( C , D ) RAW 264 . 7 was transfected with GRASP55 siRNAs or scramble ( Scr ) siRNA control . Following 48 h of siRNA treatment , cells were transfected with RFP - GFP - LC3 plasmid ( GFP is sensitive to acidification , whereas RFP is not ) , after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified ( graph in D ) by determining the number of early autophagic organelles ( GFP + RFP + puncta ) and autolysosomal organelles ( GFP − RFP + puncta ) per cell as illustrated in fluorescent images ( yellow arrows , GFP + RFP + ; red arrows , GFP − RFP + ) . Total , yellow + red puncta per cell . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 . Figure source data can be found in Supplementary data .

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(C, D) RAW 264.7 was transfected with GRASP55 siRNAs or scramble (Scr) siRNA control. Following 48 h of siRNA treatment, cells were transfected with RFP-GFP-LC3 plasmid (GFP is sensitive to acidification, whereas RFP is not), after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified (graph in D) by determining the number of early autophagic organelles (GFP+RFP+ puncta) and autolysosomal organelles (GFP−RFP+ puncta) per cell as illustrated in fluorescent images (yellow arrows, GFP+RFP+; red arrows, GFP−RFP+). Total, yellow+red puncta per cell. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.


( A ) Atg5fl / fl Cre − and Atg5fl / fl Cre + bone marrow ‐ derived macrophages ( BMMs ) , pretreated overnight with 100 ng / ml LPS , were stimulated for 1 h with the inflammasome agonist nigericin ( 20 μM ) with ( Starvation ; EBSS ) or without ( Full ; full medium ) autophagic induction . Cell culture supernatants were assayed for murine IL ‐ 1β by ELISA . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05.


( B ) LPS ‐ pretreated Atg5fl / fl Cre − and Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting . Figure source data can be found in Supplementary data .

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(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.


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 .

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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 ) .

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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 .

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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 ) .

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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 .

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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 ) .

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

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

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 .

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


Western blot for p53 , phosphorylated p53 at Ser15 ( p53 pS15 ) , and vinculin ( vinc ) in HCT116 treated with 300 μM 5 - FU for 8 h and silenced for PHD1 , PHD2 , or PHD3 .

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

Western blot for p53, phosphorylated p53 at Ser15 (p53 pS15), and vinculin (vinc) in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1, PHD2, or PHD3.


qRT - PCR for PHD1 , PHD2 , and PHD3 in HCT116 silenced for PHD1 ( * P = 0 . 0006 toward Scr ) , PHD2 ( * P = 0 . 0004 toward Scr ) , or PHD3 ( * P = 0 . 0075 toward Scr ) . A two - tailed unpaired t - test was performed with n = 3 / group .

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

qRT-PCR for PHD1, PHD2, and PHD3 in HCT116 silenced for PHD1 (*P = 0.0006 toward Scr), PHD2 (*P = 0.0004 toward Scr), or PHD3 (*P = 0.0075 toward Scr). A two-tailed unpaired t-test was performed with n = 3/group.


Western blot of p53 pS15 , p53 , PHD1 , and vinculin in HCT116 treated with 300 μM 5 - FU for 8 h and silenced for PHD1 or a scrambled ( Scr ) control .

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

Western blot of p53 pS15, p53, PHD1, and vinculin in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1 or a scrambled (Scr) control.


qRT - PCR for PHD1 showing silencing efficacy with both siRNAs 1 ( * P = 0 . 0017 toward Scr ) and 2 ( * P = 0 . 0023 toward Scr ) compared to the scrambled control in HCT116 . A two - tailed unpaired t - test was performed with n = 3 / group .

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

qRT-PCR for PHD1 showing silencing efficacy with both siRNAs 1 (*P = 0.0017 toward Scr) and 2 (*P = 0.0023 toward Scr) compared to the scrambled control in HCT116. A two-tailed unpaired t-test was performed with n = 3/group.


Western blot for p53 , p53 pS15 , and vinc in HCT116 silenced for PHD1 with two different constructs ( constructs 1 and 2 ) upon exposure to 300 μM 5 - FU for 8 h .

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

Western blot for p53, p53 pS15, and vinc in HCT116 silenced for PHD1 with two different constructs (constructs 1 and 2) upon exposure to 300 μM 5-FU for 8 h.


Western blot for p53 pS15 , p53 , and vinculin in HCT116 silenced for PHD1 and treated with either 200 nM SN - 38 or 20 μM oxaliplatin for 8 h .

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

Western blot for p53 pS15, p53, and vinculin in HCT116 silenced for PHD1 and treated with either 200 nM SN-38 or 20 μM oxaliplatin for 8 h.


RNA levels of PHD1 in LIM1215 silenced for PHD1 . ( * P = 0 . 0099 toward Scr in a two - tailed unpaired t - test with n = 3 / group . )

O O O B-MEASURED_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

RNA levels of PHD1 in LIM1215 silenced for PHD1. (*P = 0.0099 toward Scr in a two-tailed unpaired t-test with n = 3/group.)


Western blot for p53 pS15 , p53 , and vinc in LIM1215 upon silencing of PHD1 and treatment for 8 h with 200 μM 5 - FU .

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

Western blot for p53 pS15, p53, and vinc in LIM1215 upon silencing of PHD1 and treatment for 8 h with 200 μM 5-FU.


A Western blot for p53 , cleaved caspase - 3 ( cleaved casp3 ) , cleaved parp , and vinculin ( vinc ) in p53wt / wt and p53 − / − HCT116 silenced for PHD1 upon exposure to 300 μM 5 - FU for 24 h , showing an increased apoptotic response to 5 - FU treatment in PHD1 - silenced cells compared to the scrambled control .

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

A Western blot for p53, cleaved caspase-3 (cleaved casp3), cleaved parp, and vinculin (vinc) in p53wt/wt and p53−/− HCT116 silenced for PHD1 upon exposure to 300 μM 5-FU for 24 h, showing an increased apoptotic response to 5-FU treatment in PHD1-silenced cells compared to the scrambled control.


B Confirmation of these results by FACS analysis of propidium iodide - stained PHD1 - silenced p53wt / wt and p53 − / − HCT116 cells exposed for 26 h to 300 - μM 5 - FU treatment . * P = 0 . 002 toward p53wt / wt HCT116 shScr 5 - FU - treated ( two - tailed unpaired t - test ) with n = 6 for non - treated p53wt / wt HCT116 and n = 3 in all other groups .

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

B Confirmation of these results by FACS analysis of propidium iodide-stained PHD1-silenced p53wt/wt and p53−/− HCT116 cells exposed for 26 h to 300-μM 5-FU treatment. *P = 0.002 toward p53wt/wt HCT116 shScr 5-FU-treated (two-tailed unpaired t-test) with n = 6 for non-treated p53wt/wt HCT116 and n = 3 in all other groups.


C Detection of cleaved casp3 , cleaved parp , and vinc in HCT116 silenced with a second siRNA for PHD1 .

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

C Detection of cleaved casp3, cleaved parp, and vinc in HCT116 silenced with a second siRNA for PHD1.


D , E Apoptosis as detected by Western blot for cleaved casp3 and parp in PHD1 - silenced HCT116 treated for 20 h with 200 nM SN - 38 ( D ) or for 24 h with 20 μM oxaliplatin ( E ) .

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

D, E Apoptosis as detected by Western blot for cleaved casp3 and parp in PHD1-silenced HCT116 treated for 20 h with 200 nM SN-38 (D) or for 24 h with 20 μM oxaliplatin (E).


F Western blot for cleaved casp3 , parp , and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5 - FU .

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

F Western blot for cleaved casp3, parp, and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5-FU.


G Similar apoptosis levels were detected by Western blot for cleaved casp3 and parp with vinc as a loading control in p53S15A → p53 − / − HCT116 silenced for PHD1 or scrambled ( Scr ) control and treated for 20 h with 300 μM 5 - FU .

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

G Similar apoptosis levels were detected by Western blot for cleaved casp3 and parp with vinc as a loading control in p53S15A→p53−/− HCT116 silenced for PHD1 or scrambled (Scr) control and treated for 20 h with 300 μM 5-FU.


Representative images and quantification of the colony formation capacity of p53wt / wt HCT116 cells transduced with a doxycycline - inducible shScr or shPHD1 silencing construct , treated for 24 h with doxycycline with or without additional treatment for 8 h with 300 μM 5 - FU . * P = 0 . 008 toward shScr , two - tailed unpaired t - test and n = 3 / 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 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

Representative images and quantification of the colony formation capacity of p53wt/wt HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct, treated for 24 h with doxycycline with or without additional treatment for 8 h with 300 μM 5-FU. *P = 0.008 toward shScr, two-tailed unpaired t-test and n = 3/group.


Tumor volume of p53wt / wtHCT116 cells transduced with a doxycycline - inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5 - FU . * P = 0 . 045 toward shScr 5 - FU - treated mice by two - way ANOVA with n = 6 for p53wt / wtHCT116 shScr , n = 7 for p53wt / wtHCT116 shPHD1 and p53wt / wtHCT116 shScr 5 - FU - treated , and n = 8 for p53wt / wtHCT116 shPHD15 - FU - treated . Tumor volume of p53 − / − HCT116 cells transduced with a doxycycline - inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5 - FU . n = 6 for p53 − / − HCT116 shScr , p53 − / − HCT116 shPHD1 , and p53 − / − HCT116 shScr 5 - FU - treated and n = 8 for p53 − / − HCT116 shPHD15 - FU - treated .

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

Tumor volume of p53wt/wtHCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. *P = 0.045 toward shScr 5-FU-treated mice by two-way ANOVA with n = 6 for p53wt/wtHCT116 shScr, n = 7 for p53wt/wtHCT116 shPHD1 and p53wt/wtHCT116 shScr 5-FU-treated, and n = 8 for p53wt/wtHCT116 shPHD15-FU-treated.Tumor volume of p53−/−HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. n = 6 for p53−/−HCT116 shScr, p53−/−HCT116 shPHD1, and p53−/−HCT116 shScr 5-FU-treated and n = 8 for p53−/−HCT116 shPHD15-FU-treated.


Western blot for p53 pS15 , p53 , and vinculin ( vinc ) in PHD1 - silenced HCT116 upon treatment with 300 μM 5 - FU for 8 h with or without 0 . 5 mM DMOG for 26 h . Vinculin ( vinc ) is used as a loading control .

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

Western blot for p53 pS15, p53, and vinculin (vinc) in PHD1-silenced HCT116 upon treatment with 300 μM 5-FU for 8 h with or without 0.5 mM DMOG for 26 h. Vinculin (vinc) is used as a loading control.


Western blot detection of PHD1 - Flag and p53 in whole cell extracts ( WCE ) or after immunoprecipitation against the Flag - tag of overexpressed PHD1 - Flag in HEK293T cells .

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

Western blot detection of PHD1-Flag and p53 in whole cell extracts (WCE) or after immunoprecipitation against the Flag-tag of overexpressed PHD1-Flag in HEK293T cells.


Western blot detection of PHD1 - Flag and p53 after immunoprecipitation against the Flag - tag of recombinant PHD1 - Flag incubated with recombinant p53 .

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

Western blot detection of PHD1-Flag and p53 after immunoprecipitation against the Flag-tag of recombinant PHD1-Flag incubated with recombinant p53.


Western blot for p53 and PHD1 in WCE or after immunoprecipitation of endogenous p53 in p53wt / wt HCT116 cells , silenced with a Scr control , siPHD1 , or sip53 , or after immunoprecipitation of p53wt / wt HCT116 cell lysates with an IgG control .

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

Western blot for p53 and PHD1 in WCE or after immunoprecipitation of endogenous p53 in p53wt/wt HCT116 cells, silenced with a Scr control, siPHD1, or sip53, or after immunoprecipitation of p53wt/wt HCT116 cell lysates with an IgG control.


Western blot for p53 pS15 , p53 , and vinculin ( vinc ) in HCT116 treated with 300 μM 5 - FU upon silencing of PHD1 and p38α , alone or in combination . Vinculin ( vinc ) is used as a loading control .

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

Western blot for p53 pS15, p53, and vinculin (vinc) in HCT116 treated with 300 μM 5-FU upon silencing of PHD1 and p38α, alone or in combination. Vinculin (vinc) is used as a loading control.


Detection of p53 pS15 and p53 after in vitro phosphorylation by p38α , p38β , or p38γ of p53immunoprecipitated from cells silenced for a control ( SIMA ) or PHD1 .

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

Detection of p53 pS15 and p53 after in vitro phosphorylation by p38α, p38β, or p38γ of p53immunoprecipitated from cells silenced for a control (SIMA) or PHD1.


Detection by Western blot of p53 and p38 from whole cell extracts ( WCE ) or after immunoprecipitation of p53 from cell silenced for a Scr control or siPHD1 and treated for 1 h with 300 μM 5 - FU .

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

Detection by Western blot of p53 and p38 from whole cell extracts (WCE) or after immunoprecipitation of p53 from cell silenced for a Scr control or siPHD1 and treated for 1 h with 300 μM 5-FU.


Transcription analysis of CDKN1A , GADD45 , MDM2 , BAX , PUMA in PHD1 - silenced p53wt / wt HCT116 either untreated or treated for 8 h with 300 μM 5 - FU .

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

Transcription analysis of CDKN1A, GADD45, MDM2, BAX, PUMA in PHD1-silenced p53wt/wt HCT116 either untreated or treated for 8 h with 300 μM 5-FU.


Western blot for cleaved parp and vinculin ( vinc ) in PHD1 - silenced HCT116 treated for 20 h with 300 μM 5 - FU alone or in combination with 3 μg / ml α - amanitin .

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

Western blot for cleaved parp and vinculin (vinc) in PHD1-silenced HCT116 treated for 20 h with 300 μM 5-FU alone or in combination with 3 μg/ml α-amanitin.


PHD1 mRNA levels in p53R248 / − HCT116 silenced for PHD1 . * P = 0 . 0005 toward scrambled control with a two - tailed unpaired t - test and n = 3 / group .

B-MEASURED_VAR 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

PHD1 mRNA levels in p53R248/− HCT116 silenced for PHD1. *P = 0.0005 toward scrambled control with a two-tailed unpaired t-test and n = 3/group.


Western blot for p53 pS15 , p53 , and vinculin in PHD1 - silenced p53R248 / − HCT116 upon 8 - h treatment with 300 μM 5 - FU .

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

Western blot for p53 pS15, p53, and vinculin in PHD1-silenced p53R248/− HCT116 upon 8-h treatment with 300 μM 5-FU.


Western blot for cleaved casp3 and vinculin in p53R248 / − HCT116 silenced for PHD1 and treated with 300 μM 5 - FU for 24 h .

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Western blot for cleaved casp3 and vinculin in p53R248/− HCT116 silenced for PHD1 and treated with 300 μM 5-FU for 24 h.


Western blot for pH2AX and vinculin ( vinc ) in p53wt / wt and p53 − / − HCT116 cells after 24 - h treatment with 300 μM 5 - FU .

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Western blot for pH2AX and vinculin (vinc) in p53wt/wt and p53−/− HCT116 cells after 24-h treatment with 300 μM 5-FU.


Detection of pH2AX and vinc in p53S15A → p53 − / − HCT116 cells silenced for PHD1 and treated with 300 μM 5 - FU for 24 h .

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Detection of pH2AX and vinc in p53S15A→p53−/− HCT116 cells silenced for PHD1 and treated with 300 μM 5-FU for 24 h.


Detection of pH2AX and vinc in HCT116 silenced with a second silencing construct of PHD1 and treated for 24 h with 300 μM 5 - FU .

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Detection of pH2AX and vinc in HCT116 silenced with a second silencing construct of PHD1 and treated for 24 h with 300 μM 5-FU.


Western blot for pH2AX and vinc in HCT116 treated for 20 h with 200 nM SN - 38 or 24 h with 20 μM oxaliplatin .

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Western blot for pH2AX and vinc in HCT116 treated for 20 h with 200 nM SN-38 or 24 h with 20 μM oxaliplatin.


Western blot for pH2AX and vinc in p53R248 / − HCT116 cells silenced for PHD1 upon treatment with 300 μM 5 - FU for 24 h .

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Western blot for pH2AX and vinc in p53R248/− HCT116 cells silenced for PHD1 upon treatment with 300 μM 5-FU for 24 h.


Detection by Western blot of p53 and XPB from whole cell extracts ( WCE ) or after immunoprecipitation of p53 from cells silenced for Scr control or siPHD1 and treated for 4 or 8 h with 300 μM 5 - FU .

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Detection by Western blot of p53 and XPB from whole cell extracts (WCE) or after immunoprecipitation of p53 from cells silenced for Scr control or siPHD1 and treated for 4 or 8 h with 300 μM 5-FU.


Western blot for pH2AX and vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5 - FU for 20 h .

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Western blot for pH2AX and vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5-FU for 20 h.


Determination of serum testosterone ( B ) and 17β - estradiol levels , in post - pubescent male , castrated male ( CtM ) , and female Hanwoo ( n = 5 in each group ) .

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Determination of serum testosterone (B) and 17β-estradiol levels, in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


Determination of the body weight , in post - pubescent male , castrated male ( CtM ) , and female Hanwoo ( n = 5 in each group ) .

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Determination of the body weight , in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


D . PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix .

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D. PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix.


E . Discriminant taxa of the male and CtM rectal microbiota , as determined by the linear discriminant analysis effect size ( LEfSe ) . The different abundances are represented by LDA scores .

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E. Discriminant taxa of the male and CtM rectal microbiota, as determined by the linear discriminant analysis effect size (LEfSe). The different abundances are represented by LDA scores.


F . The serum metabolome profiles were analyzed using GC - TOF - MS and clustered by PCoA based on the Bray - Curtis dissimilarity matrix .

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F. The serum metabolome profiles were analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix.


G . The relative abundances of serum metabolites displayed as bar graphs ( n = 5 in each group ) .

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G. The relative abundances of serum metabolites displayed as bar graphs (n = 5 in each group).


H . The BCAA serum levels quantified enzymatically , as described in the materials and methods section .

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H. The BCAA serum levels quantified enzymatically, as described in the materials and methods section.


A . Serum testosterone levels in the adult male and CtM Hanwoo ( n = 10 in each group ) . The scattered dots in the bar graph represent the individual replicates .

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A. Serum testosterone levels in the adult male and CtM Hanwoo (n = 10 in each group). The scattered dots in the bar graph represent the individual replicates.


PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the rumen , ileum , and colon , shown for the different segments of the gastrointestinal tract ( B )

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PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the rumen, ileum, and colon, shown for the different segments of the gastrointestinal tract (B)


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data or the rumen , ileum , and colon , combined data ( C ) . D . Microbial dissimilarity ( calculated based on the weighted UniFrac distance matrix , 100 values in each group ) between the male and CtM groups in the different intestinal compartments . The lines , boxes , and whiskers in the box plot diagrams represent the median , first and third quartiles , and min - to - max distribution of replicate values , respectively .

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C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data or the rumen, ileum, and colon, combined data (C). D. Microbial dissimilarity (calculated based on the weighted UniFrac distance matrix, 100 values in each group) between the male and CtM groups in the different intestinal compartments. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively.


E . The relative abundances of abundant bacterial taxa ( > 0 . 5 % of the mean abundance ) .

E. The relative abundances of abundant bacterial taxa (> 0.5% of the mean abundance).


B . The intestinal branched - chain amino acid ( BCAA ) levels in the adult male and CtM Hanwoo ( n = 10 in each group ) . BCAA levels in the luminal contents of the ileum , cecum , and colon were quantified by an enzymatic method using a BCAA assay kit . The lines , boxes , and whiskers in the box plot diagrams represent the median , first and third quartiles , and min - to - max distribution of replicate values , respectively . The scattered dots in the box plot diagrams represent the individual replicates .

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B. The intestinal branched-chain amino acid (BCAA) levels in the adult male and CtM Hanwoo (n = 10 in each group). BCAA levels in the luminal contents of the ileum, cecum, and colon were quantified by an enzymatic method using a BCAA assay kit. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates.


A . Representative images of dressed bodies of the adult male and CtM Hanwoo ( n = 10 in each group ) .

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A. Representative images of dressed bodies of the adult male and CtM Hanwoo (n = 10 in each group).


B . Dressed body weight , thickness of the dorsal subcutaneous fat , total weight of the extramuscular fat ( posterior subcutaneous fat , mesenteric fat , and retroperitoneal fat ) , and serum levels of the branched - chain amino acids ( BCAAs ) .

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B. Dressed body weight, thickness of the dorsal subcutaneous fat, total weight of the extramuscular fat (posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat), and serum levels of the branched-chain amino acids (BCAAs).


C . Representative images of fresh striploin muscle from the adult male and CtM carcasses ( n = 5 in each group ) .

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C. Representative images of fresh striploin muscle from the adult male and CtM carcasses (n = 5 in each group).


D . The intramuscular fat area in size - normalized muscle .

D. The intramuscular fat area in size-normalized muscle.


E . The intramuscular metabolome profiles analyzed using GC - TOF - MS and clustered by PCoA based on the Bray - Curtis dissimilarity

E. The intramuscular metabolome profiles analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix


F , G . The relative abundances of intramuscular metabolites ( F ) and β - hydroxybutyrate ( 3 - HB ) ( G ) in the male and CtM samples .

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F, G. The relative abundances of intramuscular metabolites (F) and β-hydroxybutyrate (3-HB) (G) in the male and CtM samples.


H . The serum and mucle ketone body levels , quantified enzymatically , as described in the Materials and Methods section ( n = 10 in each group ) .

H. The serum and mucle ketone body levels, quantified enzymatically, as described in the Materials and Methods section (n = 10 in each group).


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon , shown according to the different diets and antibiotic treatment .

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C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon, shown according to the different diets and antibiotic treatment.


D . The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot .

D. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


E . The serum branched - chain amino acid ( BCAA ) levels quantified enzymatically , as described in the Materials and Methods section .

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E. The serum branched-chain amino acid (BCAA) levels quantified enzymatically, as described in the Materials and Methods section.


F . The total weight of the extramuscular fat , including posterior subcutaneous fat , mesenteric fat , and retroperitoneal fat .

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F. The total weight of the extramuscular fat, including posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat.


G , H . Representative images ( G ) and weight ( H ) of the hindlimb intermuscular fat . The images are from a longitudinal section of the hind leg . The fat weight data are presented as a percentage of the body weight .

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G, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat. The images are from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum and hindlimb muscle ketone body levels quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels quantified enzymatically.


B . The serum testosterone levels in the Sham - R and castrated male ( CtM ) - R mice .

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B. The serum testosterone levels in the Sham-R and castrated male (CtM)-R mice.


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon .

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C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon.


E . The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot .

E. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


F . Weights of the posterior subcutaneous fat , epididymal fat , mesenteric fat , and retroperitoneal fat in the Sham - R and CtM - R mice .

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F. Weights of the posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat in the Sham-R and CtM-R mice.


G , H . Representative images ( G ) and weight ( H ) of the hindlimb intermuscular fat in the Sham - R and CtM - R mice . Images were obtained from a longitudinal section of the hind leg . The fat weight data are presented as a percentage of the body weight .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images (G) and weight (H) of the hindlimb intermuscular fat in the Sham-R and CtM-R mice. Images were obtained from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum and hindlimb muscle ketone body levels , quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


B . Body weight gain in response to the different levels of dietary BCAAs .

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B. Body weight gain in response to the different levels of dietary BCAAs.


C . Representative images of gross anatomy .

C. Representative images of gross anatomy.


D . The total weight of the extramuscular fat ( posterior subcutaneous fat , epididymal fat , mesenteric fat , and retroperitoneal fat ) in the mice fed high - fat diet ( HFD ) with or without BCAAs .

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D. The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high-fat diet (HFD) with or without BCAAs.


E . The serum BCAA levels in the mice fed HFD with or without BCAAs .

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E. The serum BCAA levels in the mice fed HFD with or without BCAAs.


F . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown . The body weight gain data are presented as a percentage of the initial body weight . The fat weight data are presented as a percentage of the body weight .

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F. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.


G . Serum and hindlimb muscle ketone body levels , quantified enzymatically .

G. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


( B , Sequential dissociation of EI24 and BiP from IRE1 elicits a two - phase activation of IRE1 in response to ER stress . Cells were treated with 4 μM tunicamycin ( TM ) for the indicated times . The interactions of EI24 and BiP with IRE1 were analyzed by

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(B, Sequential dissociation of EI24 and BiP from IRE1 elicits a two-phase activation of IRE1 in response to ER stress. Cells were treated with 4 μM tunicamycin (TM) for the indicated times. The interactions of EI24 and BiP with IRE1 were analyzed by immunoprecipitation


( D ) EI24 knockout promotes the activation of IRE1 , but not PERK or ATF6 . Control ( sgGFP ) and EI24 knockout ( sgEI24 ) HeLa cells were treated with 4 μM TM for the indicated times . Protein expression was analyzed by immunoblotting .

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(D) EI24 knockout promotes the activation of IRE1, but not PERK or ATF6. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were treated with 4 μM TM for the indicated times. Protein expression was analyzed by immunoblotting.


( G ) Expression of Flag - EI24 in EI24 knockout cells inhibits IRE1 activity . Flag - Metap2 served as the control . Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr . Protein expression was analyzed by immunoblotting .

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(G) Expression of Flag-EI24 in EI24 knockout cells inhibits IRE1 activity. Flag-Metap2 served as the control. Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr. Protein expression was analyzed by immunoblotting.


( B ) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress . HeLa cells stably expressing Flag - EI24 were treated with or without 4 μM tunicamycin ( TM ) for 2 hr . Colocalization of EI24 with ER was analyzed . KDEL - mCherry , ER marker . Scale bar , 10 μm .

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(B) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress. HeLa cells stably expressing Flag-EI24 were treated with or without 4 μM tunicamycin (TM) for 2 hr. Colocalization of EI24 with ER was analyzed. KDEL-mCherry, ER marker. Scale bar, 10 μm.


( D ) The C - terminus of EI24 is required for IRE1 binding . HeLa cells transfected with the indicated EI24 mutants were treated as in ( B ) , after which protein - protein interactions were analyzed by immunoprecipitation ( IP ) . The star represents IgG light chains . ( E ) The C - terminus of EI24 binds to IRE1 in a persistent fashion . HeLa cells transfected with the indicated EI24 domains were treated as in ( B ) . Protein - protein interactions were analyzed by IP . The star represents IgG light chains . (

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(D) The C-terminus of EI24 is required for IRE1 binding. HeLa cells transfected with the indicated EI24 mutants were treated as in (B), after which protein-protein interactions were analyzed by immunoprecipitation (IP). The star represents IgG light chains. (E) The C-terminus of EI24 binds to IRE1 in a persistent fashion. HeLa cells transfected with the indicated EI24 domains were treated as in (B). Protein-protein interactions were analyzed by IP. The star represents IgG light chains. (


( I ) EI24 overexpression cannot prevent the activation of the phospho - mimicking ( 3SE ) IRE1 mutant . Wild - type and IRE1 3SE knock - in HeLa cells were transfected with empty vector or Flag - EI24 . Transfected cells were treated as in ( B ) . Protein expressions were analyzed by immunoblotting .

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(I) EI24 overexpression cannot prevent the activation of the phospho-mimicking (3SE) IRE1 mutant. Wild-type and IRE1 3SE knock-in HeLa cells were transfected with empty vector or Flag-EI24. Transfected cells were treated as in (B). Protein expressions were analyzed by immunoblotting.


( L ) Expression of full - length EI24 or the △ TM34 or △ C mutant in EI24 knockout HeLa cells for comparison of their functions . HeLa cells transfected with the indicated EI24 mutants were treated as in ( B ) . Protein expressions were analyzed by immunoblotting .

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(L) Expression of full-length EI24 or the △TM34 or △C mutant in EI24 knockout HeLa cells for comparison of their functions. HeLa cells transfected with the indicated EI24 mutants were treated as in (B). Protein expressions were analyzed by immunoblotting.


( B , C ) EI24 knockout causes resting - state ER calcium reduction ( B ) and cytosolic calcium elevation ( C ) under ER stress . Control ( sgGFP ) and EI24 knockout ( sgEI24 ) HeLa cells were first perfused in TM - free buffer , and switched to the buffer containing 4 μM TM to induce ER stress where indicated . Resting state calcium levels in the ER ( B ) and cytosol ( C ) with or without TM are displayed in columns . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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(B, C) EI24 knockout causes resting-state ER calcium reduction (B) and cytosolic calcium elevation (C) under ER stress. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were first perfused in TM-free buffer, and switched to the buffer containing 4 μM TM to induce ER stress where indicated. Resting state calcium levels in the ER (B) and cytosol (C) with or without TM are displayed in columns. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( G ) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol . The measurements were similarly performed as in ( E ) , and were from the same batch of cells . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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(G) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( I ) Wild - type EI24 , but not the TM34 - or C - terminus - deleted mutant , preserves ER calcium stores in EI24 knockout cells . The measurements were similarly performed as in ( E ) , and were from the same batch of cells . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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(I) Wild-type EI24, but not the TM34- or C-terminus-deleted mutant, preserves ER calcium stores in EI24 knockout cells. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( B ) Knockdown of IP3R1 prevents ER stress - induced apoptosis in EI24 knockout cells . Cells were cultured with or without 4 μM TM for 8 hr . Percentages of early apoptotic cells were measured by flow cytometry . Data information : N ≈ 10000 cells for one biological replicate . Data are mean ± SEM ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t -

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(B) Knockdown of IP3R1 prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test


( D ) Knockdown of IP3R1 prevents ER stress - induced apoptosis . Cells were treated with 4 μM TM for the indicated times . Protein expressions were analyzed by immunoblotting .

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(D) Knockdown of IP3R1 prevents ER stress-induced apoptosis. Cells were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting.


( F - H ) Reintroduction of full - length EI24 , but not the TM34 - or C - terminus - deleted mutant ( F , G ) or the C - terminus alone ( H ) , inhibited ER stress - induced apoptosis in EI24 knockout cells . Cells treated with or without TM were analyzed for apoptosis by flow cytometry ( F , H ) or by immunoblotting ( G ) . Arrows in ( H ) denote decreased or increased apoptotic levels . Data information : N ≈ 10000 cells for one biological replicate . Data are mean ± SEM ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test for F ) , one - tailed Student ' s t - test for ( H ) .

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(F-H) Reintroduction of full-length EI24, but not the TM34- or C-terminus-deleted mutant (F, G) or the C-terminus alone (H), inhibited ER stress-induced apoptosis in EI24 knockout cells. Cells treated with or without TM were analyzed for apoptosis by flow cytometry (F, H) or by immunoblotting (G). Arrows in (H) denote decreased or increased apoptotic levels. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test for F), one-tailed Student's t-test for (H).


C . Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm , identified by mass - spectrometry ( schematics are based on ( Hirokawa et al , 2009 ) . Biotinylated A3 peptide served as a control . Mean spectral counts ( normalized plus pseudocounts ) ± SEM , n = 3 independent biological repeats . All proteins shown were found to significantly purify with R3 , but not A3 , with probability > 0 . 95 and a mean fold change > 2 by SAINTexpress analysis

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C. Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm, identified by mass-spectrometry (schematics are based on (Hirokawa et al, 2009). Biotinylated A3 peptide served as a control. Mean spectral counts (normalized plus pseudocounts) ± SEM, n = 3 independent biological repeats. All proteins shown were found to significantly purify with R3, but not A3, with probability > 0.95 and a mean fold change > 2 by SAINTexpress analysis


C . Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm , identified by mass - spectrometry ( schematics are based on ( Hirokawa et al , 2009 ) . Biotinylated A3 peptide served as a control . Mean spectral counts ( normalized plus pseudocounts ) ± SEM , n = 3 independent biological repeats . All proteins shown were found to significantly purify with R3 , but not A3 , with probability > 0 . 95 and a mean fold change > 2 by SAINTexpress

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A . GAR domain deletion or mutation perturbs binding to Kif5a . Co - immunoprecipitation analysis of Kif5a and HA - Dendra2 - tagged nucleolin ( both overexpressed in HEK - 293 cells ) . IP was performed with HA antibody and probed in Western blot with anti - Kif5a and anti - HA antibodies . Ncl FL - HA - Dendra2 - full length nucleolin ; Ncl ΔGAR - HA - Dendra2 - nucleolin with a GAR domain deletion ; Ncl GAR ( N ) - HA - Dendra2 - nucleolin with all 10 arginines in the GAR domain mutated to asparagines ; DEN - Dendra2 . Note that HA input blots for HA - Dendra2 ( Dendra ) and the other three constructs are from the same membrane , but shown discontinuously owing to the different migration of these proteins in PAGE .

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A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA -Dendra2- full length nucleolin; Ncl ΔGAR - HA-Dendra2- nucleolin with a GAR domain deletion; Ncl GAR(N) - HA-Dendra2- nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN - Dendra2. Note that HA input blots for HA-Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE.


A . GAR domain deletion or mutation perturbs binding to Kif5a . Co - immunoprecipitation analysis of Kif5a and HA - Dendra2 - tagged nucleolin ( both overexpressed in HEK - 293 cells ) . IP was performed with HA antibody and probed in Western blot with anti - Kif5a and anti - HA antibodies . Ncl FL -

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A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA


D . Uptake of TAMRA - labeled R4 and N4 peptides into cultured DRG neurons . Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4 ° C , washed , fixed and analyzed by confocal microscopy . PC - phase contrast . Scale bar - 10 µm . E . Quantification of ( D ) . n = 3 independent biological repeats ; means ± SEM ; * p < 0 . 05 , paired Student ' s t - test a . u . - arbitrary units .

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D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm. E. Quantification of (D). n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test a. u. - arbitrary units.


D . Uptake of TAMRA - labeled R4 and N4 peptides into cultured DRG neurons . Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4 ° C , washed , fixed and analyzed by confocal microscopy . PC - phase contrast . Scale bar - 10 µm .

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D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm.


G . HEK - 293 cells were transfected with plasmids expressing HA - Dendra2 - full length nucleolin ( Ncl FL ) , HA - Dendra2 - nucleolin with a GAR domain deletion ( Ncl ΔGAR ) or HA - Dendra2 - nucleolin with all 10 arginines in the GAR domain mutated to asparagines ( Ncl GAR ( N ) ) and processed as in ( F ) .

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G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl FL), HA-Dendra2-nucleolin with a GAR domain deletion (Ncl ΔGAR) or HA-Dendra2-nucleolin with all 10 arginines in the GAR domain mutated to asparagines (Ncl GAR(N)) and processed as in (F).


G . HEK - 293 cells were transfected with plasmids expressing HA - Dendra2 - full length nucleolin (

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G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl


A . GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons , transduced with the peripheral neuron specific AAV - PHP . S vector expressing full length HA - Dendra2 - nucleolin ( Ncl FL ) or respective ΔGAR mutant ( Ncl ΔGAR ) upon seeding and analyzed by epifluorescence microscopy 9 - 10 days later . Shown are representative Dendra2 fluorescence images collected from the non - activated ( green ) emission line . Hoechst 33342 ( 10 µM ) was used to outline nuclei . Superimposed phase contrast and Dendra2 fluorescence images are shown on the right . Scale bar - 25 µm . B . Quantification of the nucleolar / nuclear intensity ratios in DRG cells shown in ( A ) . n = 38 - 54 cells per sample in 3 independent biological repeats ; means ± SEM ; * * * * p < 0 . 0001 , Mann - Whitney test

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A. GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons, transduced with the peripheral neuron specific AAV-PHP.S vector expressing full length HA-Dendra2-nucleolin (Ncl FL) or respective ΔGAR mutant (Ncl ΔGAR) upon seeding and analyzed by epifluorescence microscopy 9-10 days later. Shown are representative Dendra2 fluorescence images collected from the non-activated (green) emission line. Hoechst 33342 (10 µM) was used to outline nuclei. Superimposed phase contrast and Dendra2 fluorescence images are shown on the right. Scale bar - 25 µm. B. Quantification of the nucleolar/nuclear intensity ratios in DRG cells shown in (A). n = 38-54 cells per sample in 3 independent biological repeats; means ± SEM; **** p<0.0001, Mann-Whitney test


C . Time - lapse live imaging of nuclei in DRG neurons transduced with AAV - PHP . S HA - Dendra2 - nucleolin ( full length and ΔGAR mutant ) , after a hypotonic challenge with double - distilled water ( ddH20 ) . Shown are epifluorescence images ( Dendra2 green emission line ) taken at indicated times after medium replacement with ddH20 . Scale bar - 10 µm . D . Quantification of nucleolar Dendra2 signal in ( C ) with intensity values immediately before medium replacement set as 100 % . n = 17 - 24 cells per sample in 3 biological repeats ; means ± SEM ; Two - way ANOVA with Sidák ' s post - test detected a significant time - dependent reduction of nucleolar signal ( p < 0 . 0001 ) and no difference between full length and ΔGAR mutant nucleolin ( p > 0 . 05 )

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C. Time-lapse live imaging of nuclei in DRG neurons transduced with AAV-PHP.S HA-Dendra2-nucleolin (full length and ΔGAR mutant), after a hypotonic challenge with double-distilled water (ddH20). Shown are epifluorescence images (Dendra2 green emission line) taken at indicated times after medium replacement with ddH20. Scale bar - 10 µm. D. Quantification of nucleolar Dendra2 signal in (C) with intensity values immediately before medium replacement set as 100%. n = 17-24 cells per sample in 3 biological repeats; means ± SEM; Two-way ANOVA with Sidák's post-test detected a significant time-dependent reduction of nucleolar signal (p < 0.0001) and no difference between full length and ΔGAR mutant nucleolin (p > 0.05)


B . Western blot analysis of nucleolin in DRG neurons from wildtype ( WT ) and GAR + / - mice cultured in Boyden chambers . Tuj1 was used as a loading control . The higher nucleolin band in GAR + / - corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion ( corresponding to about 4 kDa reduction in protein size ) . C . Quantification of ( B ) , total nucleolin levels ( sum of both bands where applicable ) were normalized to Tuj1 and expressed as GAR + / - / WT ratios . n = 3 independent biological repeats ; means ± SEM ; * p < 0 . 05 , 1 sample t - test .

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B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size). C. Quantification of (B), total nucleolin levels (sum of both bands where applicable) were normalized to Tuj1 and expressed as GAR+/- / WT ratios. n = 3 independent biological repeats; means ± SEM; * p < 0.05, 1 sample t-test.


B . Western blot analysis of nucleolin in DRG neurons from wildtype ( WT ) and GAR + / - mice cultured in Boyden chambers . Tuj1 was used as a loading control . The higher nucleolin band in GAR + / - corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion ( corresponding to about 4 kDa reduction in protein size ) .

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B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size).


A , B . Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament ( NF ) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR + / - mice shown in ( A ) . Upper panels show total nucleolin stain . Middle panels show merged image of nucleolin ( gray ) , NF and Tuj1 ( magenta ) , and DAPI ( blue ) . Lower panels show nucleolin overlaps with NF and Tuj1 signals as the " axon only " signal . B shows quantification of nucleolin immunofluorescence with approximately a 50 % reduction in nucleolin in the axons of GAR + / - mice in vivo . n = 3 WT , n = 4 GAR + / - ; means ± SEM ; * p < 0 . 05 , unpaired Student ' s t - test . Scale bar - 10 µm .

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A, B. Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament (NF) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR+/- mice shown in (A). Upper panels show total nucleolin stain. Middle panels show merged image of nucleolin (gray), NF and Tuj1 (magenta), and DAPI (blue). Lower panels show nucleolin overlaps with NF and Tuj1 signals as the "axon only" signal. B shows quantification of nucleolin immunofluorescence with approximately a 50% reduction in nucleolin in the axons of GAR+/- mice in vivo. n = 3 WT, n = 4 GAR+/-; means ± SEM; * p < 0.05, unpaired Student's t-test. Scale bar - 10 µm.


C - F . Representative , exposure matched maximum projection confocal images of FISH for Kpnb1 ( C ) or mTOR ( E ) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT ( left ) or GAR + / - mice . Upper panels for each show total mRNA signal . Middle panels show mRNA ( gray ) signals merged with NF plus Tuj1 ( magenta ) and DAPI ( blue ) . Lower panels show mRNA signal that overlap with NF plus Tuj1 signal ( labeled " axon only " signal ) . Quantification of axonal Kpnb1 ( D ) and mTOR ( F ) mRNA signals compared to the negative control , DapB mRNA , show a significant reduction in these axonal mRNAs in the GAR + / - mice . n = 3 WT , n = 3 GAR + / - ; means ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 unpaired Student ' s t - test . Scale bar - 10 µm .

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C-F. Representative, exposure matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR+/- mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled "axon only" signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR+/- mice. n = 3 WT, n = 3 GAR+/-; means ± SEM; ** p < 0.01, *** p < 0.001, ****p < 0.0001 unpaired Student's t-test. Scale bar - 10 µm.


D . Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer , replated and grown for 18h . FISH signal is shown in gray , cell somata ( left ) and axons ( right ) are visualized by neurofilament immunostaining ( magenta ) . Scr - scrambled FISH probe served as a negative control . Scale bar - 10 µm .

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D. Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer, replated and grown for 18h. FISH signal is shown in gray, cell somata (left) and axons (right) are visualized by neurofilament immunostaining (magenta). Scr - scrambled FISH probe served as a negative control. Scale bar - 10 µm.


A , B . Representative exposure - matched FISH / IF images for DRG neurons co - transfected with control vs . nucleolin ( Ncl ) siRNA plus siRNA - resistant wild type ( WT ; A ) or ∆ GAR ( B ) Ncl cDNAs . Axonal Kpnb1 mRNA is decreased with the Ncl knockdown , and this is not rescued by expression of ∆ GAR Ncl mutant ( scale bars - 10 µm ) .

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A, B. Representative exposure-matched FISH/IF images for DRG neurons co-transfected with control vs. nucleolin (Ncl) siRNA plus siRNA-resistant wild type (WT; A) or ∆GAR (B) Ncl cDNAs. Axonal Kpnb1 mRNA is decreased with the Ncl knockdown, and this is not rescued by expression of ∆GAR Ncl mutant (scale bars - 10 µm).


A . Cultured DRG neurons from wild type ( WT ) Thy1 - YFP mice and Thy1 - YFP / GAR + / - mice ( YFP signal is shown in green ) . Cells were imaged every hour for a period of 48 h . Scale bar - 100 μm . B . Quantification of the time - lapse imaging experiment shown in ( A ) . Growth rate of the longest neurite of each cell was calculated from the time point of starting growth . n = 4 independent biological repeats ; means ± SEM , * * * * p < 0 . 0001 , two - way ANOVA .

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A. Cultured DRG neurons from wild type (WT) Thy1-YFP mice and Thy1-YFP / GAR+/- mice (YFP signal is shown in green). Cells were imaged every hour for a period of 48 h. Scale bar - 100 μm. B. Quantification of the time-lapse imaging experiment shown in (A). Growth rate of the longest neurite of each cell was calculated from the time point of starting growth. n = 4 independent biological repeats; means ± SEM, **** p < 0.0001, two-way ANOVA.


A . Cultured DRG neurons from wild type ( WT ) Thy1 - YFP mice and Thy1 - YFP / GAR + / - mice ( YFP signal is shown in green ) . Cells were imaged every hour for a period of 48 h . Scale bar - 100 μm .

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A. Cultured DRG neurons from wild type (WT) Thy1-YFP mice and Thy1-YFP / GAR+/- mice (YFP signal is shown in green). Cells were imaged every hour for a period of 48 h. Scale bar - 100 μm.


C . Fluorescence images of cultured DRG neurons from adult C57BL / 6 mice infected with AAV - PHP . s expressing HA - Dendra fused with the wild type nucleolin GAR domain ( GAR WT ) or with the GAR domain with all 10 arginines substituted with asparagines , GAR ( N ) . Neurons were re - plated 9 - 10 days after AAV infection . 24h after re - plating cells were fixed and stained with anti - HA ( grey ) and anti - NFH ( magenta ) antibodies . Scale bar - 100 μm . D . Quantification of total axonal outgrowth and mean process length in HA - positive cells in ( C ) . Outgrowth measurements were based on NFH staining . n = 6 independent biological repeats ; means ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 ( Student ' s - test ) .

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C. Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV-PHP.s expressing HA-Dendra fused with the wild type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were re-plated 9-10 days after AAV infection. 24h after re-plating cells were fixed and stained with anti-HA (grey) and anti-NFH (magenta) antibodies. Scale bar - 100 μm. D. Quantification of total axonal outgrowth and mean process length in HA-positive cells in (C). Outgrowth measurements were based on NFH staining. n = 6 independent biological repeats; means ± SEM; * p < 0.05, *** p < 0.001 (Student's-test).


C . Fluorescence images of cultured DRG neurons from adult C57BL / 6 mice infected with AAV - PHP . s expressing HA - Dendra fused with the wild type nucleolin GAR domain ( GAR WT ) or with the GAR domain with all 10 arginines substituted with asparagines , GAR ( N ) . Neurons were re - plated 9 - 10 days after AAV infection . 24h after re - plating cells were fixed and stained with anti - HA ( grey ) and anti - NFH ( magenta ) antibodies . Scale bar - 100 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 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 B-MEASURED_VAR O O O O O O O O O O O

C. Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV-PHP.s expressing HA-Dendra fused with the wild type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were re-plated 9-10 days after AAV infection. 24h after re-plating cells were fixed and stained with anti-HA (grey) and anti-NFH (magenta) antibodies. Scale bar - 100 μm.


( C ) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin - II probe Sqh : : GFP and the plasma membrane marker GAP43 : : mCherry . Scale bar , 5

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(C) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin-II probe Sqh::GFP and the plasma membrane marker GAP43::mCherry. Scale bar, 5 μm


( D ) Movie ( still frames ) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus : : βH - spectrin imaged in a cross section using two - photon microscopy . βH - spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination ( arrowheads ) . Scale bar , 50 μm .

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(D) Movie (still frames) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin imaged in a cross section using two-photon microscopy. βH-spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination (arrowheads). Scale bar, 50 μm.