EMBO/soda-nel huggingface dataset:
Autoimmunity Gene IRGM Suppresses cGAS-STING and RIG-I-MAVS Signaling to control Interferon Response (2020)
Figure 7

Figure 7. Both cGAS-STING and RIG-I/MDA5-MAVS pathways contribute to enduring type 1 IFN response via JAK/STAT signaling in IRGM-depleted cells.

(A) qRT-PCR analysis with total RNA isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG-I siRNA or cGAS siRNA and RIG-I siRNA as indicated. Mean ± SD, n = 3 (biological replicates), *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.

(B) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG-I siRNA or transfected with three siRNAs as indicated. Mean ± SD, n = 3 (biological replicates), *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.

(C) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated. Mean ± SD, n = 3 (biological replicates), *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.

(D) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated. Mean ± SD, n = 3 (biological replicates), *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.

(E) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated. Mean ± SD, n = 3 (biological replicates), *p < 0.05, ***p < 0.0005, Student's unpaired t-test.

(F) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated. Mean ± SD, n = 3 (biological replicates), *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test

Expanded View Figure Legends
Biogenic Amine Neurotransmitters Promote Eicosanoid Production And Protein Homeostasis (2020)
Figure 5

Figure 5. The cytochrome P450 CYP-34A4 promotes UPS activity

A Schematic of the cyp-34A4 gene. Large boxes indicate coding sequences. Small boxes indicate UTRs. The location and nature of individual mutations is shown. Sequence deleted in od107 is shown with a horizontal black bar.

B Relative abundance of mRNA for the cyp-34A4 gene. Values are based on qRT-PCR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ***P<0.001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM.

C Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. ****P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.

D Quantified fluorescence of UbG76V-GFP normalized to mRFP, as in (C). ****P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.

E Expression of the cyp-34A4::gfp transgene. Scale bar, 100 microns.

F Expression of the cyp-34A4::gfp transgene (green). Intestinal autofluorescence (red) can be distinguished. Scale bar, 100 microns.
Biogenic Amine Neurotransmitters Promote Eicosanoid Production And Protein Homeostasis (2020)
Figure 6

Figure 6. PUFAs modulate UPS activity

A Schematic representation of the PUFA and eicosanoid synthesis pathways in C. elegans. Carbon chain length, double bond number, and double bond location along the chain are indicated for each PUFA. The gene encoding each biosynthesis enzyme in the pathway is indicated next to the arrow representing its catalyzed reaction.

B,C Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at the indicated time point. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.

D Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from lysates of the indicated genotype and time point. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=3 trials. Error bars indicate SEM.

E Quantified fluorescence as in (B) for animals of the indicated genotype at the indicated time point. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.

F Quantified fluorescence as in (B) for animals exposed to the indicated RNAi vector or empty vector (EV) control, and in either a wild type (WT) or fat-1(wa9) genetic background. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.

G Quantified fluorescence as in (B) for animals of the indicated genotype at the indicated time point. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.
Osmotic stress-induced phosphorylation by NLK at Ser 128 activates YAP (2016)
Figure 1

Figure 1. Osmotic stress induces YAP phosphorylation and LATS activation but does not inhibit YAP

A. Sorbitol stimulates YAP phosphorylation. HEK293A cells were cultured in the presence or absence of serum, and treated with 0.2 M sorbitol for the indicated time points. Phos-tag gel was used to assess total YAP phosphorylation based on mobility shift. Note that YAP and TAZ are recognized by the same antibody. l.e. denotes long exposure of YAP and TAZ.

B. Osmotic stress induces LATS phosphorylation. HEK293A cells were treated with 0.2 M sorbitol for 30 or 60 minutes in the presence or absence of serum. LATS phosphorylation at the hydrophobic motif (HM) was detected with the phospho-specific pLATS antibody.

C. Osmotic stress increases LATS kinase activity. HEK293A cells were treated with 0.2 M sorbitol for 30 minutes. Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP was determined by immunoblotting with phospho-YAP (S127) antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).

D. Osmotic stress does not lead to YAP cytoplasmic localization. HEK293A cells were treated with 0.2 M sorbitol for 1 hr in the presence of serum. YAP subcellular localization was determined by immunofluorescence staining (red). DAPI (blue) was used to stain for DNA. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields, each with approximately 100 cells.

E. Osmotic stress does not reduce YAP target gene expression. HEK293A cells were treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. n.s means p > 0.05 (two tailed student's t-test, n = 3).

F. Osmotic stress does not disrupt the interaction between YAP and TEAD1. HEK293A cells were treated with 0.2 M sorbitol for 1 hr. Endogenous YAP was immunoprecipitated with YAP antibody, and TEAD1 and YAP were detected by Western blot. Cells were serum starved for 1 hr as indicated (- serum).

G. Osmotic stress does not increase the interaction between YAP and 14-3-3. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the co-precipitated Flag-YAP was detected.
Osmotic stress-induced phosphorylation by NLK at Ser 128 activates YAP (2016)
Figure 2

Figure 2. Osmotic stress induces YAP nuclear translocation and activation

A. Osmotic stress induces YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. YAP/TAZ (red, upper panels) or YAP (red, lower panels) were stained with two specific antibodies. DAPI (blue) was used to visualize cell nuclei. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields.

B. Osmotic stress induces TAZ nuclear translocation. YAP knockout (KO) HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. TAZ localization was determined by immunofluorescencece staining with the YAP/TAZ antibody (red). Scale bar: 20 μm.

C. Osmotic stress induces a transient YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for the indicated time points.

D. Osmotic stress induces YAP target gene expression. Wild type (WT) or YAP/TAZ double knockout (Y/T KO) HEK293A cells were serum starved for 1 hr and treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).

E. Osmotic stress decreases YAP and 14-3-3 interaction. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, and were serum starved for 16 hr. Cells were then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the associated Flag-YAP was detected by Western. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).
Osmotic stress-induced phosphorylation by NLK at Ser 128 activates YAP (2016)
Figure 3

Figure 3. NLK mediates osmotic stress signal to induce YAP nuclear localization

A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Quantification of YAP/TAZ subcellular staining is shown. Scale bar: 20 μm. Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK-2 and JNK substrate c-Jun.

B. NLK knockout blocks osmotic stress-induced YAP nuclear localization. HEK293A cells were transiently transfected with CRISPR/Cas9 to knockout NLK. Wild type (WT) cells and the NLK KO cell pool were treated with 0.2 M sorbitol for 1 hr in the absence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining. Scale bar: 20 μm.

C. NLK induces YAP nuclear translocation. HEK293A cells were co-transfected with Flag-YAP together with vector control, Myc-NLK-WT (wild type NLK), or NLK-KN (kinase negative mutant). Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.

D. NLK induces YAP phosphorylation. HA-YAP was co-transfected with NLK-WT or NLK-KN in HEK293A cells. The phos-tag gel showed NLK-WT but not NLK-KN caused a significant mobility shift of YAP.

E. NLK phosphorylates YAP in vitro. NLK-WT and NLK-KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate in the presence of ATP-γ-S. Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody, which identifies the alkylated thiophosphorylation on YAP.
Osmotic stress-induced phosphorylation by NLK at Ser 128 activates YAP (2016)
Figure 4

Figure 4. Osmotic stress induces YAP Ser 128 phosphorylation and inhibits its 14-3-3 binding

A. NLK induces YAP Ser 128 phosphorylation. Flag-YAP WT or S128A mutant was co-transfected with or without Myc-NLK. Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody.

B. NLK phosphorylates YAP Ser 128 in vitro. NLK-WT or NLK-KN was transfected into HEK293A cells and immunoprecipitated. An in vitro NLK kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho-YAP (S128) antibody.

C. Osmotic stress induces YAP phosphorylation at Ser 128. In the left panel, HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated. In the right panel, Flag-YAP was transfected into HEK293A cells. Flag-YAP was immunoprecipitated, YAP Ser 128 phosphorylation was detected by a phosphospecific antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).

D. NLK deficiency reduces YAP S128 phosphorylation. Two CRISPR/CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A. WT cells and two pools of NLK CRISPR/CAS9 transfected cells were treated with 0.2 M sorbitol for the indicated time points. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 4).

E. The S128D phosphomimetic mutant abolishes YAP interaction with 14-3-3. Flag-YAP WT and mutant constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs. 14-3-3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody.

F. YAP Ser 128 phosphorylation is required for disruption of YAP-14-3-3 interaction by osmotic stress. Flag-YAP WT or Flag-YAP S128A constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs, and treated with 0.2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr. Refreshing medium served as a control to disrupt YAP and 14-3-3 binding.14-3-3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).
Osmotic stress-induced phosphorylation by NLK at Ser 128 activates YAP (2016)
Figure 5

Figure 5. YAP Ser 128 phosphorylation affects YAP subcellular localization.

A. NLK does not induce nuclear translocation of the S128A mutant YAP. HEK293A cells were co-transfected with Myc-NLK and Flag-YAP WT or S128A mutant. Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.

B. YAP Ser 128 phosphorylation is required for osmotic stress-induced YAP nuclear localization. HEK293A YAP KO cells reconstituted with Flag-YAP WT, YAP S128D, or YAP S128A mutant were treated with 0.2 M sorbitol for 1 hr. YAP subcellular localization was determined by Flag immunofluorescence staining (green). Scale bar: 20 μm.
Osmotic stress-induced phosphorylation by NLK at Ser 128 activates YAP (2016)
Figure 6

Figure 6. YAP Ser 128 phosphorylation protects cells from hyperosmotic stress

A. YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions. YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D were cultured in the absence (left panel) or presence of 0.2M sorbitol (right panel) for the indicated amount of time. Cell numbers were counted and normalized to day 0. Data are presented as mean ± SEM, n = 3.

B. YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment. Cell cycle analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. Propidium iodine (PI) was used for DNA staining (Fig EV5). Quantification of sub-G1 cells is shown. Data are presented as mean ± SEM. *p < 0.05 (two tailed student’s t-test, n = 3).

C. YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions. Annexin V analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. PE-Annexin V and 7-AAD stained for phospholipid phosphatidylserine (PS) and DNA, respectively. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).

D. A proposed model for osmotic stress regulation of YAP via NLK Kinase. See Discussion for details.
Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. (2015)
Figure 1

Podocyte-specific Phb2 knockout mice develop albuminuria and die prematurely

A Measurement of urinary albumin/creatinine by ELISA (albumin-to-creatinine ratio: day 14: Phb2fl/fl 0.09 ± 0.03 mg/mg, n = 4, versus Phb2pko 0.37 ± 0.21 mg/mg, n = 4, P = 0.2350; day 21: Phb2fl/fl 0.65 ± 0.16 mg/mg, n = 4, versus Phb2pko 193.10 ± 26.81 mg/mg, n = 4, ***P = 0.003; day 28: Phb2fl/fl 0.57 ± 0.07 mg/mg, n = 4, versus Phb2pko 470.40 ± 131.30 mg/mg, n = 4, *P = 0.0117).

B Appearance of Phb2fl/fl and Phb2pko mice at day 28.

C Analysis of body weight from postnatal day 14 until day 32 (P-values for Phb2fl/fl or Phb2het versus Phb2pko: *P = 0.0372 at day 27, *P = 0.0311 at day 28, *P = 0.0157 at day 29, **P = 0.008 at day 30, **P = 0.0013 at day 31, **P = 0.0035 at day 32, n = 3 for Phb2fl/fl and n = 4 for Phb2het and Phb2pko).

D Measurement of serum creatinine levels of mice in their fifth week of life (Phb2fl/fl/het 14.74 ± 3.75 μmol/l versus Phb2pko 74.73 ± 2.20 μmol/l, n = 3 for both groups; ***P = 0.0002).

E Measurement of serum urea levels of mice in their fifth week of life (Phb2fl/fl/het 44.00 ± 7.02 mg/dl versus Phb2pko 493.70 ± 45.34 mg/dl, n = 3 for both groups; ***P = 0.006).

F Kaplan–Meier survival curve (n = 5 for Phb2fl/fl/het and n = 6 for Phb2pko).

Data information: Results are presented as means ± SEM. (A, C–E) Student's t-test.
Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. (2015)
Figure 2

Phb2pko mice develop glomerulosclerosis

A PAS staining of kidney sections (scale bar: 20 μm).

B Immunohistochemistry for podocin on kidney sections (scale bar: 20 μm).

C Analysis of podocyte foot processes in electron micrographs (scale bar: 0.7 μm). FP, podocyte foot processes; GBM, glomerular basement membrane; EC, endothelial cells.

D Analysis of mitochondrial morphology in electron micrographs (arrows; scale bar: 0.3 μm).

E High-power view of mitochondria in electron micrographs (scale bar: 0.1 μm).

Data information: Genotypes and time points as indicated in the figure.
Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. (2015)
Figure 3

The phenotype of Phb2pko mice is not developmental

A PAS staining of tPod–Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).

B Immunohistochemistry for podocin on kidney sections of tPod–Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).

C Coomassie stain of urinary samples of tPod–Phb2pko and control mice.
Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. (2015)
Figure 4

Podocyte-specific knockout of the insulin receptor and IGF-1 receptor prolongs survival of Phb2pko mice

A Kaplan–Meier survival curve showed that an additional knockout of the insulin receptor (Insr) prolonged survival of Phb2pko mice (n = 7 for Phb2pko/Insrpko, n = 4 for Phb2pko/Insrhet, and n = 6 for Phb2pko).

B Kaplan–Meier survival curve revealed that the survival time of Phb2pko mice is not changed by an additional Igf1r deficiency (n = 6 for Phb2pko/Igf1rpko, n = 4 for Phb2pko/Igf1rhet, and n = 6 for Phb2pko).

C Statistical analysis comparing all genotypes with Phb2pko mice.

D Kaplan–Meier survival curve revealed prolonged survival of Phb2pko/Insrpko/Igf1rpko and Phb2pko/Insrpko/Igf1rhet mice (n = 19 for Phb2pko/Insrpko/Igf1rpko, n = 9 for Phb2pko/Insrhet/Igf1rpko, n = 11 for Phb2pko/Insrpko/Igf1rhet, n = 9 for Phb2pko/Insrhet/Igf1rhet, and n = 6 for Phb2pko).

E Statistical analysis comparing all genotypes with Phb2pko mice.
Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. (2015)
Figure 5

Loss of the insulin and IGF-1 receptor from Phb2pko mice does not improve mitochondrial ultrastructure

A Serum creatinine and urea levels in the fifth week of life (left graph: Phb2pko 74.73 ± 2.20 μmol/l versus Phb2pko/Insrpko/Igf1rpko 37.36 ± 8.83 μmol/l, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0169; right graph: Phb2pko 493.70 ± 45.34 mg/dl versus Phb2pko/Insrpko/Igf1rpko 272.80 ± 49.29 mg/dl, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0247).

B Abundance of deleted Phb2 gene in isolated glomeruli of Phb2fl/fl, Phb2het, Phb2pko and Phb2pko/Insrpko/Igf1rpko mice compared to the floxed Phb2 gene (left graph; quantified by qPCR: n = 3 for all groups, P = 0.83 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko) and compared to a reference gene (right graph; quantified by qPCR: n = 3 for all groups, P = 0.58 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko).

C Analysis of mitochondrial morphology in electron micrographs (left image: scale bar, 1 μm; right image: scale bar, 0.1 μm).

Data information: Results are presented as means ± SEM. Student's t-test.
Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. (2015)
Figure 6

Loss of PHB2 leads to changes in mitochondrial morphology

A Immunofluorescence staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2-deficient podocytes (scale bar: 10 μm).

B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in (A) (mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed).

C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).

D Morphometric image analysis showed a decrease in branch numbers in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).

E Morphometric image analysis detected a decrease in mitochondrial size in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).

F Measurements of mitochondrial respiration with complex II substrates (succinate and glycerol-3-phosphate) did not show significant differences between Phb2-deficient and control podocytes (n = 6).

G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2-deficient podocytes compared to control podocytes (n = 3).

H FACS analysis of MitoTracker- and MitoSOX-stained Phb2-deficient podocytes and control podocytes.

I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2-deficient podocytes compared to control podocytes (n = 3, P = 0.7782 for MitoTracker, P = 0.1088 for MitoSOX).

Data information: In (C–G, I), bars represent mean ± SEM. Student's t-test; ****P = 0.0001, ***P = 0.0002. In (C–E, G), average value for scrambled shRNA podocytes was leveled to 100%.