Figure 3 ( C ) . Relative miRNA expression of let - 7a in HEK - 293 cells infected with tetracycline - inducible expressing lentiviruses for TruB1 , mt1 , mt2 or GFP 5 days after doxycycline treatment , as determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD . ( D ) . Northern blotting for let - 7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP , 5 days after doxycycline treatment . ( G ) . Location of pseudouridine sites detected by the CMC primer extension method . Total RNA purified from HEK - 293FT cells were treated with CMC . CMC treated RNA were reverse - transcribed with RI - labelled specific primers for tRNAphe or pri - let - 7b . ddATP was used for sequence control . Pseudouridines are indicated by black arrows .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.(D). Northern blotting for let-7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment.(G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNAphe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows.


Figure 4 ( A ) . RIP analysis of pri - let - 7a1 and Flag - TruB1 from HEK - 293FT cells . RNA was extracted from IP material and analyzed by qRT - PCR . HEK - 293FT cells were infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP and treated with doxycycline . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . ( B ) . EMSA of 32p - ATP - labelled pri - let - 7a1 or pri - let - 7a1 loop mt mixed with recombinant TruB1 , mt1 or mt2 at several doses . RNP : Ribonucleoprotein complexes . ( E ) . Tag densities represented as dots . Density of miRNA clusters in HITS - CLIP were normalized by miRNA expression as determined by TaqMan array .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.(B). EMSA of 32p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes.(E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array.


Figure 5 ( A ) . In vitro processing assay for RI - labelled pri - let - 7a1 . Autoradiographs of gels showing pri - let - 7a1 treated with whole cell lysate ( WCL ) from HEK - 293FT cells transfected with GFP , TruB1 , mt1 , or mt2 ( overexpression , left ) , and TruB1 KD or ctrl ( siRNA , right ) . Dicer KD was used to show the height of pre - let - 7a1 . ( B ) RI intensities of ( A ) were quantified and normalized to ctrl . Relative processing rate of pri - let - 7a1 into pre - and mature - are shown . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD . ( C ) . RIP assay of pri - let - 7a1 and DGCR8 from HEK - 293FT cells . Western blotting for input or immunoprecipitate ( IP ) using anti - DGCR8 antibody and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . ( D ) . RIP assay of pri - let - 7a1 and Flag - tagged TruB1 from HEK - 293FT cells with Lin28B KD or ctrl ( siRNA ) . Western blotting for input or IP material using anti - Flag antibody , anti - Lin28B antibody , and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). Dicer KD was used to show the height of pre-let-7a1. (B) RI intensities of (A) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.(C). RIP assay of pri-let-7a1 and DGCR8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. (D). RIP assay of pri-let-7a1 and Flag-tagged TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show


Figure 6 ( B ) . Relative luciferase activity of KRAS reporter or Ctrl ( empty ) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline - inducible TruB1 , mt1 , mt2 , or GFP 5 days after doxycycline treatment . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD . ( D ) . Relative expression of let - 7 families determined by qRT - PCR in HEK - 293FT cells with KD of let - 7 family members or ctrl ( using 2 ' - O - methylated antisense inhibitor ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD . ( E ) . Real - time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline - inducible TruB1 or GFP , 5 days after doxycycline treatment , with or without KD of let - 7 family members . GFP : GFP without let - 7 KD . WT : overexpression of wild type TruB1 without let - 7 KD . WT + let7 KD : WT : overexpression of wild type TruB1 with let - 7 KD . GFP + let - 7KD : GFP with let - 7 KD . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.(D). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2'-O-methylated antisense inhibitor). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.(E). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. GFP: GFP without let-7 KD. WT: overexpression of wild type TruB1 without let-7 KD. WT + let7 KD: WT: overexpression of wild type TruB1 with let-7 KD. GFP + let-7KD: GFP with let-7 KD. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


Figure 1RT - qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR + / + and LXR − / − primary BMMs . Fold changes are shown relative to LXR + / + 0 h . Error bars represent ± SEM for n = 4 - 8 ( * * P = 0 . 001 at 8 h and * * P = 0 . 00004 at 16 h versus LXR − / − ; * * P = 0 . 0003 at 0 h versus LXR + / + ) ; bottom , proposed model explaining the ligand ‐ dependent induction of Abca1 . Following ligand stimulation , LXR - corepressor complexes are exchanged for LXR - coactivator complexes , which promote gene expression . The LXR - cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1RT-qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR+/+ and LXR−/− primary BMMs. Fold changes are shown relative to LXR+/+ 0 h. Error bars represent ± SEM for n = 4-8 (**P = 0.001 at 8 h and **P = 0.00004 at 16 h versus LXR−/−; **P = 0.0003 at 0 h versus LXR+/+); bottom, proposed model explaining the ligand‐dependent induction of Abca1. Following ligand stimulation, LXR-corepressor complexes are exchanged for LXR-coactivator complexes, which promote gene expression. The LXR-cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown.


Figure 2Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length SND1 , SART1 , or HMBOX1 . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . A diagram of the reporter construct is shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 9 ( * * P = 0 . 001 , * P = 0 . 011 for SND1 , * P = 0 . 046 for SART1 ) . Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length NCOA5 or an NCOA5 mutant lacking the NH2 ‐ terminus . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . Diagrams of the reporter constructs are shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 6 - 12 ( * * P 0 . 001 , * P = 0 . 035 ) . RT - qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus . LXR ligand stimulations were performed with 1 μM T0901317 for 18 h . Fold changes are shown relative to vehicle ‐ stimulated control . Error bars represent ± SEM for n = 4 ( * P = 0 . 029 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length SND1, SART1, or HMBOX1. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. A diagram of the reporter construct is shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 9 (**P = 0.001, *P = 0.011 for SND1, *P = 0.046 for SART1).Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length NCOA5 or an NCOA5 mutant lacking the NH2‐terminus. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. Diagrams of the reporter constructs are shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 6-12 (**P 0.001, *P = 0.035).RT-qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus. LXR ligand stimulations were performed with 1 μM T0901317 for 18 h. Fold changes are shown relative to vehicle‐stimulated control. Error bars represent ± SEM for n = 4 (*P = 0.029).


Figure 3In vitro pulldown assays using recombinant GST ‐ LXRα to isolate the indicated in vitro translated NCOA5 constructs . Assays were performed for 2 h in the presence or absence of 2 μM T0901317 . Samples were immunoblotted as indicated . Note the requirement of the NCOA5 NH2 ‐ terminus for interacting with LXRα . Immunoprecipitation of Protein C ‐ tagged LXRα from stable RAW 264 . 7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h , followed by immunoblotting for endogenous NCOA5 or over ‐ expressed LXRα . Protein immunoblots of nuclear extracts are shown below . NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the ligand ‐ stimulated association of NCOA5 . Error bars represent ± SEM for n = 4 - 9 ( * P = 0 . 02 ) . NCOA5 ChIP assays from LXR − / − BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 ( * * P = 0 . 0003 for vehicle and * * P = 0 . 002 for T0901317 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3In vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs. Assays were performed for 2 h in the presence or absence of 2 μM T0901317. Samples were immunoblotted as indicated. Note the requirement of the NCOA5 NH2‐terminus for interacting with LXRα.Immunoprecipitation of Protein C‐tagged LXRα from stable RAW 264.7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h, followed by immunoblotting for endogenous NCOA5 or over‐expressed LXRα. Protein immunoblots of nuclear extracts are shown below.NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the ligand‐stimulated association of NCOA5. Error bars represent ± SEM for n = 4-9 (*P = 0.02).NCOA5 ChIP assays from LXR−/− BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6 (**P = 0.0003 for vehicle and **P = 0.002 for T0901317).


Figure 4RT - qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non ‐ silencing or Ncoa5 ‐ specific shRNA . Fold changes are shown relative to shControl 0 h . Note the elevated Abca1 expression at 24 - 30 h following loss of NCOA5 . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0007 at 24 h and * * P = 0 . 004 at 30 h , * P = 0 . 02 ) . Quantification of ABCA1 immunoblots from primary BMMs infected as in ( A ) . Representative immunoblot is shown in Supplementary Fig S7E . LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h . Error bars represent ± SEM for n = 2 ( * * P = 0 . 007 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4RT-qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non‐silencing or Ncoa5‐specific shRNA. Fold changes are shown relative to shControl 0 h. Note the elevated Abca1 expression at 24-30 h following loss of NCOA5. Error bars represent ± SEM for n = 4-6 (**P = 0.0007 at 24 h and **P = 0.004 at 30 h, *P = 0.02).Quantification of ABCA1 immunoblots from primary BMMs infected as in (A). Representative immunoblot is shown in Supplementary Fig S7E. LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h. Error bars represent ± SEM for n = 2 (**P = 0.007).


Figure 5A , B RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 alone or together with 6 μg / ml PolyIC ( A ) or 10 ng / ml LPS ( B ) . Fold changes are shown relative to vehicle ‐ stimulated shControl . Note only the loss of TLR3 ‐ mediated repression following Ncoa5 silencing . Error bars represent ± SEM for n = 4 - 10 ( * * P = 0 . 0003 for A , * * P = 0 . 0004 for shControl in B , * * P = 0 . 0002 for shNcoa5 in B versus T0901317 ) . C , D RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h unstimulated , or with 6 μg / ml PolyIC ( C ) or 10 ng / ml LPS ( D ) . Fold changes are shown relative to unstimulated shControl . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0006 for C , * * P = 0 . 00000001 for shControl in D , * * P = 0 . 000001 for shNcoa5 in D , * P = 0 . 013 versus T0901317 ) . E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the increased occupancy only in the presence of both lipid and inflammatory ligands . Error bars represent ± SEM for n = 8 - 12 ( * * P = 0 . 0007 versus T0901317 ) . F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg / ml PolyIC or left unstimulated . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 . G Cholesterol efflux assays to APOA1 from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Agonist stimulations were performed for 6 h . Error bars represent ± SEM for n = 3 ( * P = 0 . 02 versus T0901317 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, B RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317 alone or together with 6 μg/ml PolyIC (A) or 10 ng/ml LPS (B). Fold changes are shown relative to vehicle‐stimulated shControl. Note only the loss of TLR3‐mediated repression following Ncoa5 silencing. Error bars represent ± SEM for n = 4-10 (**P = 0.0003 for A, **P = 0.0004 for shControl in B, **P = 0.0002 for shNcoa5 in B versus T0901317).C, D RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h unstimulated, or with 6 μg/ml PolyIC (C) or 10 ng/ml LPS (D). Fold changes are shown relative to unstimulated shControl. Error bars represent ± SEM for n = 4-6 (**P = 0.0006 for C, **P = 0.00000001 for shControl in D, **P = 0.000001 for shNcoa5 in D, *P = 0.013 versus T0901317).E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the increased occupancy only in the presence of both lipid and inflammatory ligands. Error bars represent ± SEM for n = 8-12 (**P = 0.0007 versus T0901317).F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg/ml PolyIC or left unstimulated. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6.G Cholesterol efflux assays to APOA1 from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Agonist stimulations were performed for 6 h. Error bars represent ± SEM for n = 3 (*P = 0.02 versus T0901317).


Figure 6A , B Unmodified / pSer5 RNAPII ( A ) or RNAPII pSer2 ( B ) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 TSS . Error bars represent ± SEM for n = 6 - 9 ( * * P = 0 . 0001 in A , * * P = 0 . 002 in B versus T0901317 ) . C , D RNAPII ChIP assays as in ( A , B ) but performed from LXR − / − BMMs . Error bars represent ± SEM for n = 5 - 9 ( * * P = 0 . 000000001 , * P = 0 . 02 versus T0901317 ) . E RT - qPCR of Abca1 expression from LXR + / + versus LXR − / − BMMs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 together with 6 μg / ml PolyIC . Fold changes are shown relative to vehicle ‐ stimulated LXR + / + . Error bars represent ± SEM for n = 4 ( * * P = 0 . 004 , * P = 0 . 012 versus T0901317 ) . F RNAPII pSer2 ChIP assays from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 + 6 μg / ml PolyIC . qPCR was performed for the Abca1 TSS . Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs ( * * P = 0 . 004 versus baseline ) . Error bars represent ± SEM for n = 3 from 11 mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 TSS. Error bars represent ± SEM for n = 6-9 (**P = 0.0001 in A, **P = 0.002 in B versus T0901317).C, D RNAPII ChIP assays as in (A, B) but performed from LXR−/− BMMs. Error bars represent ± SEM for n = 5-9 (**P = 0.000000001, *P = 0.02 versus T0901317).E RT-qPCR of Abca1 expression from LXR+/+ versus LXR−/− BMMs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 together with 6 μg/ml PolyIC. Fold changes are shown relative to vehicle‐stimulated LXR+/+. Error bars represent ± SEM for n = 4 (**P = 0.004, *P = 0.012 versus T0901317).F RNAPII pSer2 ChIP assays from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 + 6 μg/ml PolyIC. qPCR was performed for the Abca1 TSS. Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs (**P = 0.004 versus baseline). Error bars represent ± SEM for n = 3 from 11 mice.


Figure 4The C - terminal domains of IFT52 and IFT46 co - purify with the IFT81460 - C / 74460 - C / 27 / 251 - 136 protein complex ( C ) The SEC fractions indicated on the right with 1 - 3 were analysed by SDS - PAGE and stained with Coomassie to evaluate the protein composition . The C - terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623 - C of IFT81 and 615 - C of IFT74 ( D ) . The SEC fractions indicated on the right with 1 - 3 were analysed by SDS - PAGE and stained with Coomassie to evaluate the protein composition . ( G ) Coomassie stained SDS PAGE gel of a GST - pulldown using the CrIFT88His - GST / 70 / 521 - 430 / 46 complex immobilized on GST beads as bait and the CrIFT811 - 387 - GFP / 74135 - 420 complex used as prey . The band highlighted by an * corresponds to pulled down CrIFT74135 - 420 . CrIFT811 - 387 - GFP runs on top of IFT70 and is not visible in the pull - down .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4The C-terminal domains of IFT52 and IFT46 co-purify with the IFT81460-C/74460-C/27/251-136 protein complex (C) The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.The C-terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623-C of IFT81 and 615-C of IFT74 (D). The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.(G) Coomassie stained SDS PAGE gel of a GST-pulldown using the CrIFT88His-GST/70/521-430/46 complex immobilized on GST beads as bait and the CrIFT811-387-GFP/74135-420 complex used as prey. The band highlighted by an * corresponds to pulled down CrIFT74135-420. CrIFT811-387-GFP runs on top of IFT70 and is not visible in the pull-down.


Figure 6 ( B ) Pull - down experiments with purified IFT80Venus immobilized on GFP - binder beads and either IFT172 or IFT172968 - C . IFT80 pulls down full length IFT172 but not the truncated version lacking the N - terminal 967 residues . ( F ) GST tagged CrIFT1721 - 968 pulls down the CH - domain of CrIFT571 - 234 but not the mutated CH - domain of CrIFT57 where E77 and K84 residues were replaced by alanines .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Pull-down experiments with purified IFT80Venus immobilized on GFP-binder beads and either IFT172 or IFT172968-C. IFT80 pulls down full length IFT172 but not the truncated version lacking the N-terminal 967 residues.(F) GST tagged CrIFT1721-968 pulls down the CH-domain of CrIFT571-234 but not the mutated CH-domain of CrIFT57 where E77 and K84 residues were replaced by alanines.


Figure 1 ( a ) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV - OAT at the doses of 1x1013 ( n = 9 ) and 3x1013 genome copies ( gc ) / Kg ( n = 6 ) , or AAV - GFP at the dose of 1x1013 gc / Kg ( n = 9 ) . Normal range was established on wild - type mice ( n = 7 ) and average ± 2SD was calculated . Data are shown as averages ± SEM . * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Two - way ANOVA test ) . ( c ) Plasma ornithine and lysine concentrations at 12 months of age ( 10 - months post - injection ) in Oatrhg mice injected with AAV - OAT ( n = 3 ) or AAV - GFP ( n = 3 ) ; WT mouse controls ( n = 5 ) are also shown ; each sample was analyzed as triplicates , data are shown as averages ± SEM . One - way ANOVA test with Tukey correction ; * p < 0 . 05 ; * * * * p < 0 . 0001 . ( e ) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV - GFP or AAV - OAT . A WT mouse liver was included as control . Vinculin was used as loading control . ( g ) Liver immunohistochemistry showing approximately 10 % of OAT - positive cells in Oatrhg mice injected with AAV - OAT . Livers of WT and Oatrhg mice injected with AAV - GFP are shown as controls . 5x ( images on the left side ) and 20x ( images on the right side ) fields of view are shown . ( h ) Representative H & E staining of the retinas of 11 - month - old Oatrhg mice injected with AAV - GFP or AVV - OAT at the doses of 1x1013 or 3x1013 gc / Kg ; age - matched WT mice and uninjected Oatrhg mice were included as controls ( 40X ) . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; INL , inner nuclear layer .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(a) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV-OAT at the doses of 1x1013 (n=9) and 3x1013 genome copies (gc)/Kg (n=6), or AAV-GFP at the dose of 1x1013 gc/Kg (n=9). Normal range was established on wild-type mice (n=7) and average ± 2SD was calculated. Data are shown as averages ± SEM. ***p<0.001, ****p<0.0001 (Two-way ANOVA test).(c) Plasma ornithine and lysine concentrations at 12 months of age (10-months post-injection) in Oatrhg mice injected with AAV-OAT (n=3) or AAV-GFP (n=3); WT mouse controls (n=5) are also shown; each sample was analyzed as triplicates, data are shown as averages ± SEM. One-way ANOVA test with Tukey correction; *p <0.05; ****p<0.0001.(e) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV-GFP or AAV-OAT. A WT mouse liver was included as control. Vinculin was used as loading control.(g) Liver immunohistochemistry showing approximately 10% of OAT-positive cells in Oatrhg mice injected with AAV-OAT. Livers of WT and Oatrhg mice injected with AAV-GFP are shown as controls. 5x (images on the left side) and 20x (images on the right side) fields of view are shown.(h) Representative H&E staining of the retinas of 11-month-old Oatrhg mice injected with AAV-GFP or AVV-OAT at the doses of 1x1013 or 3x1013 gc/Kg; age-matched WT mice and uninjected Oatrhg mice were included as controls (40X). Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


Figure 2 ( a ) Representative electron micrographs of the mouse retina of an AAV - GFP injected Oatrhg mouse showing retinal pigmented epithelium ( RPE ) and adjacent abnormal outer segment ( OS ) disk membranes ( black arrows ) . Phagolysosomes containing undigested OS disc membranes are present ( asterisks ) . ( b ) Retina of AAV - OAT injected Oatrhg mice showing photoreceptor disk membranes ( white arrows ) and RPE ultrastructure comparable to age matched WT mice ( c , white arrows ) . ( c ) Control retina of WT mouse is shown as comparison .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(a) Representative electron micrographs of the mouse retina of an AAV-GFP injected Oatrhg mouse showing retinal pigmented epithelium (RPE) and adjacent abnormal outer segment (OS) disk membranes (black arrows). Phagolysosomes containing undigested OS disc membranes are present (asterisks). (b) Retina of AAV-OAT injected Oatrhg mice showing photoreceptor disk membranes (white arrows) and RPE ultrastructure comparable to age matched WT mice (c, white arrows). (c) Control retina of WT mouse is shown as comparison.


Figure 3 ( a ) Plasma ornithine concentrations in AAV - GFP - ( n = 3 ) and AAV - OAT - injected ( n = 3 ) OatΔ mice . Normal range of wild - type mice is also shown . Each value plotted represents mean ± SEM ; Two - way ANOVA test , * p < 0 . 05 , * * p < 0 . 01 . ( c ) Retinal H & E ( 40X ) of OatΔ mice injected with either AAV - GFP or AAV - OAT . Representative of three mice per groups are shown ; WT mice were included as control . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; INL , inner nuclear layer .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(a) Plasma ornithine concentrations in AAV-GFP- (n=3) and AAV-OAT-injected (n=3) OatΔ mice. Normal range of wild-type mice is also shown. Each value plotted represents mean ± SEM; Two-way ANOVA test, *p <0.05, **p<0.01.(c) Retinal H&E (40X) of OatΔ mice injected with either AAV-GFP or AAV-OAT. Representative of three mice per groups are shown; WT mice were included as control. Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


Figure 1 ( A ) WT or Mfn2 KO MEFs ( Mfn2 KO cells ) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology . Scale bar : 1 μm . ( B ) EM images of Tg ‐ treated Mfn2 KO cells show accumulation of ER membrane stacking . Scale bar : 1 μm . ( C ) WT , Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β ‐ GFP plasmid and treated with 1 μM Tg for 24 h . Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg . Scale bar : 10 μm . Insets show × 10 zoomed images . Scale bar : 5 μm . ( D ) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A ‐ bodipy to stain ER and Golgi . Representative flow ‐ cytometry histograms ( upper panel ) . Mean fluorescence intensity was used to quantify ER expansion ( n = 5 ) ( lower panel ) . Data are mean ± s . e . m . * P0 . 05 versus WT group .

O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm.(B) EM images of Tg‐treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm.(C) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β‐GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm.(D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A‐bodipy to stain ER and Golgi. Representative flow‐cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P0.05 versus WT group.


Figure 2 ( A ) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h . Total and cleaved caspase 3 levels were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( B , C ) WT and Mfn2 KO cells were treated with 0 . 5 μg / ml tunicamycin ( Tm ) , 100 ng / ml brefeldin A ( Bref ) , or 1 μM Tg for 24 h . Total and cleaved caspase 3 levels were detected by western blot ( B ) and caspase activity ( C ) by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( D ) Flow ‐ cytometry analysis of the Sub G1 DNA fragmentation in methanol ‐ fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( E ) Scr ( stably expressing scrambled shRNA ) and Mfn2 knockdown ( KD ) ( stably expressing shRNA directed against Mfn2 ) 3T3 ‐ L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h . Total and cleaved caspase 3 were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( F ) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h . Lactate dehydrogenase ( LDH ) release was analyzed by flow cytometry to assess necrotic cell death . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( G ) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z ‐ VAD ‐ fmk and stained for annexin V / PI . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT + Tg group . ( H ) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h ( 3 h of pre ‐ incubation with CHX ) . Florescence microscopy images show that CHX prevents cytoplasmic vacuolization . Scale bar : 10 μm . ( I ) WT ( black circles ) and Mfn2 KO cells ( white circles ) were incubated with 1 μM Tg for varying times , and ALIX protein was detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on the online supplementary information page .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.(B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.(D) Flow‐cytometry analysis of the Sub G1 DNA fragmentation in methanol‐fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.(E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3‐L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.(F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.(G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z‐VAD‐fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P0.05 versus WT+Tg group.(H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm.(I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


Figure 3 ( A , upper panel ) WT ( black circles ) or Mfn2 KO cells ( white circles ) were treated with 1 μM Tg for a range of times . ( A , lower ) Densitometric quantification . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group ; # P0 . 05 versus WT non ‐ Tg ‐ treated group . ( B , upper panel ) WT or Mfn2 KO cells were treated with Tg for 0 , 6 or 12 h , in the presence or absence of Bafilomycin ( Baf , 100 nM ) . LC3b ‐ I and LC3b ‐ II expression was measured by western blot . ( B , lower panel ) Densitometric quantification of LC3b ‐ II levels ( relative to tubulin ) . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT + Baf + Tg group . ( C ) WT or Mfn2 KO cells stably expressing mCherry ‐ GFP ‐ LC3b were treated with 1 μM Tg , 100 ng / ml Brefeldin ( Bref ) , or 0 . 5 μg / ml tunicamycin ( Tm ) for 24 h and examined by confocal microscopy . Scale bar : 10 μm . ( D ) Quantification of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red puncta per cell ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and group ) . * P0 . 05 versus WT group . ( E ) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( F ) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions . ( G ) Expression of Beclin ‐ 1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on the online supplementary information page .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P0.05 versus WT group; #P0.05 versus WT non‐Tg‐treated group.(B, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b‐I and LC3b‐II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b‐II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P0.05 versus WT+Baf+Tg group.(C) WT or Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm.(D) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus WT group.(E) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.(F) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions.(G) Expression of Beclin‐1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


Figure 4 ( B ) Immunodetection of p ‐ PERK , PERK , GADD34 , p ‐ eIF2α , eIF2α , ATF4 , CHOP , and XBP ‐ 1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated . ( C ) Densitometric quantifications . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( D ) Immunodetection of p ‐ eIF2α , eIF2α , and ATF4 in WT and Mfn2 KO cells treated with 0 . 5 μg / ml Tunicamycin ( Tm ) for the times indicated . ( E ) Transcriptional activity driven by ATF6 . WT and Mfn2 KO cells were co ‐ transfected with the 5xATF6 ‐ GL3 and TK ‐ Renilla plasmids and treated with 1 μM Tg for 24 h after transfection . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group . Source data for this figure is available on the online supplementary information page .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Immunodetection of p‐PERK,PERK, GADD34, p‐eIF2α, eIF2α, ATF4, CHOP, and XBP‐1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated.(C) Densitometric quantifications. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.(D) Immunodetection of p‐eIF2α, eIF2α, and ATF4 in WT and Mfn2 KO cells treated with 0.5 μg/ml Tunicamycin (Tm) for the times indicated.(E) Transcriptional activity driven by ATF6. WT and Mfn2 KO cells were co‐transfected with the 5xATF6‐GL3 and TK‐Renilla plasmids and treated with 1 μM Tg for 24 h after transfection. Data are mean±s.e.m. (n=4). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


Figure 5 ( A ) Immunodetection of PERK in WT and Mfn2 KO cells stably expressing a scrambled shRNA ( Scr ) or a shRNA directed against PERK ( PERK KD ) . ( B ) Caspase activity was detected by measurement of DEVD - AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group . ( C , D ) Total and cleaved caspase 3 levels were detected by western blot . Densitometric quantification is shown in D . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group . ( E , F ) Immunodetection of LC3b ‐ I and ‐ II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric quantification is shown in F . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group . Source data for this figure is available on the online supplementary information page .

O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Immunodetection of PERK in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed against PERK (PERK KD).(B) Caspase activity was detected by measurement of DEVD-AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.(C, D) Total and cleaved caspase 3 levels were detected by western blot. Densitometric quantification is shown in D. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.(E, F) Immunodetection of LC3b‐I and ‐II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


Figure 6 ( A ) Immunodetection of XBP ‐ 1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA ( Scr ) or a shRNA directed to XBP ‐ 1 ( XBP ‐ 1 KD ) , and treated with Tg for 6 h . ( B ) Caspase activity was detected by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . # P0 . 05 versus Scr group . ( C , D ) Total and cleaved caspase 3 levels were detected by western blot . WT and Mfn2 KO cells subjected or not to XBP ‐ 1 silencing ( Scr , and KD ) and treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . ( E , F ) Immunodetection of LC3b ‐ I and ‐ II in Scr and XBP ‐ 1 KD WT or Mfn2 KO cells treated as indicated ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric quantification is shown in F . Data are mean ± s . e . m . ( n = 3 ) . # P0 . 05 versus Scr group . ( G ) Mfn2 KO cells stably expressing mCherry ‐ GFP ‐ LC3b and subjected or not to XBP ‐ 1 silencing ( Scr or KD ) were treated with 1 μM Tg , 100 ng / ml Bref , or 0 . 5 μg / ml Tm for 24 h and examined by confocal microscopy . Scale bar : 10 μm . ( H ) Quantification of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red puncta per cell ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and group ) . # P0 . 05 versus Scr group . Source data for this figure is available on the online supplementary information page .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Immunodetection of XBP‐1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed to XBP‐1 (XBP‐1 KD), and treated with Tg for 6 h.(B) Caspase activity was detected by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. #P0.05 versus Scr group.(C, D) Total and cleaved caspase 3 levels were detected by western blot. WT and Mfn2 KO cells subjected or not to XBP‐1 silencing (Scr, and KD) and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.(E, F) Immunodetection of LC3b‐I and ‐II in Scr and XBP‐1 KD WT or Mfn2 KO cells treated as indicated (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). #P0.05 versus Scr group.(G) Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b and subjected or not to XBP‐1 silencing (Scr or KD) were treated with 1 μM Tg, 100 ng/ml Bref, or 0.5 μg/ml Tm for 24 h and examined by confocal microscopy. Scale bar: 10 μm.(H) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


Figure 7 ( A ) Immunodetection of p ‐ PERK and p ‐ eIF2α in Mfn2 KO MEFs , Mfn2 knockdown 3T3 ‐ L1 fibroblasts , and Mfn2 knockdown C2C12 myoblasts . ( B ) Densitometric quantification of p ‐ PERK and p ‐ eIF2α in MEFs . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT cells . ( C , D ) Immunodetection of p ‐ eIF2α , eIF2α , CHOP , XBP ‐ 1s , LC3b , and p62 in Mfn2 ‐ deficient liver ( C ) or skeletal muscle ( D ) from tissue ‐ specific KO mice . ( E ) Co ‐ immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK ‐ myc or an empty vector ( pBABE ‐ myc ; negative control ) . ( F ) Co ‐ immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates . PERK was immunoprecipitated with a C ‐ terminal antibody and co ‐ immunoprecipitation of Mfn2 was detected by western blot . Source data for this figure is available on the online supplementary information page .

O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Immunodetection of p‐PERK and p‐eIF2α in Mfn2 KO MEFs, Mfn2 knockdown 3T3‐L1 fibroblasts, and Mfn2 knockdown C2C12 myoblasts.(B) Densitometric quantification of p‐PERK and p‐eIF2α in MEFs. Data are mean±s.e.m. (n=3). *P0.05 versus WT cells.(C, D) Immunodetection of p‐eIF2α, eIF2α, CHOP, XBP‐1s, LC3b, and p62 in Mfn2‐deficient liver (C) or skeletal muscle (D) from tissue‐specific KO mice.(E) Co‐immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK‐myc or an empty vector (pBABE‐myc; negative control).(F) Co‐immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates. PERK was immunoprecipitated with a C‐terminal antibody and co‐immunoprecipitation of Mfn2 was detected by western blot.Source data for this figure is available on the online supplementary information page.


Figure 8 ( A ) Representative confocal images of mitochondrial morphology in scrambled ( Scr ) and PERK KD Mfn2 KO cells stained with MitoTracker Green . ( B ) Mitochondrial morphology quantification . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and group ) . * P0 . 05 versus Scr group . ( C , D ) Representative confocal images of mitochondrial morphology in scrambled ( Scr ) and PERK KD WT cells ( C ) or scrambled ( Scr ) and PERK KD Mfn1 KO cells ( D ) stained with MitoTracker Green . ( E ) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4 ‐ phenyl butyric acid for 6 h and stained with MitoTracker Green . ( F ) Flow ‐ cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO cells ( G ) or Scr and PERK KD WT and Mfn1 cells using DHR1 , 2 , 3 . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group , # P0 . 05 versus Scr group . Scale bars : 10 μm .

O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD Mfn2 KO cells stained with MitoTracker Green.(B) Mitochondrial morphology quantification. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus Scr group.(C, D) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD WT cells (C) or scrambled (Scr) and PERK KD Mfn1 KO cells (D) stained with MitoTracker Green.(E) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4‐phenyl butyric acid for 6 h and stained with MitoTracker Green.(F) Flow‐cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO cells(G) or Scr and PERK KD WT and Mfn1 cells using DHR1,2,3. Data are mean±s.e.m. (n=4). *P0.05 versus WT group, #P0.05 versus Scr group. Scale bars: 10 μm.


Figure 9 ( A ) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells . Cells were loaded with Rhod ‐ 2 and then treated with 2 . 5 mM CaCl2 ( left ) . Calcium uptake was monitored by confocal microscopy . Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod ‐ 2 and incubated with 2 . 5 mM CaCl2 for 5 min ( right ) . Scale bars : 10 μm . ( B - E ) Mitochondrial oxygen consumption ( OCR ) was measured in Scr and PERK KD Mfn2 KO cells ( B ) , Scr and PERK KD WT cells ( C ) , Scr and PERK KD Mfn1 KO cells ( D ) , and WT cells stably expressing PERK ‐ myc ( E ) . Data are mean of three independent experiments . The following parameters were measured : oxygen consumption under routine conditions ( DMEM with 5 . 5 mM glucose ) , maximal respiratory capacity reached after uncoupling with FCCP , and respiratory leak , measured after inhibition of ATP synthase with oligomycin . Data are mean ± s . e . m . * P0 . 05 versus Scr group or versus the empty plasmid group . ( F , G ) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK ‐ myc stained with MitoTracker Green . Scale bars : 10 μm . Quantitative analysis of mitochondrial morphology is shown in G . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus the empty plasmid group .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9(A) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells. Cells were loaded with Rhod‐2 and then treated with 2.5 mM CaCl2 (left). Calcium uptake was monitored by confocal microscopy. Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod‐2 and incubated with 2.5 mM CaCl2 for 5 min (right). Scale bars: 10 μm.(B-E) Mitochondrial oxygen consumption (OCR) was measured in Scr and PERK KD Mfn2 KO cells (B), Scr and PERK KD WT cells (C), Scr and PERK KD Mfn1 KO cells (D), and WT cells stably expressing PERK‐myc (E). Data are mean of three independent experiments. The following parameters were measured: oxygen consumption under routine conditions (DMEM with 5.5 mM glucose), maximal respiratory capacity reached after uncoupling with FCCP, and respiratory leak, measured after inhibition of ATP synthase with oligomycin. Data are mean±s.e.m. *P0.05 versus Scr group or versus the empty plasmid group.(F, G) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK‐myc stained with MitoTracker Green. Scale bars: 10 μm. Quantitative analysis of mitochondrial morphology is shown in G. Data are mean±s.e.m. (n=3). *P0.05 versus the empty plasmid group.


Figure 1A . Ncl and Pus7 mRNA levels ( normalized to TBP ) at different time points after LPS stimulation in c - mycwt / wt and c - myc ∆ / ∆ cells . Data are presented as mean ± SD ; n = 3 . B . To quantify RNA synthesis , we exposed our cultures to a brief pulse of ethyl - uridine ( EU ) and measured its incorporation into RNA by light microscopy . The scatter plots show the nuclear Area as detected in the 2D plane ( x axis , in pixels ) versus the EU signal ( y axis , as arbitrary units , AU ) for each single cell identified by DAPI staining in c - mycwt / wt and c - myc ∆ / ∆ populations at the indicated time points after LPS treatment . One representative experiment out of 5 is shown . C . Variation of each expressed mRNA at 4h of LPS treatment ( log2FC , relative to time 0 ) for c - mycwt / wt ( x - axis ) and c - myc ∆ / ∆ cells ( y - axis ) , as measured by RNA - seq . Regulatory groups 1 - 6 and Grey Zone ( GZ ) are as defined in the text . D . Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation . E . Variations ( log2FC ) in mature and precursor RNAs ( as measured by RNA - seq ) and in the modeled rates ( RNA synthesis , processing and degradation ) for Myc dependent and independent genes . The grey scale represents the starting level for each parameter in unstimulated cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Ncl and Pus7 mRNA levels (normalized to TBP) at different time points after LPS stimulation in c-mycwt/wt and c-myc∆/∆ cells. Data are presented as mean ± SD; n = 3.B. To quantify RNA synthesis, we exposed our cultures to a brief pulse of ethyl-uridine (EU) and measured its incorporation into RNA by light microscopy. The scatter plots show the nuclear Area as detected in the 2D plane (x axis, in pixels) versus the EU signal (y axis, as arbitrary units, AU) for each single cell identified by DAPI staining in c-mycwt/wt and c-myc∆/∆ populations at the indicated time points after LPS treatment. One representative experiment out of 5 is shown.C. Variation of each expressed mRNA at 4h of LPS treatment (log2FC, relative to time 0) for c-mycwt/wt (x-axis) and c-myc∆/∆ cells (y-axis), as measured by RNA-seq. Regulatory groups 1-6 and Grey Zone (GZ) are as defined in the text.D. Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation.E. Variations (log2FC) in mature and precursor RNAs (as measured by RNA-seq) and in the modeled rates (RNA synthesis, processing and degradation) for Myc dependent and independent genes. The grey scale represents the starting level for each parameter in unstimulated cells.


Figure 2A . Overlap between Myc ChIP - seq peaks . For each time - point indicated at the bottom , the percentage of peaks overlapping over ≥ 1 bp with any other time - point ( Reference sample ) is reported . Peaks showed consistent distributions along the time - course , almost all those called in untreated cells being included in LPS - stimulated samplesB , C . Distribution of Myc at promoters ( B ) and distal sited ( C ) in LPS - treated B - cells ( 0 , 2 , 4 8h ) and in B - cells in vivo , in either control c - mycwt / wt ( C ) , pre - tumoral Eµ - myc mice ( P ) or tumors ( T ) 4 [ ] . Each row represents a promoter - associated Myc peak within a 6 kb genomic interval ( centered on the midpoint of the peak ) . The heatmap includes every annotated promoter in chromosome 1 ( as representative of the whole genome ) that was called as Myc - associated by ChIP - seq in at least one sample . For the same intervals , we also show the distribution of H3K4me1 , H3K4me3 , and H3K27ac in the in vivo control sample , and gene annotations ( exons in red , introns in pink ; + sense , - antisense strand ) . D , E . Numbers of Myc peaks at promoters ( - 2kb , + 1kb ) ( D ) and distal sites ( E ) at different time points after LPS stimulation . The shadings mark the subsets of peaks associated with H3K4me3 ( D ) or H3K4me1 ( E ) , with or without H3K27ac . F . Percentage of active promoters ( positive for H3K4me3 and H3K27ac ) or distal sites ( H3K4me1 and H3K27ac ) that overlap with at least a Myc peak at the different time points after LPS stimulation ( 0 , 2 , 4 , 8h ) or in vivo ( C , P , T1 , T2 , T3 ) . G . Quantitative heatmap showing Myc signal intensities ( read counts normalized by library size ) at each bound region in chromosome 1 , ranked based on the signal in the control sample ( 0h LPS ) . H . Percentage of Myc peaks at promoters ( top ) or distal sites ( bottom ) that contain either canonical ( CACGTG ) or variant E - boxes ( CACGCG , CATGCG , CACGAG , CATGTG ) 83637 [ , , ] in a 200 bp window centered on the peak summit . I . Myc enrichment by ChIP - seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs . In the boxplot , the central tendency marks the median , the box ranges mark the upper and lower quartiles , and the whiskers extend to the upper and lower extreme datapoints which are no more than 1 . 5 times the interquartile range from the box . The Myc ChIP - seq time - course was performed on a unique pool of cells , obtained from > 10 mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Overlap between Myc ChIP-seq peaks. For each time-point indicated at the bottom, the percentage of peaks overlapping over ≥1 bp with any other time-point (Reference sample) is reported. Peaks showed consistent distributions along the time-course, almost all those called in untreated cells being included in LPS-stimulated samplesB, C. Distribution of Myc at promoters (B) and distal sited (C) in LPS-treated B-cells (0, 2, 4 8h) and in B-cells in vivo, in either control c-mycwt/wt (C), pre-tumoral Eµ-myc mice (P) or tumors (T) 4[]. Each row represents a promoter-associated Myc peak within a 6 kb genomic interval (centered on the midpoint of the peak). The heatmap includes every annotated promoter in chromosome 1 (as representative of the whole genome) that was called as Myc-associated by ChIP-seq in at least one sample. For the same intervals, we also show the distribution of H3K4me1, H3K4me3, and H3K27ac in the in vivo control sample, and gene annotations (exons in red, introns in pink; + sense, - antisense strand).D, E. Numbers of Myc peaks at promoters (-2kb, +1kb) (D) and distal sites (E) at different time points after LPS stimulation. The shadings mark the subsets of peaks associated with H3K4me3 (D) or H3K4me1 (E), with or without H3K27ac.F. Percentage of active promoters (positive for H3K4me3 and H3K27ac) or distal sites (H3K4me1 and H3K27ac) that overlap with at least a Myc peak at the different time points after LPS stimulation (0, 2, 4, 8h) or in vivo (C, P, T1, T2, T3).G. Quantitative heatmap showing Myc signal intensities (read counts normalized by library size) at each bound region in chromosome 1, ranked based on the signal in the control sample (0h LPS).H. Percentage of Myc peaks at promoters (top) or distal sites (bottom) that contain either canonical (CACGTG) or variant E-boxes (CACGCG, CATGCG, CACGAG, CATGTG) 83637[,,] in a 200 bp window centered on the peak summit.I. Myc enrichment by ChIP-seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs. In the boxplot, the central tendency marks the median, the box ranges mark the upper and lower quartiles, and the whiskers extend to the upper and lower extreme datapoints which are no more than 1.5 times the interquartile range from the box. The Myc ChIP-seq time-course was performed on a unique pool of cells, obtained from > 10 mice.


Figure 3A . Scatter plots correlating the variations in Myc share at promoters ( averaged between the 2 , 4 and 8h time points ) and in RNA synthesis ( at 8h ) , relative to untreated cells , for Myc - dependent and independent genes . The Spearman correlation between the two parameters is reported . B . The ability of the Myc share in predicting the transcriptional outcome can be represented in terms of a Receiver operating characteristic ( ROC ) curve , which represents the Sensitivity ( true positive rate ) and Specificity ( true negative rate ) of a predictor ( i . e . Myc share ) in discriminating between two classes ( i . e . up - and down - regulated genes ) , using different thresholds . The largest the area under the curve ( AUC ) , the largest is the predictive power , in a scale from 0 . 5 ( random classification : grey line ) to 1 ( perfect predictor ) . For each system , the dot corresponds to the variation of Myc at which promoters begin increasing their share of Myc binding . C . Fold - change in Myc levels at each bound promoter ( at 4h LPS , relative to time zero ) as a function of the initial binding intensity at time zero ( expressed as log2 of the coverage in a 200 bp window around the summit of the peak ) . Peaks containing a canonical E - box are highlighted in black . D . Percentage of Myc - bound promoters within the indicated transcriptionally regulatory categories that contains a canonical or variant E - box . The overall chi - square is 207 . 7 with a p - value < 0 . 00001 . The contribution to the chi - square of each category is reported above the corresponding bar in the plot .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Scatter plots correlating the variations in Myc share at promoters (averaged between the 2, 4 and 8h time points) and in RNA synthesis (at 8h), relative to untreated cells, for Myc-dependent and independent genes. The Spearman correlation between the two parameters is reported.B. The ability of the Myc share in predicting the transcriptional outcome can be represented in terms of a Receiver operating characteristic (ROC) curve, which represents the Sensitivity (true positive rate) and Specificity (true negative rate) of a predictor (i.e. Myc share) in discriminating between two classes (i.e. up- and down-regulated genes), using different thresholds. The largest the area under the curve (AUC), the largest is the predictive power, in a scale from 0.5 (random classification: grey line) to 1 (perfect predictor). For each system, the dot corresponds to the variation of Myc at which promoters begin increasing their share of Myc binding.C. Fold-change in Myc levels at each bound promoter (at 4h LPS, relative to time zero) as a function of the initial binding intensity at time zero (expressed as log2 of the coverage in a 200 bp window around the summit of the peak). Peaks containing a canonical E-box are highlighted in black.D. Percentage of Myc-bound promoters within the indicated transcriptionally regulatory categories that contains a canonical or variant E-box. The overall chi-square is 207.7 with a p-value <0.00001. The contribution to the chi-square of each category is reported above the corresponding bar in the plot.


Figure 4B , C . Boxplots reporting LPS - induced changes ( Log2FC ) for each of the four RNAPII kinetic rates in c - mycwt / wt ( wt ) and c - myc ∆ / ∆ cells for Myc - dependent ( B ) and Myc - independent ( C ) induced and repressed genes . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported . The RNAPII ChIP - seq time - course was performed on two biological replicates ( each on pools of cells from > 10 mice ) , and reads were merged after sequencing . D , E . Heatmaps showing the variations in RNAPII kinetic rates ( Log2FC ) on Myc - dependent ( D ) and Myc - independent ( E ) genes during the LPS time - course . The grey scale represents the starting level for each parameter in unstimulated cells . The first column on the left reports the changes in Myc share for the same genes . Genes were grouped using hierarchical clustering . F . Boxplots reporting LPS - induced changes ( Log2FC ) for each of the four RNAPII kinetic rates in c - mycwt / wt ( wt ) and c - myc ∆ / ∆ cells for genes of clusters 1 and 9 . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported . G . Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B, C. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for Myc-dependent (B) and Myc-independent (C) induced and repressed genes. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported. The RNAPII ChIP-seq time-course was performed on two biological replicates (each on pools of cells from > 10 mice), and reads were merged after sequencing.D, E. Heatmaps showing the variations in RNAPII kinetic rates (Log2FC) on Myc-dependent (D) and Myc-independent (E) genes during the LPS time-course. The grey scale represents the starting level for each parameter in unstimulated cells. The first column on the left reports the changes in Myc share for the same genes. Genes were grouped using hierarchical clustering.F. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for genes of clusters 1 and 9. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.G. Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.


Figure 1A Heart rate ( HR ) of WT and Adcy9 - / - mice averaged over 5 min of non - active versus sustained activity on home cage running wheels . Student ' s t - test P values for indicated comparisons are shown . Circles represent data from individual animals . The boxplot central band is the median while whiskers represent the 10th and 90th percentiles . B , C HR and R - R interval of WT ( black bars ) and Adcy9 - / - mice ( red bars ) before and after ISO ( 1 μg / g ) injectionD Representative ECG recordings of 7 mo WT and Adcy9 - / - mice , ~ 55 min post ISO - injection . E , F HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences ( RMSSD ; panel ( E ) ) and percentage of sequential R - R intervals differing by > 6 ms ( pNN6 ; panel ( F ) ) methods .

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Figure 1A Heart rate (HR) of WT and Adcy9-/- mice averaged over 5 min of non-active versus sustained activity on home cage running wheels. Student's t-test P values for indicated comparisons are shown. Circles represent data from individual animals. The boxplot central band is the median while whiskers represent the 10th and 90th percentiles.B,C HR and R-R interval of WT (black bars) and Adcy9-/- mice (red bars) before and after ISO (1 μg/g) injectionD Representative ECG recordings of 7 mo WT and Adcy9-/- mice, ~55 min post ISO-injection.E,F HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences (RMSSD; panel (E)) and percentage of sequential R-R intervals differing by >6 ms (pNN6; panel (F)) methods.


Figure 2A , B PLA assay performed in neonatal cardiomyocytes expressing GFP - Flag or AC9 - Flag using antibodies against Flag and endogenous POPDC1 , POPDC2 or Gβγ . Images ( A ) ; scale bar is 20 um ; nuclear staining by DAPI shown in blue ) and quantitation ( B ) of PLA signal are shown . ( D ) Quantification of COS7 cells expressing BiFC constructs for AC9 , POPDC 1 - 3 . The expressed proteins tagged with VN ( top line ) and VC ( bottom line ) are shown . ( E ) Representative live - cell images of indicated BiFC combinations in HEK293 cells ( n > 20 cells ; scale bar : 10 μm ) . Quantification of POPDC1 - VN : AC9 - VC is shown in Fig 3 and 7 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A,B PLA assay performed in neonatal cardiomyocytes expressing GFP-Flag or AC9-Flag using antibodies against Flag and endogenous POPDC1, POPDC2 or Gβγ. Images (A); scale bar is 20 um; nuclear staining by DAPI shown in blue) and quantitation (B) of PLA signal are shown.(D) Quantification of COS7 cells expressing BiFC constructs for AC9, POPDC 1-3. The expressed proteins tagged with VN (top line) and VC (bottom line) are shown.(E) Representative live-cell images of indicated BiFC combinations in HEK293 cells (n>20 cells; scale bar: 10 μm). Quantification of POPDC1-VN:AC9-VC is shown in Fig 3 and 7.


Figure 3A HEK293 cells expressing Flag - AC9 in the presence or absence of Myc - tagged POPDC1 or - 2 were subjected to co - immunoprecipitation ( Co - IP ) with anti - MYC and assayed for AC activity with 300 nM Gαs - GTPγS . Kruskal - Wallis One Way ANOVA analysis on Ranks was performed ( n = 6 experiments , P = 0 . 003 between all groups ) with multiple comparisons to AC9 control by Dunn ' s method and Mann - Whitney Rank Sum Test ( * * P = 0 . 002 ) . B A portion of the lysates and Co - IP from ( A ) were subjected to western blot ( WB ) analysis . Sf9 cells expressing Flag - AC9 served as a positive WB control . Molecular weight markers are denoted as M . Quantitation of Flag - AC9 WB by One Way ANOVA , n = 3 - 4 experiments , with comparisons by Tukey test , * * P = 0 . 003 , * * * P = 0 . 001 . ( C ) HEK293 cells expressing Flag - AC9 + / - POPDC1 - Myc or POPDC2 - Myc were subjected to Co - IP with anti - FLAG and subjected to WB analysis with anti - MYC and anti - FLAG . Quantitation of anti - MYC WB by One Way ANOVA , n = 4 experiments , with comparisons by Tukey test , * * P = 0 . 003 , * * * P < 0 . 001 ) . ( D ) HEK293 cells expressing POPDC1 - Myc + / - GFP - tagged AC9 and control TM proteins EGFR , or LAMP1 were subjected to Co - IP with anti - MYC . Western blotting of lysates and Co - IPs for GFP ( top ) and MYC ( bottom ) are shown ( n = 3 experiments ) . ( F ) BiFC of AC9 and indicated POPDC1 truncations in COS - 7 cells . Kruskal - Wallis One Way ANOVA analysis was performed ( n = 4 experiments ) with multiple comparisons by Tukey test , * * * P < 0 . 001 compared to VN control , * * P = 0 . 008 . ( G ) Co - IP with anti - MYC in COS - 7 cells expressing Flag - AC9 and indicated Myc - tagged POPDC1 truncations . Western blotting with anti - AC9 and anti - MYC ( POPDC1 ) of Co - IP and lysates is shown . Kruskal - Wallis One Way ANOVA analysis was performed ( n = 3 - 5 experiments , P = 0 . 006 ) , with multiple comparisons by Dunn ' s method ( * P = 0 . 021 , * * P = 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or -2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=6 experiments, P=0.003 between all groups) with multiple comparisons to AC9 control by Dunn's method and Mann-Whitney Rank Sum Test (**P=0.002).B A portion of the lysates and Co-IP from (A) were subjected to western blot (WB) analysis. Sf9 cells expressing Flag-AC9 served as a positive WB control. Molecular weight markers are denoted as M. Quantitation of Flag-AC9 WB by One Way ANOVA, n=3-4 experiments, with comparisons by Tukey test, **P=0.003, ***P=0.001.(C) HEK293 cells expressing Flag-AC9 +/- POPDC1-Myc or POPDC2-Myc were subjected to Co-IP with anti-FLAG and subjected to WB analysis with anti-MYC and anti-FLAG. Quantitation of anti-MYC WB by One Way ANOVA, n=4 experiments, with comparisons by Tukey test, **P=0.003, ***P<0.001).(D) HEK293 cells expressing POPDC1-Myc +/- GFP-tagged AC9 and control TM proteins EGFR, or LAMP1 were subjected to Co-IP with anti-MYC. Western blotting of lysates and Co-IPs for GFP (top) and MYC (bottom) are shown (n=3 experiments).(F) BiFC of AC9 and indicated POPDC1 truncations in COS-7 cells. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments) with multiple comparisons by Tukey test, ***P<0.001 compared to VN control, **P=0.008.(G) Co-IP with anti-MYC in COS-7 cells expressing Flag-AC9 and indicated Myc-tagged POPDC1 truncations. Western blotting with anti-AC9 and anti-MYC (POPDC1) of Co-IP and lysates is shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=3-5 experiments, P=0.006), with multiple comparisons by Dunn's method (*P=0.021, **P=0.01).


Figure 4A Co - localization of CFP - TREK - 1 with the BiFC signal from AC9 - VN : AC9 - VC homodimer ( top ) and POPDC1 - VN : AC9 - VC complex ( bottom ) in HEK293 cells . B The lifetime distribution of Cerulean - tagged proteins + / - the indicated YFP - tagged proteins expressed in HEK293 cells . Mann - Whitney Rank Sum Test was performed ( * * * P < 0 . 001 ; n = cell number indicated on each bar ) . C Co - IP of Myc - tagged POPDC1 ( anti - MYC ) pulled down both Flag - AC9 and YFP - tagged TREK - 1 ( n = 3 experiments ) . One - way ANOVA was performed with multiple comparisons by Holm - Sidak method ( # P = 0 . 007 compared to control ) . D Co - IP of TREK - 1 ( anti - GFP ) pulls down Flag - AC9 and endogenous POPDC1 . AKAP79 is not pulled down in complex ( n = 3 experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Co-localization of CFP-TREK-1 with the BiFC signal from AC9-VN:AC9-VC homodimer (top) and POPDC1-VN:AC9-VC complex (bottom) in HEK293 cells.B The lifetime distribution of Cerulean-tagged proteins +/- the indicated YFP-tagged proteins expressed in HEK293 cells. Mann-Whitney Rank Sum Test was performed (***P<0.001; n=cell number indicated on each bar).C Co-IP of Myc-tagged POPDC1 (anti-MYC) pulled down both Flag-AC9 and YFP-tagged TREK-1 (n=3 experiments). One-way ANOVA was performed with multiple comparisons by Holm-Sidak method (#P=0.007 compared to control).D Co-IP of TREK-1 (anti-GFP) pulls down Flag-AC9 and endogenous POPDC1. AKAP79 is not pulled down in complex (n=3 experiments).


Figure 5A IP - AC assay with IgG versus anti - TREK - 1 in heart homogenates from WT and Adcy9 - / - mice ; AC activity stimulated with 300 nM Gαs - GTPγS . IP - AC with anti - AKAP150 is shown as a positive control . Two - way ANOVA ( overall P < 0 . 001 , n = 3 mice per genotype ) with multiple comparisons by Holm - Sidak test ; # P < 0 . 001 for indicated comparison or to respective IgG controls . IgG versus anti - TREK - 1 in Adcy9 - / - was analyzed by Student ' s t - test ( * * P = 0 . 009 , n = 3 ) . A portion of the co - IP was subjected to western blotting with anti - TREK - 1 and anti - AKAP150 . B IP - AC assay with IgG versus anti - TREK in WT and Popdc1 - / - mouse heart homogenates ; AC activity stimulated as in ( A ) . Statistics as in ( A ) , n = 4 mice per genotype , # P < 0 . 001 . Paired t - test within Popdc1 - / - , P = 0 . 123 . C Total AC activity in heart homogenates from WT or Popdc1 - / - ( P1 - / - ) mice stimulated with 300 nM Gαs - GTPγS or 100 μM calcium and 300 nM calmodulin . D IP - AC assay with anti - MYC from cell lysates expressing AC1 or AC8 in the presence or absence of Myc - tagged POPDC1 . AC activity stimulated with 100 μM calcium and 300 nM calmodulin . Student ' s t - test ( n = 3 experiments , * * P = 0 . 0011 and * P = 0 . 012 ) . E IP - AC assay with IgG versus anti - TREK - 1 in WT and Popdc1 - / - mouse heart homogenates ; AC activity stimulated as in ( D ) . Student ' s t - test ( * P = 0 . 03 ; * * P = 0 . 005 , n = 4 mice per genotype ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A IP-AC assay with IgG versus anti-TREK-1 in heart homogenates from WT and Adcy9-/- mice; AC activity stimulated with 300 nM Gαs-GTPγS. IP-AC with anti-AKAP150 is shown as a positive control. Two-way ANOVA (overall P<0.001, n=3 mice per genotype) with multiple comparisons by Holm-Sidak test; #P<0.001 for indicated comparison or to respective IgG controls. IgG versus anti-TREK-1 in Adcy9-/- was analyzed by Student's t-test (**P=0.009, n=3). A portion of the co-IP was subjected to western blotting with anti-TREK-1 and anti-AKAP150.B IP-AC assay with IgG versus anti-TREK in WT and Popdc1-/- mouse heart homogenates; AC activity stimulated as in (A). Statistics as in (A), n=4 mice per genotype, #P<0.001. Paired t-test within Popdc1-/-, P=0.123.C Total AC activity in heart homogenates from WT or Popdc1-/- (P1-/-) mice stimulated with 300 nM Gαs-GTPγS or 100 μM calcium and 300 nM calmodulin.D IP-AC assay with anti-MYC from cell lysates expressing AC1 or AC8 in the presence or absence of Myc-tagged POPDC1. AC activity stimulated with 100 μM calcium and 300 nM calmodulin. Student's t-test (n=3 experiments, **P=0.0011 and *P=0.012).E IP-AC assay with IgG versus anti-TREK-1 in WT and Popdc1-/- mouse heart homogenates; AC activity stimulated as in (D). Student's t-test (*P=0.03; **P=0.005, n=4 mice per genotype).


Figure 6A Treatment of HEK293 cells for 10 min in the presence of 10 µM ISO reduced the amount of Flag - AC9 and POPDC1 - Myc pulled down by TREK - 1 co - IP . B , C Quantification ( IP / input ) of Flag - AC9 ( B ) and POPDC1 - Myc ( C ) between ISO treatment groups by two - tailed paired Student ' s t - test ( * P = 0 . 028 , * * P = 0 . 007 ; n = 3 biological replicates ) .

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Figure 6A Treatment of HEK293 cells for 10 min in the presence of 10 µM ISO reduced the amount of Flag-AC9 and POPDC1-Myc pulled down by TREK-1 co-IP.B, C Quantification (IP/input) of Flag-AC9 (B) and POPDC1-Myc (C) between ISO treatment groups by two-tailed paired Student's t-test (*P=0.028, **P=0.007; n=3 biological replicates).


Figure 7 , B BiFC signal between the indicated VN - and VC - tagged proteins ( POPDC1 , P1 ) . B Percent decrease of BiFC signal in ( A ) with ISO treatment ( 10 μM , 10 min at 37 ° C ) . Paired t - test for each condition ( vehicle versus ISO ) using raw data prior to calculation of percent of vehicle control ( n = 6 ; * P < 0 . 05 ; * * P < 0 . 01C ISO dose - response curves for BiFC interactions between POPDC1 : AC9 , TREK - 1 : AC9 , and TREK - 1 with a catalytically inactive AC9 ( TREK - 1 : AC9D ) . D TREK - 1 : AC9 BiFC signal is absent in COS - 7 cells lacking detectable POPDC1 . TREK - 1 : TREK - 1 BiFC is a positive control . One - way ANOVA with multiple comparisons by Holm Sidak method ( n = 3 experiments ; * * * P < 0 . 001 ) . E Overexpression of POPDC1 truncation of Popeye domain ( POPDC1 - Δ172 ) , but not WT POPDC1 , abolishes ISO reduction of TREK - 1 : AC9 BiFC signal . One - way ANOVA with multiple comparisons by Holm Sidak method ( n = 3 - 4 experiments ; * * * P < 0 . 001 compared to control ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7, B BiFC signal between the indicated VN- and VC-tagged proteins (POPDC1, P1). B Percent decrease of BiFC signal in (A) with ISO treatment (10 μM, 10 min at 37°C). Paired t-test for each condition (vehicle versus ISO) using raw data prior to calculation of percent of vehicle control (n=6; *P<0.05; **P<0.01C ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9D).D TREK-1:AC9 BiFC signal is absent in COS-7 cells lacking detectable POPDC1. TREK-1:TREK-1 BiFC is a positive control. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3 experiments; ***P<0.001).E Overexpression of POPDC1 truncation of Popeye domain (POPDC1-Δ172), but not WT POPDC1, abolishes ISO reduction of TREK-1:AC9 BiFC signal. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4 experiments; ***P<0.001 compared to control).