose was analyzed in an in - vitro assay . After 1 h nearly half of GDP - D - mannose was transformed to GDP - fucose in the control cell line , whereas the patient ´ s fibroblasts were nearly unable to catalyze the reaction . Data were obtained from fibroblasts ; n = 1 .

C Conversion of GDP-D-mannose into GDP-L-fucose. The conversion GDP-D-[14C]mannose to GDP-L-[14C]fucose was analyzed in an in-vitro assay. After 1 h nearly half of GDP-D-mannose was transformed to GDP-fucose in the control cell line, whereas the patient´s fibroblasts were nearly unable to catalyze the reaction. Data were obtained from fibroblasts; n = 1.


D Lectin binding studies and quantification in fibroblasts . Aleuria aurantia lectin ( AAL ) was used to address the fucosylation level in fibroblasts . AAL staining was performed before and after treatment of the patient cell line with 100 µM L - fucose . Before sugar supplementation a significant loss of AAL signal strength was measured , whereas after treatment a significant re - fucosylation was detected . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 4 times , for statistics an unpaired t test was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars and error bars represent mean ± SD .

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D Lectin binding studies and quantification in fibroblasts. Aleuria aurantia lectin (AAL) was used to address the fucosylation level in fibroblasts. AAL staining was performed before and after treatment of the patient cell line with 100 µM L-fucose. Before sugar supplementation a significant loss of AAL signal strength was measured, whereas after treatment a significant re-fucosylation was detected. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


E Complementation study with patient - derived fibroblasts . Viral infection was used to introduce an empty cloning vector and the wild - type GFUS cDNA in patient cells , respectively . For analysis lysates of a control cell - line , a control cell - line supplemented with 100 µM fucose , patient cells , patient cells supplemented with 100 µM fucose , patient cells transfected with an empty vector and patient cells transfected with wild type GFUS were analyzed by AAL staining clearly indicating the disease - causing influence of the defective GFUS in the patient . Western blot analysis against GFUS further showed expression of wildtype - GFUS protein in the infected patient derived fibroblasts . Data were obtained from fibroblasts ; n = 4 ; experiment was independently repeated 2 times , for statistics an one - way ANOVA was performed . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Bars and error bars represent mean ± SD .

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E Complementation study with patient-derived fibroblasts. Viral infection was used to introduce an empty cloning vector and the wild-type GFUS cDNA in patient cells, respectively. For analysis lysates of a control cell-line, a control cell-line supplemented with 100 µM fucose, patient cells, patient cells supplemented with 100 µM fucose, patient cells transfected with an empty vector and patient cells transfected with wild type GFUS were analyzed by AAL staining clearly indicating the disease-causing influence of the defective GFUS in the patient. Western blot analysis against GFUS further showed expression of wildtype-GFUS protein in the infected patient derived fibroblasts. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 2 times, for statistics an one-way ANOVA was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.


A Aleuria aurantia lectin ( AAL ) was used to address the fucosylation level in sera of controls and the patient ( left ) which revealed significant loss of fucose residues in case of the patient . Data were obtained from serum ; n = 6 ; experiment was independently repeated 3 times , for statistics an unpaired t test was performed . Data information : * * p < 0 . 01 . Bars and error bars represent mean ± SD . Exact p ‑ values are reported in the results part .

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A Aleuria aurantia lectin (AAL) was used to address the fucosylation level in sera of controls and the patient (left) which revealed significant loss of fucose residues in case of the patient. Data were obtained from serum; n = 6; experiment was independently repeated 3 times, for statistics an unpaired t test was performed. Data information: **p < 0.01. Bars and error bars represent mean ± SD. Exact p‑values are reported in the results part.


B FACS analysis of PBMCs . Results of flow cytometry analysis of patient ( red ) and control ( green ) PBMCs stained with 1 µg / ml FAA lectin without ( dark ) or with 200 mM L - fucose ( light ) before the start of fucose therapy . C FACS analysis of PBMCs . Results of flow cytometry analysis of patient ( red ) and control ( green ) PBMCs stained with 1 µg / ml FAA lectin without ( dark ) or with 200 mM L - fucose ( light ) 8 weeks after therapy start

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B FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) before the start of fucose therapy. C FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) 8 weeks after therapy start


A - G Hematoxylin and eosin ( H & E ) staining of the frontal sections of miniature pig mandible slices showing morphological changes during the permanent canine ( PC ) initiation stage from embryonic day 50 ( E50 ) to postnatal day 10 ( PN10 ) ; ( A ' - F ' ) are magnifications of boxed regions in their corresponding figure panel . DC , deciduous canine ; PC , permanent canine ; SDL , successional dental lamina ; n = 3 . Scale bars = 100 µm .

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A-G Hematoxylin and eosin (H&E) staining of the frontal sections of miniature pig mandible slices showing morphological changes during the permanent canine (PC) initiation stage from embryonic day 50 (E50) to postnatal day 10 (PN10); (A'-F') are magnifications of boxed regions in their corresponding figure panel. DC, deciduous canine; PC, permanent canine; SDL, successional dental lamina; n = 3. Scale bars = 100 µm.


H In situ hybridization ( ISH ) showing the expression pattern of Sox2 in the initiation stage from E50 to E90 ; right figure panels are magnifications of boxed regions in left panels . Dashed lines mark the position of the successional dental lamina ( SDL ) ; green arrowhead indicates positive staining of Sox2 at the tip of the SDL . n = 3 . Scale bars = 100 µm .

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H In situ hybridization (ISH) showing the expression pattern of Sox2 in the initiation stage from E50 to E90; right figure panels are magnifications of boxed regions in left panels. Dashed lines mark the position of the successional dental lamina (SDL); green arrowhead indicates positive staining of Sox2 at the tip of the SDL. n = 3. Scale bars = 100 µm.


I Immunofluorescence ( IF ) of Ki67 at the SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm .

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I Immunofluorescence (IF) of Ki67 at the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.


J Semi - quantification and comparison of Ki67 expression levels during E60 , E70 , and E90 stages . n = 3 . Data information : Data represent the means ± SEM . * P < 0 . 05 ( one - way ANOVA and Newman - Keuls post - hoc tests ) .

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J Semi-quantification and comparison of Ki67 expression levels during E60, E70, and E90 stages. n = 3. Data information: Data represent the means ± SEM. *P < 0.05 (one-way ANOVA and Newman-Keuls post-hoc tests).


K TUNEL assay showing the apoptosis status of the SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm .

K TUNEL assay showing the apoptosis status of the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.


A Three - dimensional reconstruction of serial H & E frontal sections of miniature pig mandibles at embryonic day 60 ( E60 ) and E90 ; deciduous canine ( DC ) in purple , permanent canine ( PC ) in yellow , and alveolar socket in green . The red , blue , and green arrows indicate the width of the labial alveolar socket , lingual alveolar socket , and DC , respectively . n = 3 .

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A Three-dimensional reconstruction of serial H&E frontal sections of miniature pig mandibles at embryonic day 60 (E60) and E90; deciduous canine (DC) in purple, permanent canine (PC) in yellow, and alveolar socket in green. The red, blue, and green arrows indicate the width of the labial alveolar socket, lingual alveolar socket, and DC, respectively. n = 3.


B DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90 . n = 3 . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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B DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.


C The proportion of DC width relative to the total alveolar socket width during E60 and E90 . n = 3 . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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C The proportion of DC width relative to the total alveolar socket width during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.


D Micro - CT imaging of the whole mandible at E60 ; boxed region is magnified in D ' ( top panel ) . ( D ' ) Mandible slice isolated with Geomagic software . White dashed lines mark DC . E 3 - D color map ( left ) after alignment of mandible slices before and after ( sham ) surgery showing a comparison of the surface points . Coronal sections through cusp tips ( transparent squares , left ) were selected for 2 - D comparisons ( middle ) ; right panels are magnifications of yellow - boxed regions . The solid purple contour and dotted black contour indicate the pre - and post - surgery shapes , respectively . The distance between the two contours are the colored line segments showing the distance and direction of the movement . The colored ball in the 2 - D comparison marks the position of the maximum displacement . The PC position is indicated in pink . n = 3 .

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D Micro-CT imaging of the whole mandible at E60; boxed region is magnified in D' (top panel). (D') Mandible slice isolated with Geomagic software. White dashed lines mark DC. E 3-D color map (left) after alignment of mandible slices before and after (sham) surgery showing a comparison of the surface points. Coronal sections through cusp tips (transparent squares, left) were selected for 2-D comparisons (middle); right panels are magnifications of yellow-boxed regions. The solid purple contour and dotted black contour indicate the pre- and post-surgery shapes, respectively. The distance between the two contours are the colored line segments showing the distance and direction of the movement. The colored ball in the 2-D comparison marks the position of the maximum displacement. The PC position is indicated in pink. n = 3.


F Dissected mandible slice with a " U " shape .

F Dissected mandible slice with a "U" shape.


A Diagram of the dissected miniature pig mandible slice illustrating the deciduous ( DC ) and permanent ( PC ) canine and application of compression in vitro using Flexcell FX - 5000 Compression System .

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A Diagram of the dissected miniature pig mandible slice illustrating the deciduous (DC) and permanent (PC) canine and application of compression in vitro using Flexcell FX-5000 Compression System.


B - D H & E staining of canine frontal sections from embryonic day 60 ( E60 ) ( B ) , after culturing for two days without stress ( 0 kPa ) ( C ) , or with stress ( 3 kPa ) ( D ) . ( B ' - D ' ) are magnifications of boxed regions in their corresponding figure panels . Scale bars = 50 µm . n = 6 / 6 in E60 group ; n = 12 / 12 in 0 - kPa group ; n = 4 / 8 in 3 - kPa group .

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B-D H&E staining of canine frontal sections from embryonic day 60 (E60) (B), after culturing for two days without stress (0 kPa) (C), or with stress (3 kPa) (D). (B'-D') are magnifications of boxed regions in their corresponding figure panels. Scale bars = 50 µm. n = 6/6 in E60 group; n = 12/12 in 0-kPa group; n = 4/8 in 3-kPa group.


F RT - qPCR of several mechanosensitive proteins ( TGFB1 , NFKB1 , EGR1 , and RUNX2 ) and of four mechanoreceptors ( ITGB1 , ITGB3 , ITGAV and ITGA2 ) in PC and surrounding soft tissue at E60 and after culturing for two days with ( 3 kPa ) or without extra mechanical stress ( 0 kPa ) . n = 3 for each group . Data represent the means ± SEM . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant . =

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F RT-qPCR of several mechanosensitive proteins (TGFB1, NFKB1, EGR1, and RUNX2) and of four mechanoreceptors (ITGB1, ITGB3, ITGAV and ITGA2) in PC and surrounding soft tissue at E60 and after culturing for two days with (3 kPa) or without extra mechanical stress (0 kPa). n = 3 for each group. Data represent the means ± SEM. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups), *P < .05, **P < .01, ***P < .001; NS, not significant. =


A - L IF of integrin β1 , ERK1 , and RUNX2 in miniature pig canine frontal sections at embryonic day 60 ( E60 ) , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days ; ( A ' - L ' ) are magnifications of boxed regions in the corresponding figure panels . M Relative IF expression levels of integrin β1 , ERK1 , and RUNX2 during E60 , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days . Data information : Data represent the means ± SEM . n = 3 . Scale bars = 25 µm ( A ' , G ' ) and 50 µm ( other panels ) . Unpaired t - tests for M , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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A-L IF of integrin β1, ERK1, and RUNX2 in miniature pig canine frontal sections at embryonic day 60 (E60), E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days; (A'-L') are magnifications of boxed regions in the corresponding figure panels. M Relative IF expression levels of integrin β1, ERK1, and RUNX2 during E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. Scale bars = 25 µm (A', G') and 50 µm (other panels). Unpaired t-tests for M , *P < .05, **P < .01, ***P < .001; NS, not significant.


N Morphology of the human PC at weeks 18 - 19 ( H & E staining ) ; ( N ' ) is magnification of the boxed region in ( N ) . DC , deciduous canine ; PC , permanent canine ; IEE , inner enamel epithelium ; OEE , outer enamel epithelium . Mesenchyme between DC and PC is indicated with a green arrow .

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N Morphology of the human PC at weeks 18-19 (H&E staining); (N') is magnification of the boxed region in (N). DC, deciduous canine; PC, permanent canine; IEE, inner enamel epithelium; OEE, outer enamel epithelium. Mesenchyme between DC and PC is indicated with a green arrow.


O - Q ISH expression patterns of ITGB1 , ERK1 , and RUNX2 in human PCs . Green arrows indicate the mesenchyme between DC and PC .

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O-Q ISH expression patterns of ITGB1, ERK1, and RUNX2 in human PCs. Green arrows indicate the mesenchyme between DC and PC.


R IF of RUNX2 in human PC . Mesenchyme between DC and PC is indicated with a red arrow . Red arrow indicates the mesenchyme between DC and PC .

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R IF of RUNX2 in human PC. Mesenchyme between DC and PC is indicated with a red arrow. Red arrow indicates the mesenchyme between DC and PC.


S PC epithelium areas in E60 , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days . Data information : Data represent the means ± SEM . n = 3 . , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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S PC epithelium areas in E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. , *P < .05, **P < .01, ***P < .001; NS, not significant.


IF of RUNX2 and phospho - ERK1 / 2 ( p - ERK1 / 2 ) upon force loading with 0 or 1 . 0 g / cm2 for 2 h or after force was removed and the cells cultured for an additional 1 , 2 , and 4 hours .

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IF of RUNX2 and phospho-ERK1/2 (p-ERK1/2) upon force loading with 0 or 1.0 g/cm2 for 2 h or after force was removed and the cells cultured for an additional 1, 2, and 4 hours.


V Relative IF expression levels of RUNX2 and p - ERK1 / 2 between groups Data information : Data represent the means ± SEM . n = 3 . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for V , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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V Relative IF expression levels of RUNX2 and p-ERK1/2 between groups Data information: Data represent the means ± SEM. n = 3. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for V , *P < .05, **P < .01, ***P < .001; NS, not significant.


W Western blots of p - ERK1 / 2 and RUNX2 levels after 1 . 0 g / cm2 was applied for 2 h , with or without anti - integrin β1 antibody and ERK1 inhibitor ( U0126 ) . X Relative expression levels of p - ERK1 / 2 , RUNX2 , and Actin in ( W ) . Data information : Data represent the means ± SEM . n = 3 . Unpaired t - tests for M ; One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for X , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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

W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126). X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W). Data information: Data represent the means ± SEM. n = 3. Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for X, *P < .05, **P < .01, ***P < .001; NS, not significant.


A - C H & E staining of miniature pig canine frontal sections from embryonic day 60 ( E60 group ) ( A ) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag ( overexpression group ) ( B ) or overexpression control lentiviral vector with a Myc tag ( O - control group ) ( C ) ; ( A ' - C ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C H&E staining of miniature pig canine frontal sections from embryonic day 60 (E60 group) (A) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag (overexpression group) (B) or overexpression control lentiviral vector with a Myc tag (O-control group) (C); (A'-C') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


D - F Immunohistochemical ( IHC ) staining of Myc expression in mandible slices from the E60 , overexpression , and O - control groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 100 µm ( D - F )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunohistochemical (IHC) staining of Myc expression in mandible slices from the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 100 µm (D-F)


G - I IF staining of RUNX2 expression ( yellow arrows ) in mandible slices of the E60 , overexpression , and O - control groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50

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

G-I IF staining of RUNX2 expression (yellow arrows) in mandible slices of the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


J , K Relative expression levels of Myc and RUNX2 in the E60 , O - control , and overexpression groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for J , K , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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

J, K Relative expression levels of Myc and RUNX2 in the E60, O-control, and overexpression groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, K , *P < .05, **P < .01, ***P < .001.


L , M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector ( knockdown control , K - control ) ( L ) or RUNX2 shRNA lentiviral vector ( knockdown ) ( M ) ; ( L ' - M ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50 µm ( other panels ) .

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

L, M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector (knockdown control, K-control) (L) or RUNX2 shRNA lentiviral vector (knockdown) (M); (L'-M') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm (other panels).


N Relative RUNX2 expression levels between K - control and knockdown groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for N , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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

N Relative RUNX2 expression levels between K-control and knockdown groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for N , *P < .05, **P < .01, ***P < .001.


O - Q H & E staining of mandible slice sections from the K - control , knockdown + force ( 3 kPa ) , and K - control + force ( 3 kPa ) groups . ( O ' - Q ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50

O-Q H&E staining of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (O'-Q') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


R - T IF staining of RUNX2 expression ( yellow arrows ) of mandible slice sections from the K - control , knockdown + force ( 3 kPa ) , and K - control + force ( 3 kPa ) groups . ( R ' - T ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 25 µm ( R ' - T ' )

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

R-T IF staining of RUNX2 expression (yellow arrows) of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (R'-T') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 25 µm (R'-T')


U Relative RUNX2 expression levels among the three groups Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for U , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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

U Relative RUNX2 expression levels among the three groups Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for U, *P < .05, **P < .01, ***P < .001.


V PC epithelium areas in the overexpression , O - control , knockdown , knockdown + force , K - control , and K - control + force groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for V ; , * P < . 05 , * * P < . 01 , * * * P < . 001 .

V PC epithelium areas in the overexpression, O-control, knockdown, knockdown + force, K-control, and K-control + force groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for V; , *P < .05, **P < .01, ***P < .001.


A , B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 ( E60 ) and E90 . ( A ' , B ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 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

A, B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 (E60) and E90. (A', B') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


C Relative ISH expression levels of RUNX2 in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data information Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for C , * P < . 05 , * * P < . 01 , * * * P < . 001 .

C Relative ISH expression levels of RUNX2 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for C , *P < .05, **P < .01, ***P < .001.


D , E ISH of miniature pig canine frontal sections showing expression patterns of β - catenin at E60 and E90 . ( D ' , E ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 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

D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


F Relative ISH expression levels of β - catenin in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative ISH expression levels of β-catenin in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


G , H IF of Lef1 at E60 and E90 . ( G , H ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 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

G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


I Relative IF expression levels of Lef1 in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


J FISH of β - catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope . Boxed regions of mesenchyme are magnified in the right panels . Epithelium is indicated with a yellow arrow . Data information : Scale bars = 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

J FISH of β-catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope. Boxed regions of mesenchyme are magnified in the right panels. Epithelium is indicated with a yellow arrow. Data information: Scale bars = 50 µm.


K Relative FISH expression levels of β - catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60 , E90 , and E60 cultured for 12 and 24 h . Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA for K , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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

K Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60, E90, and E60 cultured for 12 and 24 h. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA for K, *P < .05, **P < .01, ***P < .001.


L , M IF of β - catenin at E60 and E90 ; The yellow arrows indicate the localization of β - catenin in the nucleus . Data information : Scale bars = 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

L, M IF of β-catenin at E60 and E90; The yellow arrows indicate the localization of β-catenin in the nucleus. Data information: Scale bars = 50 µm.


N Relative IF expression levels of β - catenin in the nucleus of epithelium and mesenchyme at E60 and E90 . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

N Relative IF expression levels of β-catenin in the nucleus of epithelium and mesenchyme at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


O , P FISH of β - catenin in mandible slices infected with RUNX2 shRNA lentivirus ( knockdown ) or scrambled shRNA lentivirus ( scramsh ) via RNAscope . Data information : Scale bars = 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, P FISH of β-catenin in mandible slices infected with RUNX2 shRNA lentivirus (knockdown) or scrambled shRNA lentivirus (scramsh) via RNAscope. Data information: Scale bars = 50 µm.


Q Relative FISH expression levels of β - catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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

Q Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


A , B IF of Lef1 in mandible slices at embryonic day 60 ( E60 ) infected with RUNX2 overexpression lentiviral vector ( overexpression ) or overexpression control lentiviral vector ( O - control ) . ( A ' - B ' ) are magnifications of boxed regions in the corresponding figure panels . Dashed lines mark the epithelium of PC . C Relative IF expression levels of Lef1 in epithelium ( Epi . ) and mesenchyme ( Mes . ) of the overexpression and O - control groups . Data information : Data represent the means ± SEM . Scale bars = 100 µm . n = 3 for all experiments . Unpaired t - tests for C , * * P < . 01 , * * * P < . 001 ; NS . not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 IF of Lef1 in mandible slices at embryonic day 60 (E60) infected with RUNX2 overexpression lentiviral vector (overexpression) or overexpression control lentiviral vector (O-control). (A'-B') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. C Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) of the overexpression and O-control groups. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for C , **P < .01, ***P < .001; NS. not significant.


D , E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days . ( D ' , E ' ) are magnifications of boxed regions in the corresponding figure panels . Dashed lines mark the epithelium of PC . F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure . Data information : Data represent the means ± SEM . Scale bars = 100 µm . n = 3 for all experiments . Unpaired t - tests for , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS . not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days. (D', E') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for , *P < .05, **P < .01, ***P < .001; NS. not significant.


G Western blots of Lef1 , non - phospho - β - catenin , and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector . ( G ' ) Relative expression levels between control vector and RUNX2 overexpression groups . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS . not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector. (G') Relative expression levels between control vector and RUNX2 overexpression groups. Data information: Data represent the means ± SEM. Unpaired t-tests , *P < .05, **P < .01, ***P < .001; NS. not significant.


H Western blots of Lef1 , non - phospho - β - catenin , and RUNX2 after DFCs were infected with scrambled shRNA ( scramsh ) or RUNX2 knockdown shRNA ( knockdown ) lentiviral vectors . ( H ' ) Relative expression levels between scramsh and knockdown groups .

H Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with scrambled shRNA (scramsh) or RUNX2 knockdown shRNA (knockdown) lentiviral vectors. (H') Relative expression levels between scramsh and knockdown groups.


I Western blots of nuclear non - phospho - β - catenin and Lamin B in DFCs of RUNX2 overexpression ( O ) , control vector ( V ) , RUNX2 knockdown ( K ) , scramsh ( S ) , compressed force ( F ; 1 . 0 g / cm2 ) , and control ( C ; 0 g / cm2 ) groups . Sample were loaded in a gradient ( 7 . 5 , 15 , 30 , and 60 μg ) showing a linear relationship for each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs of RUNX2 overexpression (O), control vector (V), RUNX2 knockdown (K), scramsh (S), compressed force (F; 1.0 g/cm2), and control (C; 0 g/cm2) groups. Sample were loaded in a gradient (7.5, 15, 30, and 60 μg) showing a linear relationship for each group.


J Western blots of nuclear non - phospho - β - catenin and Lamin B in DFCs treated with IWR - 1 - endo . Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR - 1 - endo treatment .

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

J Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs treated with IWR-1-endo. Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR-1-endo treatment.


A Coolness stimulates intracellular cyclic guanosine monophosphate ( cGMP ) accumulation in HEK ‐ 293T cells expressing GC ‐ G . Two days after transfection of empty vector or GC ‐ G ‐ encoding plasmid , cells were exposed to ambient temperatures ( 37 or 15 ° C ) for 20 min and cellular cGMP concentration was measured .

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

A Coolness stimulates intracellular cyclic guanosine monophosphate (cGMP) accumulation in HEK‐293T cells expressing GC‐G. Two days after transfection of empty vector or GC‐G‐encoding plasmid, cells were exposed to ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured.


B Temperature dependence of GC ‐ G activity . Membrane preparations from HEK ‐ 293T cells transfected with empty vector or GC ‐ G ‐ encoding plasmid were used to determine cyclase activity at the indicated temperatures for 20 min ( asterisks denote values significantly elevated compared to 37 ° C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Temperature dependence of GC‐G activity. Membrane preparations from HEK‐293T cells transfected with empty vector or GC‐G‐encoding plasmid were used to determine cyclase activity at the indicated temperatures for 20 min (asterisks denote values significantly elevated compared to 37°C).


C GC ‐ G is the only murine transmembrane GC that can be stimulated by cool temperatures . Expression plasmids encoding FLAG ‐ tagged GC subtypes GC ‐ A to GC ‐ G were transfected into HEK ‐ 293T cells . Two days after transfection , membrane protein fractions were prepared and used for cyclase activity assays at 37 or 15 ° C . Protein expression of each receptor GC was confirmed by Western blot ( WB ) analysis with anti ‐ FLAG 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 O O O O O O O O

C GC‐G is the only murine transmembrane GC that can be stimulated by cool temperatures. Expression plasmids encoding FLAG‐tagged GC subtypes GC‐A to GC‐G were transfected into HEK‐293T cells. Two days after transfection, membrane protein fractions were prepared and used for cyclase activity assays at 37 or 15°C. Protein expression of each receptor GC was confirmed by Western blot (WB) analysis with anti‐FLAG antibody.


D The H + CYC domain is critical for stimulation by coolness . The domain structure of full ‐ length ( FL ) GC ‐ G and its mutant variants is shown in the upper panel . A FLAG tag was added at the N ‐ terminus of each protein . The ΔECD + KLD mutant protein lacks amino acids 73 - 455 and 556 - 833 ; the ΔCYC variant lacks amino acids 834 - 1 , 003 . The H + CYC mutant protein contains amino acids 806 - 1 , 100 . ECD , extracellular domain ; KLD , kinase ‐ like domain ; SP , signal peptide ; TM , transmembrane region . HEK ‐ 293T cells expressing the indicated GC ‐ G constructs were incubated at 37 or 15 ° C for 20 min , then intracellular cGMP concentration was measured ( 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 B-MEASURED_VAR O O O O O O O O

D The H+CYC domain is critical for stimulation by coolness. The domain structure of full‐length (FL) GC‐G and its mutant variants is shown in the upper panel. A FLAG tag was added at the N‐terminus of each protein. The ΔECD+KLD mutant protein lacks amino acids 73-455 and 556-833; the ΔCYC variant lacks amino acids 834-1,003. The H+CYC mutant protein contains amino acids 806-1,100. ECD, extracellular domain; KLD, kinase‐like domain; SP, signal peptide; TM, transmembrane region. HEK‐293T cells expressing the indicated GC‐G constructs were incubated at 37 or 15°C for 20 min, then intracellular cGMP concentration was measured (lower panel).


In the chimeric GC ‐ A / G protein , the H + CYC domain of GC ‐ A was replaced by that of GC ‐ G ( upper panel ) . HEK ‐ 293T cells expressing the indicated proteins were exposed to ambient temperature of 37 or 15 ° C for 20 min ; then , intracellular cGMP concentration was measured . Data are mean ± SD from three experiments in triplicate . * * 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 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

In the chimeric GC‐A/G protein, the H+CYC domain of GC‐A was replaced by that of GC‐G (upper panel). HEK‐293T cells expressing the indicated proteins were exposed to ambient temperature of 37 or 15°C for 20 min; then, intracellular cGMP concentration was measured. Data are mean ± SD from three experiments in triplicate. **P 0.01.


B , C Protein expression and coolness ‐ stimulated cGMP synthesis of the above shown divergent C ‐ terminal fragments of GC ‐ G with intact or ablated hinge ( H ) region . The expression plasmids encoding the H + CYC domain or a deletion construct ( Δ1 - Δ2 ) tagged with a FLAG epitope were transfected into HEK ‐ 293T cells . Two days after transfection , cells were exposed to the ambient temperatures ( 37 or 15 ° C ) for 20 min and cellular cGMP concentration was measured ( C ) . Protein expression of each construct was confirmed by Western blot ( WB ) analysis with anti ‐ FLAG antibody ( B ) .

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 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

B, C Protein expression and coolness‐stimulated cGMP synthesis of the above shown divergent C‐terminal fragments of GC‐G with intact or ablated hinge (H) region. The expression plasmids encoding the H+CYC domain or a deletion construct (Δ1-Δ2) tagged with a FLAG epitope were transfected into HEK‐293T cells. Two days after transfection, cells were exposed to the ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured (C). Protein expression of each construct was confirmed by Western blot (WB) analysis with anti‐FLAG antibody (B).


D A recombinant protein containing the H + CYC domain of GC ‐ G acquires coolness ‐ evoked activity . Diagram of recombinant GST . GC ‐ G ‐ CYC protein containing the H + CYC domain of GC ‐ G ( amino acids 849 - 1 , 078 ) compared to the domain structure of full ‐ length GC ‐ G ( upper panel ) . The purified GST . GC ‐ G ‐ CYC protein was exposed to the indicated temperatures for 20 min , and GST . GC ‐ G ‐ CYC cyclase activity was measured ( lower panel ; asterisks denote values significantly elevated compared to 37 ° C ) .

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D A recombinant protein containing the H+CYC domain of GC‐G acquires coolness‐evoked activity. Diagram of recombinant GST.GC‐G‐CYC protein containing the H+CYC domain of GC‐G (amino acids 849-1,078) compared to the domain structure of full‐length GC‐G (upper panel). The purified GST.GC‐G‐CYC protein was exposed to the indicated temperatures for 20 min, and GST.GC‐G‐CYC cyclase activity was measured (lower panel; asterisks denote values significantly elevated compared to 37°C).


A HEK ‐ 293T cells were co ‐ transfected with plasmids encoding two differential epitope ‐ tagged ( FLAG or Myc ) versions of GC ‐ G or GC ‐ A , respectively . After 2 days , transfected cells were lysed with detergent and lysates were subjected to immunoprecipitation ( IP ) for 2 h with anti ‐ Myc antibody at two different temperatures ( 15 or 25 ° C ) , followed by Western blotting ( WB ) using anti ‐ FLAG antibody to determine the amount of dimerized / oligomerized GC proteins ( upper panel ) . These approaches demonstrated that in contrast to GC ‐ A , homomeric protein / protein association of GC ‐ G seems to be enhanced by cool temperatures ( upper panel ) . Cell lysates were also precipitated and immunoblotted with the anti ‐ Myc antibody , demonstrating that cooling does not interfere with immunoprecipitation of Myc ‐ tagged GC ‐ G via the anti ‐ Myc antibody ( middle panel ) . Likewise , precipitation and immunoblotting with the anti ‐ FLAG antibody yielded bands of similar intensity for 15 and 25 ° C , respectively ( lower panel ) . B Relative quantification of homomeric protein / protein association of GC ‐ G and GC ‐ A was performed by densitometric scanning . Each intensity value from the upper panel was subsequently normalized to the total protein levels as determined in the middle and the lower panel of Fig A ( intensity of the immunoreactive bands obtained with the anti ‐ Myc or anti ‐ FLAG antibody , respectively ) . Relative dimerization level at 15 ° C was further calculated by normalizing to values obtained at 25 ° C ; the latter were set as 1 . Results are means ± SD of three experiments , * * P 0 . 01 ( compared to 25 ° C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK‐293T cells were co‐transfected with plasmids encoding two differential epitope‐tagged (FLAG or Myc) versions of GC‐G or GC‐A, respectively. After 2 days, transfected cells were lysed with detergent and lysates were subjected to immunoprecipitation (IP) for 2 h with anti‐Myc antibody at two different temperatures (15 or 25°C), followed by Western blotting (WB) using anti‐FLAG antibody to determine the amount of dimerized/oligomerized GC proteins (upper panel). These approaches demonstrated that in contrast to GC‐A, homomeric protein/protein association of GC‐G seems to be enhanced by cool temperatures (upper panel). Cell lysates were also precipitated and immunoblotted with the anti‐Myc antibody, demonstrating that cooling does not interfere with immunoprecipitation of Myc‐tagged GC‐G via the anti‐Myc antibody (middle panel). Likewise, precipitation and immunoblotting with the anti‐FLAG antibody yielded bands of similar intensity for 15 and 25°C, respectively (lower panel).B Relative quantification of homomeric protein/protein association of GC‐G and GC‐A was performed by densitometric scanning. Each intensity value from the upper panel was subsequently normalized to the total protein levels as determined in the middle and the lower panel of Fig A (intensity of the immunoreactive bands obtained with the anti‐Myc or anti‐FLAG antibody, respectively). Relative dimerization level at 15°C was further calculated by normalizing to values obtained at 25°C; the latter were set as 1. Results are means ± SD of three experiments, **P 0.01 (compared to 25°C).


HEK ‐ 293T cells expressing CNGA3 alone ( upper panel ) or together with GC ‐ G ( full ‐ length in the middle panel and H + CYC domain in the lower panel ) were loaded with the Ca2 + indicator Fura ‐ 2 to monitor intracellular [ Ca2 + ] i concentration . Cells co ‐ expressing GC ‐ G and CNGA3 showed a rapid increase in [ Ca2 + ] i when the temperature was lowered from 37 to 15 ° C ( middle and lower panel ) ; cells only expressing CNGA3 are not responsive to cool temperatures ( top panel ) . Removal of extracellular Ca2 + ( − Ca2 + ; middle panel ) completely suppressed the response to cooling . ΔF represents changes in the ratio of the fluorescence intensity of Fura ‐ 2 at 340 / 380 nm excitation . The ratiometric Ca2 + responses are representative of 30 cells recorded from three experiments .

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HEK‐293T cells expressing CNGA3 alone (upper panel) or together with GC‐G (full‐length in the middle panel and H+CYC domain in the lower panel) were loaded with the Ca2+ indicator Fura‐2 to monitor intracellular [Ca2+]i concentration. Cells co‐expressing GC‐G and CNGA3 showed a rapid increase in [Ca2+]i when the temperature was lowered from 37 to 15°C (middle and lower panel); cells only expressing CNGA3 are not responsive to cool temperatures (top panel). Removal of extracellular Ca2+ (−Ca2+; middle panel) completely suppressed the response to cooling. ΔF represents changes in the ratio of the fluorescence intensity of Fura‐2 at 340/380 nm excitation. The ratiometric Ca2+ responses are representative of 30 cells recorded from three experiments.


A - H In situ hybridization experiments with an antisense probe for c ‐ Fos on coronal sections through the GG of wild ‐ type ( WT ; left panel ) or GC ‐ G ‐ KO ( right panel ) mouse pups ( P0 - P4 ) exposed to warm ( 35 ° C ; A - D ) or cool ( 22 ° C ; E - H ) ambient temperature for 2 h . Panels ( C , D , G , H ) show higher magnifications of the boxed areas in ( A , B , E , F ) . At 35 ° C ( A - D ) , no c ‐ Fos expression was detectable in the GG of both WT ( A , C ) and GC ‐ G ‐ KO ( B , D ) individuals . Upon exposure to 22 ° C , strong c ‐ Fos expression was observed in the GG of WT pups ( E , G ) , whereas c ‐ Fos expression was barely detectable in the GG of GC ‐ G ‐ KO animals ( F , H ) . The data shown are representative of 10 independent experiments . Pups originated from six different litters for each genotype . Scale bars : 200 μm in ( A , B , E , F ) ; 50 μm in ( C , D , G , H ) . I Quantification of c ‐ Fos ‐ positive GG cells in WT and GC ‐ G ‐ KO pups on exposure to 22 ° C for 2 h . All stained cells on every section along the rostrocaudal extent of the GG were counted ; the results shown are based on the above ‐ mentioned 10 experiments . The number of c ‐ Fos ‐ positive cells in the GG of a GC ‐ G ‐ KO mouse was determined relative to that in a concomitantly processed WT pup ; the latter number was set to 100 % . Data are mean ± SD ( n = 11 in each genotype ) , * * P 0 . 0001 .

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A-H In situ hybridization experiments with an antisense probe for c‐Fos on coronal sections through the GG of wild‐type (WT; left panel) or GC‐G‐KO (right panel) mouse pups (P0-P4) exposed to warm (35°C; A-D) or cool (22°C; E-H) ambient temperature for 2 h. Panels (C, D, G, H) show higher magnifications of the boxed areas in (A, B, E, F). At 35°C (A-D), no c‐Fos expression was detectable in the GG of both WT (A, C) and GC‐G‐KO (B, D) individuals. Upon exposure to 22°C, strong c‐Fos expression was observed in the GG of WT pups (E, G), whereas c‐Fos expression was barely detectable in the GG of GC‐G‐KO animals (F, H). The data shown are representative of 10 independent experiments. Pups originated from six different litters for each genotype. Scale bars: 200 μm in (A, B, E, F); 50 μm in (C, D, G, H).I Quantification of c‐Fos‐positive GG cells in WT and GC‐G‐KO pups on exposure to 22°C for 2 h. All stained cells on every section along the rostrocaudal extent of the GG were counted; the results shown are based on the above‐mentioned 10 experiments. The number of c‐Fos‐positive cells in the GG of a GC‐G‐KO mouse was determined relative to that in a concomitantly processed WT pup; the latter number was set to 100%. Data are mean ± SD (n = 11 in each genotype), **P 0.0001.


J - N Calcium imaging of coronal sections through the GG of olfactory marker protein ‐ green fluorescent protein ( OMP ‐ GFP ) / GC ‐ G + / + or OMP ‐ GFP / GC ‐ G ‐ KO pups ( P1 - P4 ) . High magnification image ( J ) of a tissue slice through the GG of an OMP ‐ GFP pup with GG neurons labeled by intrinsic GFP fluorescence ( GFP fluorescence was merged with the transmitted ‐ light channel ) . Cells analyzed are circled in blue ( GFP ‐ negative ) or red ( GFP ‐ positive ) . NC , nasal cavity . Scale bar : 30 μm . ( K - M ) Representative ratiometric Ca2 + transients induced by cooling from 37 to 15 ° C in GFP ‐ negative non ‐ neuronal cells ( K ) and in GFP ‐ positive GG neurons from OMP ‐ GFP / GC ‐ G + / + ( L ) and OMP ‐ GFP / GC ‐ G ‐ KO pups ( M ) . The numbers in the bottom right hand corners are the number of cells with Ca2 + transient similar to what is shown in the respective graph ( left ) and total number of measured cells ( right ) . ( N ) Quantification of coolness ‐ induced ΔF in GG neurons from OMP ‐ GFP / GC ‐ G + / + and OMP ‐ GFP / GC ‐ G ‐ KO mice . Coolness ‐ induced ΔF was calculated by subtracting the baseline fluorescence ratio ( 340 / 380 nm ) at 37 ° C from the peak fluorescence ratio ( 340 / 380 nm ) measured at 15 ° C . For OMP ‐ GFP / GC ‐ G + / + , the 29 coolness ‐ responsive neurons ( black bar ) of all 54 analyzed neurons ( gray bar ) from five slices ( obtained from different animals ) were analyzed . For OMP ‐ GFP / GC ‐ G ‐ KO , the 50 analyzed neurons ( shaded bar ) from five slices ( obtained from different animals ) were analyzed . Data are mean ± SD , * * P 0 . 01 .

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J-N Calcium imaging of coronal sections through the GG of olfactory marker protein‐green fluorescent protein (OMP‐GFP)/GC‐G+/+ or OMP‐GFP/GC‐G‐KO pups (P1-P4). High magnification image (J) of a tissue slice through the GG of an OMP‐GFP pup with GG neurons labeled by intrinsic GFP fluorescence (GFP fluorescence was merged with the transmitted‐light channel). Cells analyzed are circled in blue (GFP‐negative) or red (GFP‐positive). NC, nasal cavity. Scale bar: 30 μm. (K-M) Representative ratiometric Ca2+ transients induced by cooling from 37 to 15°C in GFP‐negative non‐neuronal cells (K) and in GFP‐positive GG neurons from OMP‐GFP/GC‐G+/+ (L) and OMP‐GFP/GC‐G‐KO pups (M). The numbers in the bottom right hand corners are the number of cells with Ca2+ transient similar to what is shown in the respective graph (left) and total number of measured cells (right). (N) Quantification of coolness‐induced ΔF in GG neurons from OMP‐GFP/GC‐G+/+ and OMP‐GFP/GC‐G‐KO mice. Coolness‐induced ΔF was calculated by subtracting the baseline fluorescence ratio (340/380 nm) at 37°C from the peak fluorescence ratio (340/380 nm) measured at 15°C. For OMP‐GFP/GC‐G+/+, the 29 coolness‐responsive neurons (black bar) of all 54 analyzed neurons (gray bar) from five slices (obtained from different animals) were analyzed. For OMP‐GFP/GC‐G‐KO, the 50 analyzed neurons (shaded bar) from five slices (obtained from different animals) were analyzed. Data are mean ± SD, **P 0.01.


A Reduced responsive rate of coolness ‐ stimulated USV in GC ‐ G ‐ KO pups . Pups were removed from their littermates and dams at postnatal ( P ) day 2 , 4 , and 6 and exposed to 15 ° C . USV was measured by use of the Anabat SD1 Bat detector . The number of responsive pups versus total number of pups tested for each WT and GC ‐ G ‐ KO group at different ages is indicated on the top of each bar . * P 0 . 05 . B , C Total number of USV calls ( B ) and response latency ( C ) in GC ‐ G ‐ KO and WT pups at P2 . The horizontal bars indicate the mean . * P 0 . 05 ; * * P 0 . 01 .

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A Reduced responsive rate of coolness‐stimulated USV in GC‐G‐KO pups. Pups were removed from their littermates and dams at postnatal (P) day 2, 4, and 6 and exposed to 15°C. USV was measured by use of the Anabat SD1 Bat detector. The number of responsive pups versus total number of pups tested for each WT and GC‐G‐KO group at different ages is indicated on the top of each bar. *P 0.05.B, C Total number of USV calls (B) and response latency (C) in GC‐G‐KO and WT pups at P2. The horizontal bars indicate the mean. *P 0.05; **P 0.01.


A NTL - 2 - foci localize in close proximity to mitochondria in young adult animals ; hypodermis is imaged ( green : NTL - 2 , red : TMRE ( Tetramethylrhodamine , ethyl ester , perchlorate ) , a mitochondrial membrane potential - dependent dye ) ( n = 3 independent experiments ) . B Localization of NTL - 2 - foci relative to mitochondria in young adult animals ; body wall muscle cells are imaged ( green : NTL - 2 , red : TOMM - 20 , an outer mitochondrial membrane ( OMM ) protein - marker of mitochondria ) ( n = 3 independent experiments ) . C EDC - 3 - foci localize in close proximity to mitochondria in young adult animals ; body wall muscle cells are imaged ( green : EDC - 3 , red : TMRE ) under control conditions ( n = 3 independent experiments ) . D Localization of EDC - 3 - foci relative to mitochondria in young adult animals ; body wall muscle cells are imaged ( red : EDC - 3 , green : mitochondrial matrix targeted by GFP ) ( n = 3 independent experiments ) .

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A NTL-2-foci localize in close proximity to mitochondria in young adult animals; hypodermis is imaged (green: NTL-2, red: TMRE (Tetramethylrhodamine, ethyl ester, perchlorate), a mitochondrial membrane potential-dependent dye) (n=3 independent experiments). B Localization of NTL-2-foci relative to mitochondria in young adult animals; body wall muscle cells are imaged (green: NTL-2, red: TOMM-20, an outer mitochondrial membrane (OMM) protein-marker of mitochondria) (n=3 independent experiments). C EDC-3-foci localize in close proximity to mitochondria in young adult animals; body wall muscle cells are imaged (green: EDC-3, red: TMRE) under control conditions (n=3 independent experiments). D Localization of EDC-3-foci relative to mitochondria in young adult animals; body wall muscle cells are imaged (red: EDC-3, green: mitochondrial matrix targeted by GFP) (n=3 independent experiments).


F Immunoblot analysis of the cytoplasmic and mitochondria - containing fractions obtained from whole animal extracts , showing that NTL - 2 , CCF - 1 , EDC - 3 and DCAP - 2 are localized in the cytoplasm and co - precipitate with mitochondria ( n = 3 independent experiments ) .

F Immunoblot analysis of the cytoplasmic and mitochondria-containing fractions obtained from whole animal extracts, showing that NTL-2, CCF-1, EDC-3 and DCAP-2 are localized in the cytoplasm and co-precipitate with mitochondria (n=3 independent experiments).


I EDC - 3 - and NTL - 2 - foci lose their specific localization close to mitochondria upon paraquat treatment of transgenic animals expressing mitochondria - targeted GFP , top : EDC - 3 - foci , bottom : NTL - 2 - foci ( n = 3 independent experiments ) .

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I EDC-3- and NTL-2-foci lose their specific localization close to mitochondria upon paraquat treatment of transgenic animals expressing mitochondria-targeted GFP , top: EDC-3-foci, bottom: NTL-2-foci (n=3 independent experiments).


L EDC - 3 - and NTL - 2 - foci lose their specific localization in the vicinity of mitochondria upon genetic inhibition of mrps - 5 , top : EDC - 3 - foci , bottom : NTL - 2 - foci ( n = 3 independent experiments ) .

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L EDC-3- and NTL-2-foci lose their specific localization in the vicinity of mitochondria upon genetic inhibition of mrps-5, top: EDC-3-foci, bottom: NTL-2-foci (n=3 independent experiments).


A Confocal images of young adult animals expressing mitochondria - targeted GFP in body wall muscle cells upon dcap - 2 or ntl - 2 RNAi treatment ( right panels indicate higher magnification images of inlays in left panels ; right panel scale bar , 10μm ) . Images were acquired using a X40 objective lens ( n = 3 independent experiments ) .

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A Confocal images of young adult animals expressing mitochondria-targeted GFP in body wall muscle cells upon dcap-2 or ntl-2 RNAi treatment (right panels indicate higher magnification images of inlays in left panels; right panel scale bar, 10μm). Images were acquired using a X40 objective lens (n=3 independent experiments).


D Quantification of intestinal mitochondrial mass upon knockdown of either dcap - 2 or ntl - 2 , in animals shown in Fig 2B & C . ( n = 3 independent experiments with at least 30 animals / experiment ; * * * * P < 0 . 0001 ; one - way analysis of variance ( ANOVA ) .

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D Quantification of intestinal mitochondrial mass upon knockdown of either dcap-2 or ntl-2, in animals shown in Fig 2B & C. (n=3 independent experiments with at least 30 animals/experiment; ****P< 0.0001; one-way analysis of variance (ANOVA).


F Representative images showing the effect of dcap - 2 and ntl - 2 genetic inhibition on the intestinal cell mitochondrial content in 7 - day - old nematodes . ( n = 3 independent experiments ; Images were acquired using a X5 objective lens ) . G Quantification of the mean GFP fluorescence intensity in the matrix of intestinal mitochondria as shown in Fig 2F ( n = 3 independent experiments with at least 57 animals / experiment ; * * * * P < 0 . 0001 ; one - way analysis of variance ( ANOVA ) ) .

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F Representative images showing the effect of dcap-2 and ntl-2 genetic inhibition on the intestinal cell mitochondrial content in 7-day-old nematodes. (n=3 independent experiments; Images were acquired using a X5 objective lens). G Quantification of the mean GFP fluorescence intensity in the matrix of intestinal mitochondria as shown in Fig 2F (n=3 independent experiments with at least 57 animals/experiment; ****P< 0.0001; one-way analysis of variance (ANOVA)).


H Quantification of the mtDNA / nDNA ratio under control conditions and upon genetic inhibition of either dcap - 2 or ntl - 2 ( n = 2 independent experiments ; * * * P < 0 . 001 , * P < 0 . 05 ; one - way analysis of variance ( ANOVA ) ) .

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H Quantification of the mtDNA/nDNA ratio under control conditions and upon genetic inhibition of either dcap-2 or ntl-2 (n=2 independent experiments; ***P< 0.001, *P< 0.05; one-way analysis of variance (ANOVA)).


A Immunoblot analysis of SKN - 1 protein levels upon the indicated genetic inhibitions . B Quantification showing the normalized SKN - 1 protein levels ( n = at least 3 independent experiments ; * P < 0 . 05 , * * P < 0 . 01 ; Welch ' s one - way analysis of variance ( ANOVA ) followed by Dunnett ' s T3 multiple comparisons test ) .

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A Immunoblot analysis of SKN-1 protein levels upon the indicated genetic inhibitions. B Quantification showing the normalized SKN-1 protein levels (n= at least 3 independent experiments; *P<0.05, **P<0.01; Welch's one-way analysis of variance (ANOVA) followed by Dunnett's T3 multiple comparisons test).


C Measurement of the promoter activity of the SKN - 1 target gene gst - 4 upon genetic inhibition of either dcap - 2 or ntl - 2 in wild - type background , daf - 2 mutant background and in a mutant background ( skn - 1 ( lax120 ) ) where SKN - 1 is constitutively active ( n = 3 independent experiments with at least 256 animals / experiment ; * * * * P < 0 . 0001 ; two - way analysis of variance ( ANOVA ) , followed by Turkey ' s multiple comparison test ) .

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C Measurement of the promoter activity of the SKN-1 target gene gst-4 upon genetic inhibition of either dcap-2 or ntl-2 in wild-type background, daf-2 mutant background and in a mutant background (skn-1(lax120)) where SKN-1 is constitutively active (n=3 independent experiments with at least 256 animals/experiment; ****P< 0.0001; two-way analysis of variance (ANOVA), followed by Turkey's multiple comparison test).


E Relative expression of skn - 1 mRNA in wild - type , DCAP - 2 - and NTL - 2 - depleted animals ( n = 4 independent experiments , P ( dcap - 2 ( RNAi ) ) = 0 . 98 , P ( ntl - 2 ( RNAi ) ) > 0 . 99 ; one - way analysis of variance ( ANOVA ) ) .

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E Relative expression of skn-1 mRNA in wild-type, DCAP-2- and NTL-2- depleted animals (n=4 independent experiments, P(dcap-2(RNAi)) =0.98, P(ntl-2(RNAi))>0.99; one-way analysis of variance (ANOVA)).


H Relative expression of aak - 2 mRNA in wild - type , DCAP - 2 - and NTL - 2 - depleted animals ( n = 2 independent experiments ; P ( dcap - 2 ( RNAi ) ) = 0 . 85 , P ( ntl - 2 ( RNAi ) ) > 0 . 99 ; one - way analysis of variance ( ANOVA ) ) .

H Relative expression of aak-2 mRNA in wild-type, DCAP-2- and NTL-2- depleted animals (n=2 independent experiments; P(dcap-2(RNAi)) =0.85, P(ntl-2(RNAi))>0.99; one-way analysis of variance (ANOVA)).


J Quantification of the mitochondrial mass in the animals shown in Fig 3I under the indicated treatments ( n = 3 independent experiments with at least 255 animals / experiment ; * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; one - way analysis of variance ( ANOVA ) ) .

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J Quantification of the mitochondrial mass in the animals shown in Fig 3I under the indicated treatments (n=3 independent experiments with at least 255 animals/experiment; *P< 0.05, **P< 0.01, ****P< 0.0001; one-way analysis of variance (ANOVA)).


D Representative images showing an increase in NTL - 2 - foci upon dcap - 2 genetic inhibition ( n = 3 independent experiments ) .

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D Representative images showing an increase in NTL-2-foci upon dcap-2 genetic inhibition (n=3 independent experiments).


E Quantification of NTL - 2 foci upon dcap - 2 genetic inhibition ( n = 3 independent experiments with at least 40 animals / experiment ; * * * * P < 0 . 0001 ; two - tailed unpaired t - test ) .

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E Quantification of NTL-2 foci upon dcap-2 genetic inhibition (n=3 independent experiments with at least 40 animals/experiment; ****P<0.0001; two-tailed unpaired t-test).


F Representative images showing an increase in the signal of DCAP - 2 - foci upon ntl - 2 genetic inhibition . Images were acquired using a X40 objective lens ( n = 3 independent experiments ) . G Respective quantification ( n = 3 independent experiments with at least 80 animals / experiment ; * * * * P < 0 . 0001 ; two - tailed unpaired t - test ) .

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F Representative images showing an increase in the signal of DCAP-2-foci upon ntl-2 genetic inhibition. Images were acquired using a X40 objective lens (n=3 independent experiments). G Respective quantification (n=3 independent experiments with at least 80 animals/experiment; ****P<0.0001; two-tailed unpaired t-test).


L Quantification of the percentage of fluorescence recovery after photobleaching ( FRAP ) to measure the rates of de novo protein synthesis upon genetic inhibition of either ntl - 2 or dcap - 2 in 6 - day - old - adult animals ; the protein synthesis inhibitor cycloheximide ( 500μM ) and let - 363 genetic inhibition where used as controls . Measurements started after animals have recovered for 2hrs post photobleaching ( n = 3 independent experiments with at least 50 animals / experiment ) .

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L Quantification of the percentage of fluorescence recovery after photobleaching (FRAP) to measure the rates of de novo protein synthesis upon genetic inhibition of either ntl-2 or dcap-2 in 6-day-old-adult animals; the protein synthesis inhibitor cycloheximide (500μM) and let-363 genetic inhibition where used as controls. Measurements started after animals have recovered for 2hrs post photobleaching (n=3 independent experiments with at least 50 animals/experiment).


A Analysis of expression of select MTPTs by real time quantitative PCR ( RT - qPCR ) following RNA immunoprecipitation ( RIP ) in anti - GFP isolates from NTL - 2 : : GFP transgenic animals , unc - 119 ( ed3 ) III ( no GFP expression ) and pgst - 4GFP ( used as an additional negative control ) counterparts ( n = 2 independent experiments , * * * P < 0 . 001 ; one - way analysis of variance ( ANOVA ) .

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A Analysis of expression of select MTPTs by real time quantitative PCR (RT-qPCR) following RNA immunoprecipitation (RIP) in anti-GFP isolates from NTL-2::GFP transgenic animals, unc-119(ed3)III (no GFP expression) and pgst-4GFP (used as an additional negative control) counterparts (n=2 independent experiments, ***P< 0.001; one-way analysis of variance (ANOVA).


B Representative images showing the localization of NTL - 2 / storage bodies relative to mitochondria upon genetic inhibition of either tomm - 20 or akap - 1 ( green : NTL - 2 , red : TMRE , a mitochondrial - membrane potential - dependent dye ; n = 3 independent experiments ) . Scale bars , 20 µm . Images were acquired using a X63 objective lens . C Quantification of the distances NTL - 2 / storage bodies acquire from mitochondria upon genetic inhibition of either tomm - 20 or akap - 1 ( n = 3 independent experiments with at least 45 animals / experiment ; * * * P < 0 . 001 ; one - way analysis of variance ( ANOVA ) ) .

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B Representative images showing the localization of NTL-2/storage bodies relative to mitochondria upon genetic inhibition of either tomm-20 or akap-1 (green: NTL-2, red: TMRE, a mitochondrial-membrane potential-dependent dye; n=3 independent experiments). Scale bars, 20 µm. Images were acquired using a X63 objective lens. C Quantification of the distances NTL-2/storage bodies acquire from mitochondria upon genetic inhibition of either tomm-20 or akap-1 (n=3 independent experiments with at least 45 animals/experiment; ***P< 0.001; one-way analysis of variance (ANOVA)).


E Immunoblot analysis in mitochondria isolates of whole animal extracts showing the protein levels of NTL - 2 contained in the isolate in control conditions and upon genetic inhibition of akap - 1 , tomm - 20 , mrps - 5 and atp - 3 ; genetic inhibition of ntl - 2 is used as a control ; NTL - 2 protein is detected by an anti - GFP antibody in transgenic animals that contain the NTL - 2 : : GFP protein fusion ( n = 2 independent experiments ) . F Quantification of NTL - 2 protein bound to mitochondria under control conditions and upon genetic inhibitions shown in E ; CTC - 1 is used as a loading control for mitochondria ( n = 2 independent experiments ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; one - way analysis of variance ( ANOVA ) followed by Dunnett ' s test ) .

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E Immunoblot analysis in mitochondria isolates of whole animal extracts showing the protein levels of NTL-2 contained in the isolate in control conditions and upon genetic inhibition of akap-1, tomm-20, mrps-5 and atp-3; genetic inhibition of ntl-2 is used as a control; NTL-2 protein is detected by an anti-GFP antibody in transgenic animals that contain the NTL-2::GFP protein fusion (n=2 independent experiments). F Quantification of NTL-2 protein bound to mitochondria under control conditions and upon genetic inhibitions shown in E; CTC-1 is used as a loading control for mitochondria (n=2 independent experiments; *P<0.05, **P<0.01, ***P<0.001; one-way analysis of variance (ANOVA) followed by Dunnett's test).


A Percent survival of wild - type , DCAP - 2 - and NTL - 2 - depleted animals subjected to heat stress for 5 hrs at 37oC and then counted every 24 hours ( n = 4 independent experiments with at least 294 animals / experiment ) . B Percent survival of wild - type , DCAP - 2 - and NTL - 2 - depleted animals following paraquat ( 8mM ) administration and counted every 24 hours ( n = 4 independent experiments with at least 144 animals / experiment ) . C Percent survival of wild - type , DCAP - 2 - and NTL - 2 - depleted animals counted every 24 hours post CCCP ( 15μM ) treatment ( n = 4 independent experiments with at least 116 animals / experiment ) .

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A Percent survival of wild-type, DCAP-2- and NTL-2- depleted animals subjected to heat stress for 5 hrs at 37oC and then counted every 24 hours (n=4 independent experiments with at least 294 animals/experiment). B Percent survival of wild-type, DCAP-2- and NTL-2- depleted animals following paraquat (8mM) administration and counted every 24 hours (n=4 independent experiments with at least 144 animals/experiment). C Percent survival of wild-type, DCAP-2- and NTL-2- depleted animals counted every 24 hours post CCCP (15μM) treatment (n=4 independent experiments with at least 116 animals/experiment).


E Knockdown of ntl - 2 shortens the lifespan of wild - type animals , while NTL - 2 overexpression extends lifespan .

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E Knockdown of ntl-2 shortens the lifespan of wild-type animals, while NTL-2 overexpression extends lifespan.


A Knockdown of akap - 1 extends the lifespan of wild - type animals , while it does not further extend the lifespan of NTL - 2 overexpressing animals . B Knockdown of akap - 1 reverses the lifespan shortening of ntl - 2 mutant worms . C Knockdown of akap - 1 ameliorates the lifespan shortening caused by ntl - 2 genetic inhibition ( double RNAi administered as 1 : 1 mixture ) .

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A Knockdown of akap-1 extends the lifespan of wild-type animals, while it does not further extend the lifespan of NTL-2 overexpressing animals. B Knockdown of akap-1 reverses the lifespan shortening of ntl-2 mutant worms. C Knockdown of akap-1 ameliorates the lifespan shortening caused by ntl-2 genetic inhibition (double RNAi administered as 1:1 mixture).


A , MC38 cells in tissue culture were treated with mock ( 0Gy ) and 10Gy irradiation . Conditioned medium ( CM ) was collected after 48 hours and assayed for the indicated cytokines . Fold - changes in the amounts of the cytokines are shown .

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A, MC38 cells in tissue culture were treated with mock (0Gy) and 10Gy irradiation. Conditioned medium (CM) was collected after 48 hours and assayed for the indicated cytokines. Fold-changes in the amounts of the cytokines are shown.


B , CSF - 1 mRNA expression was measured in MC38 and KPC cells exposed to 10Gy IR compared with mock irradiated cells . Cells were harvested at 24 , 48 and 72 hours after irradiation and RNA expression was analysed by RT - qPCR . Data are presented as mean ± SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 3 ) .

B, CSF-1 mRNA expression was measured in MC38 and KPC cells exposed to 10Gy IR compared with mock irradiated cells. Cells were harvested at 24, 48 and 72 hours after irradiation and RNA expression was analysed by RT-qPCR. Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=3).


C , CSF - 1 protein ( pg / mg total protein ) in CM collected from MC38 and KPC cells 48 hours after exposure to 10Gy IR compared to mock - irradiated cells . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 3 ) .

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C, CSF-1 protein (pg/mg total protein) in CM collected from MC38 and KPC cells 48 hours after exposure to 10Gy IR compared to mock-irradiated cells. Data are presented as mean ± SEM and analysed by Mann-Witney (n=3).


D , CSF - 1 protein ( pg / mg total protein ) measured by ELISA in the sera of naïve mice , mice bearing mock - irradiated tumours , and mice bearing irradiated tumours analysed at time points as indicated ( n = 4 / group ) . Data are presented as mean ± SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison .

D, CSF-1 protein (pg/mg total protein) measured by ELISA in the sera of naïve mice, mice bearing mock-irradiated tumours, and mice bearing irradiated tumours analysed at time points as indicated (n=4/group). Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison.


E - F , MC38 ( E ) and KPC ( F ) tumours were irradiated with 10Gy . Average tumour volume ( red line ) is shown with mean TAM infiltrate ( blue line ) for each time point . For TAM quantification , tumours were disaggregated and CD11b + / F480 + TAMs identified by flow cytometry . Data are presented as mean ± SEM for TAMs and SEM for tumour volume ( n = 6 ) .

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E-F, MC38 (E)and KPC (F) tumours were irradiated with 10Gy. Average tumour volume (red line) is shown with mean TAM infiltrate (blue line) for each time point. For TAM quantification, tumours were disaggregated and CD11b+/F480+ TAMs identified by flow cytometry. Data are presented as mean ± SEM for TAMs and SEM for tumour volume (n=6).


G - H , Immunofluorescent staining of MC38 ( G ) and KPC ( H ) tumour sections ; blue = DAPI , green = CD68 ( TAMs ) .

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G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections; blue=DAPI, green = CD68 (TAMs).


I - J , Flow cytometric analysis of immune cell populations within MC38 ( I ) and KPC ( J ) tumours 5 days following 10Gy IR compared to mock - irradiated tumours . Macrophages ( CD11b + F480 + ) , neutrophils ( CD11b + Ly6G + ) , myeloid - derived suppressor cells ( CD11b + Gr1 + ) , CD8 T cells ( CD45 + CD3 + CD8 + ) , CD4 T cells ( CD45 + CD3 + CD4 + , Natural Killer cells ( CD45 + NK1 . 1 + ) were identified . Pie charts represent the proportion of CD45 + leukocytes out of the total cells .

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I-J, Flow cytometric analysis of immune cell populations within MC38 (I) and KPC (J) tumours 5 days following 10Gy IR compared to mock-irradiated tumours. Macrophages (CD11b+ F480+), neutrophils (CD11b+ Ly6G+), myeloid-derived suppressor cells (CD11b+ Gr1+), CD8 T cells (CD45+ CD3+ CD8+), CD4 T cells (CD45+CD3+CD4+, Natural Killer cells (CD45+ NK1.1+) were identified. Pie charts represent the proportion of CD45+ leukocytes out of the total cells.


A , Quantification of iNOS expression on gated macrophages ( CD11b + F480 + ) from MC38 and KPC tumours , with representative histograms . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 6 mice / group ) .

A, Quantification of iNOS expression on gated macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).


B , Quantification of CD206 expression on macrophages ( CD11b + F480 + ) from MC38 and KPC tumours , with representative histograms . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 6 mice / group ) .

B, Quantification of CD206 expression on macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).


C , Quantification of the percentage of TAMs ( CD11b + F480 + ) in MC38 control tumours compared with irradiated tumours ( n = 6 mice / group ) .

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C, Quantification of the percentage of TAMs (CD11b+ F480+) in MC38 control tumours compared with irradiated tumours (n=6 mice/group).


D - E Quantification of iNOShi ( D ) macrophages as a percentage of total cells in MC38 tumours ( n = 6 ) .

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D-E Quantification of iNOShi (D) macrophages as a percentage of total cells in MC38 tumours (n=6).


D - E , Quantification of CD206hi ( E ) macrophages as a percentage of total cells in MC38 tumours ( n = 6 ) .

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D-E, Quantification of CD206hi (E) macrophages as a percentage of total cells in MC38 tumours (n=6).


F , The total number of iNOShi TAMs were divided by CD206hi TAMs to derive a M1 / M2 ratio in MC38 and KPC tumours receiving mock or 10Gy irradiation . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 6 mice / group ) .

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F, The total number of iNOShi TAMs were divided by CD206hi TAMs to derive a M1/M2 ratio in MC38 and KPC tumours receiving mock or 10Gy irradiation. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).


G - H , TAMs ( CD11b + F480 + ) were isolated by FACS . Expression of selected immune stimulatory and immunosuppressive genes in TAMs was determined by RT - qPCR ( n = 3 ) . Data are presented as mean fold change ± SEM compared to TAMs from mock - irradiated tumours ( n = 3 ) . Statistical significance was determined by Mann - Witney .

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G-H, TAMs (CD11b+F480+) were isolated by FACS. Expression of selected immune stimulatory and immunosuppressive genes in TAMs was determined by RT-qPCR (n=3). Data are presented as mean fold change ± SEM compared to TAMs from mock-irradiated tumours (n=3). Statistical significance was determined by Mann-Witney.


I - J , Assessment of TAM suppression of T cells was assayed by evaluation of inhibition of T cell proliferation . TAMs were isolated by magnetic bead separation using F480 microbeads and co - cultured at a 1 : 1 ratio with CFSE labelled CD8 + T cells . CFSE dilution was analysed by flow cytometry to measure proliferation . Percentages of CFSElo T cells were analysed by Kruskal - Wallis with Dunn ' s multiple comparisons test . Representative histograms are shown ( right panel ) . Experiments were repeated twice for each tumour cell line ( n = 3 mice / group ) * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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I-J, Assessment of TAM suppression of T cells was assayed by evaluation of inhibition of T cell proliferation. TAMs were isolated by magnetic bead separation using F480 microbeads and co-cultured at a 1:1 ratio with CFSE labelled CD8+ T cells. CFSE dilution was analysed by flow cytometry to measure proliferation. Percentages of CFSElo T cells were analysed by Kruskal-Wallis with Dunn's multiple comparisons test. Representative histograms are shown (right panel). Experiments were repeated twice for each tumour cell line (n=3 mice/group) *P <0.05 **P <0.01 ***P <0.001.