EMBO/SourceData huggingface dataset:
Figure legend: "(C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells."
NCBI gene IDs:
"HA: 
IKKε: 9641"
Figure legend: "(B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells."
NCBI gene IDs:
"HA: 
IKKε: 9641"
Figure legend: "(C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines, measured using Seahorse XF96e or XF24 analysis."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry."
NCBI gene IDs:
"GFP: 
HA: 
IKKε: 9641"
Figure legend: "(F) Representative western blot showing l­evel of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours)."
NCBI gene IDs:
"GFP: 
HA: 
IKKε: 9641"
Figure legend: "(G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours)."
NCBI gene IDs:
"GFP: 
HA: 
IKKε: 9641"
Figure legend: "(H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells."
NCBI gene IDs:
"GFP: 
HA: 
IKKε: 9641"
Figure legend: "(I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry."
NCBI gene IDs:
"GFP: 
HA: 
IKKε: 9641"
Figure legend: "(J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry."
NCBI gene IDs:
"GFP: 
HA: 
IKKε: 9641"
Figure legend: "(A) Representative western blot showing level of ATF4 in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours)."
NCBI gene IDs:
"HA: 
ATF4: 468
IKKε: 9641"
Figure legend: "(B) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4 biological replicates)"
NCBI gene IDs:
"β-Actin: 
GFP: 
HA: 
IKKε: 9641
PHGDH: 26227
PSAT1: 29968
PSPH: 5723"
Figure legend: "(C) Representative western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours."
NCBI gene IDs:
"GFP: 
HA: 
IKKε: 9641"
Figure legend: "(D) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in a non-silenced, non-treated control, and normalised to β-Actin (n=5 biological replicates)."
NCBI gene IDs:
"β-Actin: 
HA: 
ATF4: 468
IKKε: 9641
PHGDH: 26227
PSAT1: 29968
PSPH: 5723"
Figure legend: "(E) Representative western blot showing levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours."
NCBI gene IDs:
"HA: 
ATF4: 468
IKKε: 9641"
Figure legend: "(A) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. Data are expressed as fold changes, relative to levels in a non-silenced control of each cell line and normalised to β-Actin (n≥3 biological replicates)."
NCBI gene IDs:
"β-Actin: 
IKBKE: 9641
IKKε: 9641
PHGDH: 26227
PSAT1: 29968
PSPH: 5723"
Figure legend: "(B) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE (IKKε)-silenced ZR-75-1, T47D, MDA-MB-468 and MCF7 breast cancer cell lines."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(C-E) Levels of the SBP enzymes in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. (C) PHGDH, (D) PSAT1 and (E) PSPH levels in indicated cell lines normalised to Vinculin. Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation (n≥3 biological replicates)."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(F) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced ZR-75-1, T47D and MDA-MB-468 breast cancer cell lines. Data are expressed as fold changes, relative to levels in non-silenced control cells and normalised to β-Actin (n=3 biological replicates)."
NCBI gene IDs:
"β-Actin: 
ATF4: 468
PHGDH: 26227
PSAT1: 29968
PSPH: 5723"
Figure legend: "(G) Representative western blot showing levels of PHGDH, PSAT1 and PSPH in ATF4-silenced ZR-75-1, MDA-MB-468, MDA-MB-231, T47D and HCC1143 breast cancer cell lines."
NCBI gene IDs:
"ATF4: 468"
Figure legend: "(A) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A)."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(B) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C)."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641"
Figure legend: "(C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p&lt;0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641
PSAT1: 29968"
Figure legend: "Expression of IKBKE (IKKε) and the SBP enzymes PSAT1, in the METABRIC dataset. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p&lt;0.05, **p&lt; 0.01, ***p&lt;0.001, ****p&lt;0.0001."
NCBI gene IDs:
"IKBKE: 9641
IKKε: 9641
PSAT1: 29968"
Figure legend: "Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset. The expression of a proliferation related gene set and ATF4 are also shown. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p&lt;0.05, **p&lt; 0.01, ***p&lt;0.001, ****p&lt;0.0001."
NCBI gene IDs:
"ATF4: 468
PHGDH: 26227
PSPH: 5723"
Figure legend: "(J) Association between IKBKE (IKKε) and ATF4 mRNA overexpression evaluated by chi-squared independence test. The + sign indicates samples with significant (p&lt;0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown."
NCBI gene IDs:
"ATF4: 468
IKBKE: 9641
IKKε: 9641
PSAT1: 29968"
Figure legend: "(K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset."
NCBI gene IDs:
"ATF4: 468
IKBKE: 9641
IKKε: 9641
PSAT1: 29968"
Figure legend: "a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white dots) or either SF1670 (first row), SC-79 (second row), 740 Y-P (third row) or AS1842856 (fourth row, black dots) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=4 independent experiments performed."
NCBI gene IDs:
"IFNG: 3458
TBX21: 30009"
Figure legend: "f. IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT-shRNA control, measured at day 6 after 4 hours of stimulation with PMA and ionomycin."
NCBI gene IDs:
"Foxo1: 2308
Foxo3: 2309
IFNG: 3458
TBX21: 30009"
Figure legend: "g. Percentage of suppression of CFSE-labeled Tresp proliferation co-cultured with Tregs transduced with a non-target (white dots), Foxo1 (left diagram, black dots) or Foxo3 shRNA (right diagram, black dots) at different Treg:Tresp ratios (n=4). *p &lt; 0.05, **p &lt; 0.005, ***p &lt; 0.0005."
NCBI gene IDs:
"Foxo1: 2308
Foxo3: 2309"
Figure legend: "Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. a. IFNG, TBX21, IL10, FOXP3 gene expression after sort at day 5 (n=5)."
NCBI gene IDs:
"AKT1: 207
AKT2: 208
AKT3: 10000
FOXP3: 50943
IFNG: 3458
IL10: 3586
TBX21: 30009"
Figure legend: "Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. b. Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 (n=4)."
NCBI gene IDs:
"AKT1: 207
AKT2: 208
AKT3: 10000"
Figure legend: "Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. c. Statistical analysis of the frequency of IFN+Foxp3+ Tregs after AKT silencing (n=4)."
NCBI gene IDs:
"AKT1: 207
AKT2: 208
AKT3: 10000"
Figure legend: "Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. d. shRNA transduced cells were co-cultured with CFSE-labeled Tresp and Tresp proliferation was measured after 4 days. e. Statistical analysis of the percentage of suppression by AKT1-, AKT2- and AKT3-silenced Tregs as compared to NT-transduced cells of n=6 experiments performed. *p &lt; 0.05, **p &lt; 0.005, ***p &lt; 0.0005."
NCBI gene IDs:
"AKT1: 207
AKT2: 208
AKT3: 10000"
Figure legend: "a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white squares) and IL-12 either alone (white circles) or in the presence of LY294002 or MK2206 (black circles) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=8 independent experiments performed."
NCBI gene IDs:
"IFNG: 3458
TBX21: 30009"
Figure legend: "C: Representative images from fluorescence live cell microscopy of Dad1-GFP strains with Duo1WT-6xFlag or  Duo1∆SxIP-6xFlag. Displayed cells are in either metaphase (left) or anaphase (right). Scale bar: 2 μm. D: Quantification of signal intensities of Dad1-GFP at metaphase and anaphase kinetochore clusters of Duo1WT-6xFlag  and Duo1∆SxIP-6xFlag strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single  kinetochore cluster. Values were normalized to the mean value of  metaphase clusters of the Duo1WT-6xFlag strain. A one-way ANOVA test with Sidak's multiple comparisons test was used to calculate p values. n ≥ 76 kinetochore clusters."
NCBI gene IDs:
"Flag: 
Duo1: 852819"
Figure legend: "F: Analysis of  cell cycle progression  of yeast  strains with different  Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor  arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated  time points after release from  α factor arrest was analyzed by  FACS  (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C   DNA content, respectively. An intermediate DNA content is detected for cells in S phase."
NCBI gene IDs:
"Duo1: 852819"
Figure legend: "B: Representative images from live cell microscopy of Dam1WT and dam1-19 strains with Dad1-GFP. Displayed cells are in  metaphase (left) or anaphase (right). Scale bar: 2 μm. C: Quantification of Dad1-GFP signal intensities at metaphase and anaphase kinetochore  clusters of  Dam1WT and dam1-19 strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single  kinetochore  cluster. Values were normalized to the mean value of metaphase clusters of the  Dam1WT strain. p-values were obtained by a one-way ANOVA test with Sidak's multiple comparisons test. n ≥ 86 kinetochore clusters."
NCBI gene IDs:
"Dam1: 853010
dam1: 853010"
Figure legend: "E, F: GST-Bim1185-344 pull down assays. Binding of immobilized GST-Bim1185-344 to Duo1-6xFlag was analyzed in context of (E) selective inhibition of Mps1  (mps1-737) or overexpression of Mps1 from a galactose-inducible promotor (F). For inhibition of Mps1, cells were grown at the restrictive  temperature of 37 °C. Overexpression of Mps1 was achieved by growing cells in medium containing  galactose. GST-Bim1185-344 immobilized on  glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions. Binding of Duo1 was analyzed by  western blot. Pgk1  indicates equal protein amounts in all samples."
NCBI gene IDs:
"Mps1: 851533"
Figure legend: "G: Live cell microscopy of a Dad1-GFP strain under conditions of Mps1 overexpression. The localization of Dam1c was visualized by  Dad1-GFP in either an Mps1WT or Mps1 overexpression setup (pGal-Mps1). Ndc80-mRuby2 was used as reference point for the localization of kinetochores. Images show representative metaphase cells from both strains. The area in the white dashed box is shown as magnification in the lower left corner. White arrowheads point at nuclear microtubules  decorated by Dad1-GFP. Please note that brightness and contrast of the  Dad1-GFP image in the lower row were optimized to make the nuclear microtubules  clearly visible. Scale bar: 2 μm."
NCBI gene IDs:
"Gal: 855828
Mps1: 851533"
Figure legend: "D: Serial dilution assay  of yeast strains with different  Duo1 and Ndc80 alleles in an Ndc80-FRB-GFP background. Ndc80WT or Ndc80∆490-510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1∆SxIP allele (at DUO1 locus). Cells were spotted on  YEPD medium without and with 1 µg/ml Rapamycin and incubated at the indicated temperatures."
NCBI gene IDs:
"GFP: 
Duo1: 852819
DUO1: 852819
LEU2: 850342
Ndc80: 854662"
Figure legend: "B:  Serial dilution assay  of  yeast  strains with 6xFlag-tagged  Duo1WT or Duo1∆SxIP and Ask1WT, Ask12A or Ask12D. Cells were grown at the indicated temperatures and images were taken two days (25 °C, 30 °C and 37 °C) or four days (16 °C) after spotting."
NCBI gene IDs:
"Flag: 
Ask1: 853814
Duo1: 852819"
Figure legend: "A. Assessment of protein levels in different AIFM1 knockout clones. Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells."
NCBI gene IDs:
"AIFM1: 9131"
Figure legend: "D. Assessment of protein levels in HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA (expression of AIFM1-HA induced for one day). Different amounts of lysates from the indicated cells were analyzed by reducing SDS-PAGE and immunoblotting. While levels of MIA40 were largely unchanged, levels of many MIA40/CHCHD4 substrates were diminished in AIFM1 knockout cells. N = 3 biological replicates."
NCBI gene IDs:
"HA: 
AIFM1: 9131"
Figure legend: "F. BN-PAGE analyses of HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA or AIFM1. Complex I was resolved from isolated mitochondria with blue native electrophoresis (BN-PAGE) followed by immunoblotting or in gel determination of the complex I specific activity. Levels of complex I were decreased in AIFM1 knockout cells. N = 3 biological replicates"
NCBI gene IDs:
"HA: 
AIFM1: 9131"
Figure legend: "A. In organello import assay with NDUFS5, NDUFB7 and as control the matrix protein SOD2. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells. Non-imported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified using ImageQuantTL and the amount of imported protein was plotted. NDUFS5 import is strongly affected by loss of AIFM1. Import of NDUFB7 is less affected and SOD2 import remained unaffected. This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar."
NCBI gene IDs:
"AIFM1: 9131"
Figure legend: "B. Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analyzed by pulse-chase experiments coupled to redox state determination. To this end, cells were incubated with 35S-Met for 5 min (pulse). After removal of 35S-Met, cells were left in \"cold\" Met for different chase times. Further oxidation was stopped by addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5."
NCBI gene IDs:
"HA: 
AIFM1: 9131"
Figure legend: "C. Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells. Cells were incubated with 35S-Met for 5 min (pulse), and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells."
NCBI gene IDs:
"AIFM1: 9131"
Figure legend: "D. Interaction between endogenous MIA40/CHCHD4 and its substrates in wild type and AIFM1 knockout cells. Cells were subjected to native immunoprecipitation (IP) against endogenous MIA40/CHCHD4. Precipitates were analyzed by SDS-PAGE and immunoblotting against the indicated proteins. In AIFM1 knockout cell interaction between MIA40/CHCHD4 and its substrates is strongly affected with the interaction between MIA40/CHCHD4 and NDUFS5 almost completely abolished. This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells, indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40/CHCHD4 rather than the cause. Quantification using Image lab. Data from 2 experiments were combined and standard deviations are presented. Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells, respectively."
NCBI gene IDs:
"AIFM1: 9131"
Figure legend: "A. Assessment of MIA40/CHCHD4 variant-AIFM1 interaction. The indicated MIA40/CHCHD4-Strep variants were affinity precipitated (AP) under native conditions after stopping thiol-disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR and MIA40/CHCHD4 by reducing SDS-PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40-Strep. Both, wildtype MIA40/CHCHD4 and MTS-MIA40/CHCHD4 but not MIA40/CHCHD4∆1-40 variants coprecipitated AIFM1. All mitochondria-localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4."
NCBI gene IDs:
"AIFM1: 9131
MIA40: 131474"
Figure legend: "C. Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. AIFM1 knockout cells stably expressing AIFM1-HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and AIFM1-HA (C) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6-8 hours AP, affinity precipitation; asterisk, background band."
NCBI gene IDs:
"HA: 
AIFM1: 9131"
Figure legend: "A. Gel filtration analysis to assess the AIFM1-MIA40 complex in HEK293 cells. HEK293 cells, cells lacking AIFM1 (AIFM1 knockout, AIFM1 KO) or AIFM1 knockout cells complemented with AIFM1-HA were lysed under native conditions (triton X-100), and the cleared lysates subjected to gel filtration analysis. Eluted fractions were subjected to TCA precipitation, resuspension in loading buffer containing SDS and DTT, and subsequent immunoblotting against AIFM1 and MIA40. Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa (as judged by comparison to protein markers: apoferritin 443 kDa; β-amylase 200 kDa; alcohol dehydrogenase 150 kDa; bovine serum albumin 66 kDa; carbonic anhydrase 29 kDa). Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1-HA. B. Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (∆1-40 MIA40/CHCHD4-Strep), which constitute the AIFM1 interaction motif. Experiment was performed as described in (A). AIFM1 and endogenous MIA40/CHCHD4 migrate in a complex with a size larger than 150 kDa, while N-terminally truncated MIA40/CHCHD4 migrates as monomer. For number of biological replicates and quantifications, see 5C. C. Quantification of 5A,B and Appendix Figure S9. Quantification of the areas representing the region of monomeric MIA40/CHCHD4 and the AIFM1-MIA40/CHCHD4 complex, respectively was performed. Quantifications consistently show that AIFM1 knockout, siRNA-mediated depletion of AIFM1 and overexpression of MIA40/CHCHD4 all resulted in an increase of monomeric MIA40/CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40/CHCHD4 are tightly regulated. The numbers below the bars indicate the numbers of biological replicates performed. Averages and standard deviations are presented."
NCBI gene IDs:
"HA: 
AIFM1: 9131
CHCHD4: 131474
MIA40: 131474"
Figure legend: "F. Assessing the solubility of MIA40/CHCHD4 in the IMS. Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling. MIA40/CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1-HA. It was released from cells lacking AIFM1. This indicates that MIA40/CHCHD4 in wildtype cells is membrane-associated via AIFM1."
NCBI gene IDs:
"HA: 
AIFM1: 9131"