EMBO/soda-nel huggingface dataset:
Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling (2015)
Figure 1

Figure 1. Nox4 selectively regulates ATF4 during ER stress.

A. Tunicamycin (Tn, 2 µg/ml) increased Nox4 protein levels in H9c2 cells. Tubulin was used as a loading control. n=4-6/group. *, significant compared to baseline.

B. Thapsigargin (Tp, 1 µg/ml) increased Nox4 protein levels in H9c2 cells. n=4-6/group. *, significant compared to baseline.

C. Effect of Nox4 on the unfolded protein response. Nox4 was depleted in H9c2 cells by shRNA-mediated knockdown (Ad.shNox4) or cells were treated with a control adenovirus (Ad.Ctl). In cells with Nox4 knockdown, tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94, Grp78 and calreticulin than in conrol cells. Nuclear protein levels of ATF4 were substantially lower in Nox4-depleted cells than control cells but the levels of cleaved ATF6 (ATF6c) were similar. Histone was used as a loading control. The relative mRNA levels of Xbp1-s (a readout of IRE1 signaling) were unaltered after Nox4 knockdown. Mean data are shown in Appendix Fig S1E. Similar results were obtained with an independent siRNA approach (Appendix Fig S2A).

D. Effect of adenoviral-mediated overexpression of Nox4 (Ad.Nox4) or a control β-galactosidase protein (Ad.β-Gal) on tunicamycin responses of H9c2 cells. Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin-induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels. Mean data are shown in Appendix Fig S2B.

E,F. Effect of Nox4 knockdown or overexpression, respectively, on the tunicamycin-induced changes in mRNA levels of ATF4 target genes. n=4/group. Psat1, phosphoserine aminotransferase; Phgdh, 3-phosphoglycerate dehydrogenase; Asns, asparagine synthetase; Slc6a9, glycine transporter 1.

G. Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin-treated H9c2 cells. Scrambled siRNAs were used as a control (Ctl). Representative immunoblots shown to the top (captions at bottom of bar graphs refer also to the immunoblots); tubulin was used as a loading control. n=4/group. *, significant compared to baseline; #, significant comparing siATF4 versus corresponding siCtl.

H. Effect of ATF4 overexpression on Nox4 mRNA and protein levels. n=3/group.

All blots are representative of at least 3 independent experiments. Values above bar graphs denote the level of significance.
Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling (2015)
Figure 2

Figure 2. Nox4 selectively inhibits PP1 activity and prolongs eIF2 phosphorylation.

A. The knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin-induced eIF2α phosphorylation in H9c2 cells, with no change in phospho-Thr980-PERK (PERK-P) levels. GADD34 levels were significantly decreased after Nox4 knockdown while there was no change in PP1 protein levels.

B. Overexpression of Nox4 in H9c2 cells caused prolongation of tunicamycin-induced eIF2α phosphorylation, with minimal change in phospho-PERK levels.

C,D. Mean levels of phosphorylated eIF2α relative to total eIF2α protein in tunicamycin-treated cells after Nox4 knockdown or overexpression, respectively. n=3/group. *, significant compared to baseline; #, significant comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).

E,F. Effect of Nox4 knockdown or overexpression, respectively, on okadaic acid-resistant Ser/Thr phosphatase activity in membrane fractions of tunicamycin-treated H9c2 cells. n=4/group. *p<0.05, **p<0.01 cf. baseline; #, p<0.05, ##, p<0.01 comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).

G,H. Nox4 knockdown or overexpression, respectively, had no effect on the phosphorylation of glycogen synthase at Ser641 (GS-P) or Histone H3 at Ser57 (H3-P) in H9c2 cells.

I. Nox4-/- MEF cells (KO) showed blunted tunicamycin-induced increases in levels of phospho-eIF2α, ATF4 and ER chaperones as compared to wild-type (WT) MEFs, a response that was rescued by re-introduction of Nox4 (KO+Nox4). The latter had no effect on GS-P or H3-P levels.

All blots are representative of at least 3 independent experiments. Values above bar graphs denote the level of significance. Mean data from quantification of immunoblots are shown in Appendix Fig S3.
Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling (2015)
Figure 3

Figure 3. Nox4 binds to GADD34 to mediate spatially-localized PP1 inhibition and enhance eIF2 phosphorylation.

A. Sub-cellular localization of Nox4. Tunicamycin (Tn 2 µg/ml, 6h) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy (scale bar, 10 µm).3D SIM images (scale bars, 2 µm) showed localisation of Nox4 (green) to the ER, which was labeled with an anti-KDEL antibody (red). At higher magnification (right), yellow dots denote co-localization of Nox4 and KDEL signals. Cell nuclei were stained with DAPI (blue). 1 Z slice from 3D stack is shown.

B. Progressive enrichment of GADD34, PP1, eIF2α and Nox4 in membrane fractions of tunicamycin-treated H9c2 cells.

C. After sucrose gradient fractionation of lysates of tunicamycin-treated H9c2 cells, GADD34, PP1 and Nox4 co-eluted in fractions 12 and 13 (F12, F13).

D. Immunoprecipitation (IP) of pooled fractions 12/13 with an anti-GADD34 antibody revealed the presence of both PP1 and Nox4.

E. The association of Nox4 with GADD34 was validated in HEK293 cells co-transfected with Nox4 and either Flag-tagged or non-tagged GADD34, followed by IP with an anti-Flag antibody.

F. Co-transfection of HEK293 cells with GADD34-Flag and different myc-tagged Nox4 constructs, followed by IP with an anti-myc antibody. GADD34 binds to full-length Nox4 (FL) and the Nox4 transmembrane domain (TD) but not the C-terminal domain (CD).

G. Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto, respectively, in tunicamycin-treated MEF cells. Redox-insensitive mutant probes were used as negative controls and to exclude pH changes. Extracellular H2O2 (200 nM) was added as a positive control. The pseudocolor scale is shown along the left vertical edge of each image. KO = Nox4-/-. Scale bars, 2 µm.

H. Transfection of HEK293 cells with PP1 and GADD34 increased PP1 activity (bar graph) and decreased phospho-eIF2 levels (immunoblots). Co-transfection of full-length Nox4 reduced PP1 activity and increased phospho-eIF2 levels (captions at bottom refer both to the bar graphs and immunoblots). These effects were abrogated when either Nox4 P437H or the Nox4 transmembrane domain (TD) were transfected. Nox4 did not affect phosphorylation of glycogen synthase (GS-P) or histone H3 (H3-P). All experiments were n=3/group. Values below the immunoblots are mean ± SEM levels for phospho-eIF2/total-eIF2. *, p<0.05 comparing third and fourth lanes; Levels of significance for comparisons of PP1 activities are shown above the bar columns. See also Fig EV1 and Appendix Fig S4.
Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling (2015)
Figure 4

Figure 4. Redox inhibition of PP1 involves metal center oxidation.

A. Recombinant PP1 was inhibited by H2O2 (0.2 mM) and activity was not restored by glutathione (GSH), cysteine (Cys) or dithiothreitol (DTT). A Cys127Ser/Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild-type PP1. Values above bars denote level of significance for the inhibitory effect of H2O2.

B. Ascorbate (Asc) dose-dependently restored PP1 activity. #, significant effect of Asc compared to H2O2 alone.

C. EPR spectra of PP1 incubated with ascorbate (1 mM) alone (a) or PP1 exposed to H2O2 followed by catalase treatment, then incubation with ascorbate (b). Panel b shows a typical spectrum for the ascorbyl radical (hyperfine splitting constant, aH = 1.8G), similar to the positive control obtained by exposing ascorbate to H2O2 (c). Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1, prior to ascorbate addition.

D. Cartoon representation of the active site of H2O2-treated PP1 as in Fig EV2D. 2mFo-DFc electron density map at the 2.2 Å resolution is shown in yellow at the 1.1 level. H2O2-treatment causes an overall shrinkage of the PP1 coordination sphere by 0.12 Å compared to ascorbate-treated crystals consistent with the oxidation of the dinuclear center. This increases the energy barrier for the catalytic steps involving -OH- attack on the phosphorous center of the bridging phosphate and rupture of the P-O scissile bond with the assistance of H125 (black arrows). Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit. See also Appendix Table S1.

E. Effect of Nox4 on GADD34/PP1-mediated eIF2 dephosphorylation in transfected HEK293 cells. Nox4 increased eIF2 phosphorylation in cells transfected with GADD34 and WT PP1, and resulted in even higher phospho-eIF2α levels in cells transfected with N124D or D64N PP1 variants.

F. Effect of ascorbate (Asc, 0.5 mM) on phosphatase inhibition in tunicamycin-treated H9c2 cells with overexpression or knockdown of Nox4 (Ad.Nox4 and Ad.shNox4, respectively). In control cells, ascorbate enhanced tunicamycin-stimulated increases in phosphatase activity. Phosphatase activity was lower in Nox4-overexpressing than control cells but was normalized by ascorbate to the same level as in control cells. In Nox4 knockdown cells, tunicamycin-induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect.

All experiments are n=3/group. Values above bar graphs denote the level of significance. See also Appendix Fig EV2.
Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling (2015)
Figure 5

Figure 5. Nox4 enhances cell survival and protects hearts against I/R injury through increase in eIF2α phosphorylation.

A. H9c2 cells treated with tunicamycin (2 µg/ml, 12h) showed significantly lower survival when endogenous Nox4 was silenced (siNox4) as compared to cells treated with a scrambled siRNA (siCtl). Cell survival was restored by treatment with either guanabenz (Gbz, 5 µM) or salubrinal (Sal, 50 µM) but was unaffected by clonidine (Cld, 5 µM). n=3/group.

B. Nox4-depleted cells had lower levels of phospho-eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz (Gbz).

C. Schematic representation of the effect of Nox4 and the small molecule inhibitors, guanabenz and salubrinal, on the GADD34/PP1/eIF2 interaction.

D. Hearts from Nox4 knockout (KO) mice and WT controls were subjected to global ischemia followed by aerobic reperfusion (I/R). Infarct size assessed by triphenyltetrazolium chloride (TTC) staining was greater in Nox4 KO hearts compared to WT and was significantly reduced by guanabenz (Gbz). In the representative heart sections shown at the top, white denotes infarct area and red the viable area. Scale bars, 1 mm. Numbers of hearts are indicated within the bars.

E. Immunoblotting of heart homogenates after I/R showed lower levels of phospho-eIF2α, ATF4 and ER chaperones, and higher levels of cleaved caspase-12, in Nox4 KO compared to WT. Tubulin was used as a loading control. Treatment with guanabenz (Gbz) reversed these changes (blots shown to the right).

Values above bar graphs denote the level of significance. See also Appendix Fig S5.
Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling (2015)
Figure 6

Figure 6. Nox4 protects against acute kidney injury in vivo.

A. Plasma urea levels were elevated to a greater extent in tunicamycin-treated Nox4 KO mice than WT. Co-treatment with guanabenz (Gbz) reduced urea levels in both groups. Numbers of animals are indicated within bars.

B. 48 hours after systemic tunicamycin treatment, kidneys of Nox4 KO mice showed a marked surface pallor (bottom right).

C. TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin-treated Nox4 KO mice. n=4/group.

D. Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase-12 and cleaved PARP levels in tunicamycin-treated Nox4 KO mice compared to WT.

E. Survival curves showed that a very high proportion of Nox4 KO mice died after AKI. Guanabenz (Gbz) treatment dramatically improved survival in tunicamycin-treated KO mice. Number of animals as indicated. Levels of significance by Kaplan Meier analysis are reported to the right.

F. Schematic depicting the effect of Nox4-generated ROS on PP1 activity and the balance between eIF2α phosphorylation and dephosphorylation. Nox4 is upregulated by ATF4 and binds to GADD34. It inhibits GADD34-bound PP1 through the local generation of H2O2 and oxidation of the metal (M) center of the serine-threonine phosphatase. The consequent prolongation of eIF2α phosphorylation promotes cell survival in the face of acute protein unfolding stress. M = iron or manganese, which are oxidized from the M (II) to the M (III) species.

Values above bar graphs denote the level of significance.
Network organization of the human autophagy system (2010)
figf5

a, GFP–MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells. Scale bar, 20 &mgr;m. b, Integrated spot signal per cell (ISSC) for 344 siRNAs targeting 86 genes (n = 4). Control siRNA (siCK) is indicated by black vertical bars. c, Normalized ISSC (N-ISSC) for GFP–MAP1LC3B in U2OS cells with or without rapamycin (6 h) (4 siRNAs per gene with each bar representing one of four siRNAs). Unless noted otherwise, P  0.01 using Students t-test; *, P  0.05; white rectangles, P > 0.05. d, Anti-LC3 blots of U2OS cell extracts after depletion of the indicated proteins in the presence or absence of BafA1 (100 nM, 3 h), and re-probed with anti-PCNA. LC3-I and LC3-II represent unconjugated and PE-conjugated MAP1LC3 proteins, respectively. e, Normalized mean fluorescence intensity (MFI) of GFP–MAP1LC3B cells after depletion with the indicated siRNAs in the presence or absence of BafA1. Error bars indicate standard deviation; n = 2.
Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth (2018)
Figure 2

Figure 2. Mitochondrial Akt phosphorylates MICU1 at the Ser124 position Sequence alignment of the MICU1 protein from nine vertebrate species. The Akt consensus phosphorylation motif, R-X-R-X-X-S/T, is marked in yellow. HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm. Analysis of the number of cells, expressed as a percentage, showing obvious mitochondrial staining of activated (S473 phosphorylated) Akt (p-Akt) (n=3 independent experiments; 350-355 cells). Means ± SEMs. ****p<0.0001 (Student&quot;s t-test). Hek293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into Cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate. Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate. Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localizations. Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture. OMM: outer mitochondrial membrane; IMS: intermembrane space. Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of vehicle- or rapamycin (Rapa., 1 μM for 4 h)-treated Hek293T cell mitochondria following carbonate extraction at pH 11.5. Akt, phosphorylated (S473) Akt (p-Akt), MICU1, the established integral membrane proteins TIM23 and MCU, and the soluble protein cytochrome c (Cyt. c) was analyzed. Hek293T cells were transfected with the GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). GFP-MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphorylated (S473) Akt (p-Akt) antibodies by western blotting. Hek293T cells were transfected with either the GFP-tagged wild-type (WT) MICU1 or MICU1 S124A-GFP mutant. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting.
Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth (2018)
Figure 3

Figure 3. Mitochondrial Akt augments the basal Ca2+ levels in a MICU1-dependent manner Mitochondrial preparations of both Hek293T (left) and HeLa (right) cells transiently transfected with an HA-tagged constitutively active Akt T308D/S473D (Akt D/D) construct were analyzed by western blotting. GAPDH: cytosolic marker; TIM23: mitochondrial marker. Resting mitochondrial calcium levels in the control (ctrl) and Akt D/D-expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 44-49 cells). Schematic model of the HA-tagged constitutively active Akt form targeted to the mitochondrial intermembrane space (mt-Akt D/D). Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence. TOM20 was used as a mitochondrial marker. Merged images are indicated (merge). Scale bar 10 μm. Hek293T cells were transfected with the indicated constructs. The GFP-tagged MICU1 was immunoprecipitated from the whole-cell lysate, and the precipitated proteins were immunoblotted with the HA and phosphorylated (S473) Akt (p-Akt) antibodies. Hek293T cells were transfected with the indicated constructs. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting. Resting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells). Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m (n=3 independent experiments; 38-50 cells). (H and I) Representative kinetics (H) and analysis (I) of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [Ca2+] (n=3 independent experiments). Data information: (B, F) Means ± SEMs. ****p<0.0001 (Student&quot;s t-test); (G, I) Means ± SEMs. ****p<0.0001 (one-way ANOVA).
Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth (2018)
Figure 4

Figure 4. Akt-mediated phosphorylation of MICU1 affects protein maturation and stability Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. pre-MICU1: precursor form of MICU1; m-MICU1: mature form of MICU1. Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. Where indicated, the cells were treated with 5 μM MG132 for 4 h. pre: precursor; int: intermediate; m: mature form. Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs, following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. The cells were treated with 5 μM MG132 for 4 h before fractionation. Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionation. ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1), both WT and mutants, is represented relative to the amount at time 0. (n=3 independent experiments). ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were harvested, and total protein was extracted and subjected to western blotting analysis with the indicated antibodies. Immunoblotting was performed under reducing (with the presence of DTT) or non-reducing (non-red) conditions (without DTT). Cells were treated with 5 μM MG132 for 4 h before lysis. Quantification of HA levels in non-reducing condition showed in (E). (n=3 independent experiments). Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells). Data information: (E, G) Means ± SDs. **p<0.01; ***p<0.001; ****p<0.0001 (one-way ANOVA); (H) Means ± SEMs. **p<0.01; ***p<0.001 (one-way ANOVA). >
Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth (2018)
Figure 5

Figure 5: IMMP1L-silencing alters MICU1 processing and basal [Ca2+]m levels Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis. Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria transfected with HA-tagged MICU1 WT following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. pre: precursor; int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis. Control or IMMP1L-silenced HeLa cells were transfected with HA-tagged MICU1 WT. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1) is represented relative to the amount at time 0. (n=3 independent experiments). (F and G) Representative kinetics (F) and analysis (G) of aequorin-based [Ca2+]m measurements in control or IMMP1L-silenced HeLa cells challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments). (H) Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells). Data information: (E) Means ± SDs. *p<0.05; **p<0.01; ***p<0.001 (one-way ANOVA); (G, H) Means ± SEMs. **p<0.01; ****p<0.0001 (Student&quot;s t-test).