EMBO/soda-nel huggingface dataset:
Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2S production (2021)
Figure 1

Figure 1. Syk inhibitor R406 can significantly promote mouse chemical reprogramming.

A Schematic diagram depicting the procedure of compound screening during mouse chemical reprogramming.

B The chemical screening result evaluated by GFP+ colony number on d24.

C GFP+ colony number of samples treated with R406, R788 and Lenvatinib. n = 3.

D Fluorescence image of colonies with pOct4-GFP expression after DMSO and R406 treatments. Scale bar, 100 μm.

E-G Concentration (F) and stage (G) test of R406 (E) during reprogramming. n = 3.

H Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm.

I RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3.

J Diagram showing the procedure of shSyk virus infection at the early stage of reprogramming.

K RT-qPCR analysis of Syk expression in MEFs infected with shNC and shSyk viruses. n = 3.

L Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm.

M Schematic diagram of the procedure of establishing ciPSC lines.

N ciPSCs express key pluripotent markers Oct4, Sox2 and Nanog. Scale bar, 100 μm.

O ciPSCs have developmental potentials to form teratomas containing tissues from all three germ layers. Scale bar, 100 μm.

P Chimeric mice were generated from ciPSCs.

Data information: all data are presented as mean ± SD. Statistical significance was assessed by the two-tailed Student's t-test, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Fig EV1.
Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2S production (2021)
Figure 2

Figure 2. Inhibition of Syk-Cn-NFAT signaling pathway promotes chemical reprogramming.

A Schematic diagram showing Syk-Cn-NFAT pathway axis.

B Immunofluorescence of Sall4 in reprogramming intermediates treated with DMSO and FK506. n = 5. Scale bar, 100 μm.

C, D Concentration (C) and stage (D) test of FK506 during reprogramming. n = 3.

E RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in reprogramming intermediates treated with DMSO and FK506. n = 3.

F Diagram showing the procedure of Ppp3ca knockdown at the early stage of reprogramming.

G RT-qPCR analysis of Ppp3ca expression in MEFs infected with shNC and shPpp3ca viruses. n = 3.

H Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm.

I RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3.

J Diagram showing the procedure of shNfatc1 virus infection at the early stage of reprogramming.

K RT-qPCR analysis of Nfatc1 expression in MEFs infected with shNC and shNfatc1 viruses. n = 3.

L Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm.

M RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3.

Data information: all data are presented as mean ± SD. Statistical significance was assessed by the two-tailed Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Fig EV2.
Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2S production (2021)
Figure 3

Figure 3. Inhibition of Syk-Cn-NFAT signaling axis by R406 can transcriptionally upregulate genes involved in glycine, serine and threonine metabolism.

A Volcano plot of differentially expressed genes (DEGs) in DMSO- and R406-treated cells on d12. Red dots represent upregulated genes and blue dots represent downregulated genes in R406-treated cells. Dash lines represent 2-fold change, adjusted p-value < 0.05. Statistical significance was assessed by the method described previously (Love et al., 2014).

B Heatmap showing the normalized expression (z score) of DEGs. Red indicates up-regulated, whereas blue indicates down-regulated genes.

C KEGG analysis of upregulated genes in R406-treated reprogramming intermediates. Statistical significance was assessed by Benjamini-Hochberg method.

D Heatmap of RNA-seq data for the normalized expression (z score) of genes involved in glycine, serine and threonine metabolism. Red indicates up-regulated genes, whereas blue indicates down-regulated genes.

E, F RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3.

G Schematic of CUT&Tag experiments to map the genomic occupancy of NFATc1.

H NFAT motif enrichment and associated p-value are shown. Statistical significance was assessed by the method described previously (Heinz et al, 2010).

I NFATc1 occupancy at Gldc, Cbs and Tdh gene loci in cells treated with DMSO and R406 and no-antibody control.

J The predicted NFAT binding sites at the promoters of Gldc, Cbs and Tdh.

K qPCR assay of the binding of NFATc1 at Gldc, Cbs and Tdh gene loci in cells treated with DMSO and R406. n = 3.

Data information: all data are presented as mean ± SD. Statistical significance was assessed by the two-tailed Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Fig EV3.
Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2S production (2021)
Figure 5

Figure 5. R406 can upregulate cellular H2S level and H2S can promote chemical reprogramming.

A H2S levels in DMSO- and R406-treated reprogramming intermediates detected by monobromobimane method. n = 4.

B H2S levels in reprogramming intermediates treated with DPBS, HA and NAC. n = 3.

C, Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm.

D RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3.

E Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm.

F RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.

G H2S levels in reprogramming intermediates treated with shNC and shCbs. n = 3.

H Diagram showing the procedure of reprogramming infected with shRNA for Cbs.

I RT-qPCR analysis of Cbs expression in MEFs infected with shNC and shCbs. n = 3.

J, K Immunofluorescence of Sall4 in shNC- and shCbs-infected cells with and without R406 treatment on d12. n = 5. Scale bar, 100 μm.

L Diagram showing the procedure of reprogramming infected with Cbs-OE (pSin-Cbs) virus.

M RT-qPCR analysis of Cbs expression in MEFs infected with pSin-GFPN and pSin-Cbs viruses. n = 3.

N Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12. n = 4. Scale bar, 100 μm.

Data information: all data are presented as mean ± SD. Statistical significance was assessed by the two-tailed Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Fig EV5.
Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19 (2014)
Figure 1

Atg19 phosphorylation is required for Atg11 binding and Cvt pathway function

Domain structure of Atg19.
atg19Δ cells containing HTB‐Atg19 were grown to mid‐log phase. Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; lysine substitutions: gray; Atg11 binding region: green.
atg19Δ cells containing GFP‐Atg19 wild‐type or mutants as indicated were grown to mid‐log phase. Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild‐type. Atg19 expression was assessed by anti‐GFP Western blotting.
atg19Δ GFP‐Atg11 Atg1‐TAP cells containing myc‐Atg19 as indicated were grown to log phase. Atg11 was immunoprecipitated, and bound Atg19 was analyzed by Western blotting.
GST‐fused propeptide of Ape1, Atg19 as indicated, and a His‐tagged fragment of Atg11 (amino acids 685–1178) were purified from Escherichia coli, mixed, and propeptide‐bound proteins were isolated and analyzed by Coomassie staining. Quantification of three individual experiments is shown. Error bars represent standard deviation.

Source data are available online for this figure.
Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19 (2014)
Figure 2

Non‐phosphorylatable Atg19 is unable to bind Atg11

GFP‐Atg11 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h, and autophagy was induced by treating cells for 1 h with rapamycin. Scale bar, 5 μm.
Ape1‐mRuby atg19Δ cells containing GFP‐Atg19 wild‐type or GFP‐Atg19‐3A were analyzed in log phase. Scale bar, 5 μm.
GFP‐Atg8 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were analyzed as in (A). Scale bar, 5 μm.
Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19 (2014)
Figure 3

Atg34 phosphorylation on homologous sites is required for Ams1 transport to the vacuole

Comparison of Atg19 and Atg34 domain structures and C‐terminal amino acid sequences. Green: Atg11 binding region. Serine residues S390, S391, and S396, which are phosphorylated in Atg19, and the conserved sites in Atg34 are shown enlarged.
atg34Δ yeast cells containing HTB‐Atg34 were grown to mid‐log phase and treated with rapamycin. Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; Atg11 binding region: green; lysine substitution: gray.
Ams1‐GFP atg19Δatg34Δ cells containing protein A (Stag)‐tagged Atg34 as indicated were starved for 4 h. Processing of endogenous Ams1‐GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1‐GFP normalized to the wild‐type. Expression of Atg34 proteins was assessed by anti‐protein A Western blotting.
Alignment of the Atg1 consensus sites in the Atg11 binding region of Atg19 and Atg34 with the known Atg1 phosphorylation sites of Atg9 and Atg2.
GST, GST‐Atg19, and GST‐Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1‐TAP bound to IgG magnetic beads.
In vitro phosphorylation of recombinant Atg19, Atg19‐3A, and Atg19‐3D using wild‐type and kinase‐dead Atg1‐D211A as described in (E).

Source data are available online for this figure.
Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19 (2014)
Figure 4

Hrr25 kinase is responsible for Atg19 phosphorylation in the Atg11 binding region

Wild‐type, hrr25‐ts, and hrr25‐ts cells containing HRR25 on a centromeric plasmid were grown to mid‐log phase at 18°C followed by 2 and 4 h of log‐phase growth at 37°C. Ape1 processing was analyzed by Western blotting.
atg19Δ and atg19Δ hrr25‐ts cells containing GFP‐Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37°C.
GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg1‐D211A cells were analyzed for GFP‐Atg11 dot formation. Quantification of at least 70 cells in three individual experiments is shown. Error bars represent standard deviation. Scale bar, 3 μm.
GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg19Δ cells containing BFP‐Ape1 and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h. coloc.: Cells with GFP‐Atg11/BFP‐Ape1 co‐localization. n = 50 (wt), 84 (hrr25‐ts), 127 (atg19Δ). Scale bar, 3 μm.
Hrr25 was immunoprecipitated from Hrr25‐TAP cells and as a control from a non‐tagged wild‐type strain. In vitro phosphorylation of recombinant Atg19, Atg19‐3A, Atg34, and GST using the kinase‐bound beads was performed as described in Fig E. Both Atg19 panels are from the same gel with the same exposure.

Source data are available online for this figure.
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 1

Fig. 1. Deletion of Adcy9 results in bradycardia at rest and HR variability during recovery period after ISO injection.

A 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) injection; mean of 5 min intervals ± SE are shown.

D 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.

Data information: Two-way ANOVA Repeated measures analysis (time and genotype) with Holm-Sidak method used for pairwise comparisons for HR, RR, and HR variability with ISO; n=9 WT and n=10 Adcy9-/- mice; *P<0.05; **P<0.01; ***P<0.001.
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 2

Fig. 2. Cellular interaction of POPDC isoforms with AC9.

A,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. Unpaired two-sided Student's t-test (n=4 independent experiments, **P=0.0086) was performed for each antibody pair. Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

(C) Cartoon of AC9-POPDC BiFC complex.

(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. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=4 experiments) with multiple comparisons to control by Dunnett's method (#P<0.05). Individual comparisons were performed with two-tailed Student's t-test (**P=0.003, *P=0.018) or Mann-Whitney Rank Sum Test (^P=0.004 versus control) Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

(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.
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 3

Fig. 3. AC9 interacts with the POPDC1 transmembrane and Popeye domains.

A 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). Data are plotted as mean ± SEM.

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. Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

(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). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

(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).

(E) Schematic of POPDC1 truncations.

(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. Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

(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). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 4

Fig. 4. AC9 is part of a POPDC1-TREK-1 complex.

A 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. Scale bar: 10 µm.

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). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

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). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

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). One-way ANOVA was performed with multiple comparisons by Student-Newman-Keuls method (#P<0.05 compared to control; *P=0.013 and 0.026 comparing bars 2-3 and 3-4, respectively) or two-tailed Welch's t-test for non-equal variances (^P=0.014 and ^P=0.029). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

Data information: Quantification of Flag-AC9 WB (IP/total expression in lysate) is shown to the right of (C) and (D).
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 5

Fig. 5. TREK-1 pulls-down AC9 activity in heart which requires POPDC1 expression.

A 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. Data are plotted as mean ± SEM.

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. Data are plotted as mean ± SD.

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. Student's t-test (n=4, n.s.) Data are plotted as mean ± SD.

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). Data are plotted as mean ± SEM.

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). Data are plotted as mean ± SD.
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 6

Fig 6. Isoproterenol treatment reduces AC9 and POPDC1 interaction with TREK-1.

A 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). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 7

Fig. 7. ISO-dependent loss of TREK-1 from AC9-POPDC1 complex requires an intact Popeye domain.

A, B BiFC signal between the indicated VN- and VC-tagged proteins (POPDC1, P1). Kruskal-Wallis One-Way ANOVA on Ranks (n=5 experiments, P<0.001 between groups) with multiple comparison to VN and VC controls by Student-Newman-Keuls tests (*P<0.05). Data are plotted as mean ± SEM.

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<0.01). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range.

C ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9D). Two-way ANOVA with multiple comparisons by Bonferroni method (n=3-4; *P<0.05 for comparisons between TREK:AC9 versus TREK:AC9D and POPDC1:AC9). Data are plotted as mean ± SEM.

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). Data are plotted as mean ± SD.

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). Data are plotted as mean ± SEM.

F Model of cAMP effects on AC9-POPDC1-TREK-1 complex formation.
POPDC1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Potassium Channel TREK-1 in Heart (2022)
Figure 8

Fig 8. AC9 activity opposes the enhancement of TREK-1 currents by POPDC1.

A Representative two-electrode voltage clamp measurements in Xenopus oocytes injected with TREK-1 alone (orange) or TREK-1 co-injected with cRNAs encoding the indicated proteins. Voltage was ramped from -120 to +40 mV.

B Relative TREK-1 current amplitudes at +40 mV normalized to TREK-1 injected alone. The number of oocytes is given within the bar graphs (n=37-97). 3-5 batches of oocytes were used for each condition. Data are presented as mean ± SEM. Two-tailed Student's t-test between the indicated groups (***, P=0.00018; ****, P<1x10-5).

C Relative currents normalized to TREK-1 plus POPDC1 in the absence and presence of theophylline. Data are plotted as mean ± SEM. Two-tailed Student's t-test (**, P=0.0011; ***, P=0.0002; ****, P<1x10-5; n=40-85 biological replicates).

D, E Oocytes were treated with 0.5 mM theophylline prior to voltage-clamp measurements as in (A, B). Data are presented as mean ± SEM. Two-tailed Student's t-test between the indicated groups (**, P=0.0011; ***, P=4x10-5 ; ****, P<1x10-7).

Extended views Figure Legends