EMBO/soda-nel huggingface dataset:
L-arginine homeostasis governs adult neural stem cell activation by modulating energy metabolism in vivo (2023)
Figure 3

Figure 3. Arg-II deficiency leads to exhaustion of RGLs with age.

(A) Experimental scheme for assessing Nestin-GFP+ RGLs in 6-month-old mice.

(B) Representative images of Nestin-GFP+GFAP+Mcm2+ RGLs in the DG. Scale bar = 50 μm.

(C) Quantification of GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.001) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice.

(D) Percentage of GFAP+Nestin-GFP+Mcm2+ activated RGLs among total GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.0013) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice.

(E) Experimental scheme for assessing RGLs after a 5-month post-tamoxifen induction in 2 month-old mice.

(F) Quantification of tdT+GFAP+ RGLs (WT vs. cKO, Student's t-test, p = 0.037) in the DG of WT and cKO mice after 5-month of tamoxifen injection. WT, n = 3 mice; cKO, n = 3 mice.

(G) Percentage of tdT+GFAP+Mcm2+ activated RGLs among total tdT+GFAP+ RGLs (WT vs. cKO, Student's t-test, p = 0.007) in the DG of WT and cKO mice after 5-month of tamoxifen injection. WT, n = 3 mice; cKO, n = 3 mice.

(H) Experimental scheme for assessing adult-born neurons by a 1-month BrdU pulse chase in 6 month-old mice.

(I) Representative images of NeuN+BrdU+ adult-born neurons in the DG of 6 month-old mice. Scale bar = 50 μm.

(J) Quantification of NeuN+BrdU+ adult-born neurons (WT vs. KO, Student's t-test, p = 0.0052) in the DG of WT and KO mice at 7 months of age. WT, n = 4 mice; KO, n = 3 mice.

Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.
L-arginine homeostasis governs adult neural stem cell activation by modulating energy metabolism in vivo (2023)
Figure 5

Figure 5. Elevated L-arginine levels impair the attachment of HK1 onto mitochondria.

(A) DARTS analysis indicated HK1 as a molecular target of L-arginine in aNPCs. The aNPC lysates treated with L-arginine, digested with pronase and then analyzed by Western blot with anti-HK1 and GAPDH antibodies.

(B) Quantification of HK1 expression level in aNPCs lysates treated with L-arginine and followed by digesting with pronase (One-way ANOVA, F(3, 8)= 3.61, p = 0.043). n = 3 independent experiments.

(C and D) Surface plasmon resonance analysis for the binding of L-arginine to HK1 (C) and the dissociation constant (Kd) of L-arginine and HK1 interaction (D).

(E) Western blot analysis (left) and quantification (right, WT vs. KO, Student's t-test, p = 0.71) of HK1 expression level in WT- and KO-aNPCs. WT, n = 3 independent experiments, KO, n = 3 independent experiments.

(F) Quantification of HK activity after L-arginine treatment (One-way ANOVA, F(4, 10) = 0.11, p = 0.97. Each treatment, n = 3 independent experiments.

(G) Western blot analysis of HK1 expression in the cytoplasmic and mitochondrial fraction of WT- and KO-aNPCs.

(H) Quantification of HK1 expression level in the cytoplasmic and mitochondrial fraction of WT- and KO-aNPCs (cytoplasmic fraction, WT vs. KO, Student's t-test, p = 0.019; mitochondrial fraction, WT vs. KO, Student's t-test, p < 0.001). WT, n = 6 independent experiments; KO, n = 6 independent experiments.

(I) Co-IP of HK1 with VDAC1 in vehicle or L-arginine-treated cells.

(J) Quantification of the co-IP efficacy of HK1 and VDAC interaction in vehicle or L-arginine-treated cells (IP: HK1, vehicle vs. L-arginine, Student's t-test, p < 0.001; IP: VDAC1, vehicle vs. L-arginine, Student's t-test, p = 0.0078). Vehicle, n = 3 independent experiments; L-arginine, n = 3 independent experiments.

(K) Schematic illustration of interaction of HK1 and VDAC1 by proximity ligation assay.

(L) Representative images of proximity ligation assay in aNPCs. Scale bar, 10 μm.

(M) Quantification of total fluorescent dots of HK1 and VDAC1 interaction (WT vs. KO, Student's t-test, p < 0.001) in WT- and KO-aNPCs. WT, n = 83 cells from 3 independent experiments; KO, n = 96 cells from 3 independent experiments.

Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.
L-arginine homeostasis governs adult neural stem cell activation by modulating energy metabolism in vivo (2023)
Figure 7

Figure 7. Selective inhibition of OXPHOS rescues RGL overactivation caused by Arg-II deficiency.

(A) Experimental scheme for assessing Nestin-GFP+ RGLs and their progeny by 2-hour BrdU pulse chase in P60 mice after IACS treatment.

(B) Representative images of Nestin-GFP+GFAP+BrdU2+ RGLs in the DG. Scale bar = 20 μm.

(C) Quantification of Nestin-GFP+GFAP+ RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.99). WT + Vehicle, n = 4 mice; WT + IACS, n = 4 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 3 mice.

(D) Quantification of Nestin-GFP+GFAP+BrdU+ activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice.

(E) Quantification of Nestin-GFP+GFAP-BrdU+ progenitors (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.028) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice.

(F) Quantification of Nestin-GFP+Tbr2+BrdU+ IPCs (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.02) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 4 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 4 mice.

(G) Experimental scheme for assessing adult-born neurons by a 30-day BrdU pulse chase in P60 mice after IACS treatment.

(H) Representative images of NeuN+BrdU+ adult-born neurons in the DG. Scale bar = 20 μm.

(I) Quantification of NeuN+BrdU+ adult-born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 5 mice; WT + IACS, n = 5 mice; KO + Vehicle, n = 5 mice, KO + IACS, n = 5 mice.

Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.
Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF (2020)
Figure 6

Figure 6 | Abnormal cortical structures in offspring brains derived from aged fathers in the early postnatal period.

A. Representative images of the cerebral cortex of YFO (left) and AFO (right) at postnatal day 6. Scale bar: 1 mm.

B. Cortical thickness in the postnatal brain of YFO (n=4, from different mothers) and AFO (n=5, from different mothers).

C. Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Satb2+/Ctip2-: layer 2-4, Ctip2high: layer 5, Satb2-/Ctip2-: layer 6. Scale bar: 100 μm.

D. Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Tbr1-/Ctip2-: layer 2-4, Ctip2high: layer 5, Tbr1+/Ctip2-: layer 6. Scale bar: 100 μm.

E. Layer thickness (mm) in each cortical layer in the postnatal brain of YFO (n=4, from different mothers) and AFO (n=5, from different mothers).

F. Cell number in each cortical layer in the postnatal brain of YFO and AFO.

G. Cell density in each cortical layer in the postnatal brain of YFO and AFO.

Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.
Loss of PINK1 impairs stress-induced autophagy and cell survival. (2014)
Figure 1

Stable PINK1 knockdown in SH-SY5Y cells.

A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005. B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005. C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.
Loss of PINK1 impairs stress-induced autophagy and cell survival. (2014)
Figure 2

Reduced autophagy after PINK1 knockdown.

2A) SH-SY5Y cells were starved for 2 h in HBSS with Bafilomycin (+Baf) or left untreated in RPMI+5% FCS (- Baf). The LC3-II and actin content were determined by western blotting. A representative blot is shown on the right, showing only LC3-II bands in the Bafilomycin-treated samples. The LC3-II bands of Bafilomycin treated samples were normalized to actin and the relative LC3-II content of nt cells was set as 1. Cells with stable PINK1 knockdown exhibited a reduced LC3-II/actin ratio compared to the control (nt) cells; n = 4, p<0.005. 2B) Cortical neurons from 3 different isolations (10-20 DIV) of WT and PINK1 KO mice were either non-starved or starved for 2 h in HBSS and the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of WT and PINK1 KO mice, respectively, was set as 1. Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3-II/actin ratio compared to the WT cells; n = 5, p<0.001. 2C) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA. After 48 h the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with scrambled siRNA was set as 1. Transient PINK1 knockdown resulted in a reduced LC3-II/actin ratio compared to the cells transfected with scrambled siRNA; n = 6; p<0.005. 2D) HeLa cells were transiently transfected with PINK1-GFP, PINK1G309D-GFP or GFP. After 24 h cells were starved for 2 h in HBSS. Afterwards the LC3-II and actin content were determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with GFP was set as 1. Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3-II/actin ratio compared to cells transfected with GFP; n = 3, PINK1: p<0.01; PINK1G309D: p<0.05.
Loss of PINK1 impairs stress-induced autophagy and cell survival. (2014)
Figure 3

Reduced LAMP-2 expression after PINK1 knockdown.

3A) Regular, non-transduced SH-SY5Y cells were cultivated either in RPMI+10% FCS or starved in HBSS medium for 24 and 40 h. The LAMP-2 and actin content were determined by western blotting and the relative LAMP-2 content of untreated cells was set as 1. Starvation enhanced LAMP-2 protein levels; n = 3, p<0.05. 3B) Transduced nt and PINK1 knockdown (kd) SH-SY5Y cells were cultivated either in RPMI+5% FCS or starved for 24 h and 40 h in HBSS. The LAMP-2 and actin content were determined by western blotting. A representative blot after 40 h starvation in HBSS is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP-2/actin ratio compared to the nt cells; n = 4, p<0.05. 3C) nt and PINK1 knockdown (kd) SH-SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp-2. Micrographs were taken with constant microscopical settings. LAMP-2 positive signals in each cell were quantified with ImageJ as described in Material and Methods. Knockdown of PINK1 resulted in a decreased amount of LAMP-2 positive stainings/cell after starvation; n = 2, at least 8 fields of view/condition, 89–124 cells/condition; 24 h: p<0.001; 40 h: p<0.05. 3D) nt and PINK1 knockdown (kd) SH-SY5Y cells were stained with the photo-reactive dye MTR and either irradiated for 45 min or left untreated. 24 h and 48 h after irradiation the LAMP-2 and actin content were determined by western blotting. A representative blot 48 h after irradiation is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp-2/actin ratio compared to the control (nt) cells; n = 5, p<0.001. 3E) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown (kd) SH-SY5Y cells cultivated in RPMI medium with 5% FCS. The phosphatase activity was measured and normalized to the protein content. The phosphatase activity of nt cells was set as 1. PINK1 knockdown mediated a reduction of lysosomal activity; n = 3; p<0.05.
Loss of PINK1 impairs stress-induced autophagy and cell survival. (2014)
Figure 4

Reduced PINK1 levels influence cell metabolism and population growth.

4A) Oxygen consumption rate (OCR) was measured in nt and PINK1 knockdown (kd) SH-SY5Y cells without (nt) or with PINK1 knockdown (kd) cultivated in RPMI medium with 5% FCS. Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd, no significant changes were observed; n = 1 (quadruplicates). 4B) Energy charge (EC) of nt and PINK1 knockdown (kd) SH-SY5Y cells grown in RPMI+5% FCS was determined as detailed in Material & Methods. PINK1 knockdown resulted in a lower EC compared to nt cells; n = 5, p<0.05. 4C) nt and PINK1 knockdown (kd) SH-SY5Y cells were grown for 2 months in RPMI+5% FCS and their population doublings were determined. Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth; n = 1 (triplicates); * p<0.05 (day 12) - p<1×10−6 (day 61). 4D) SH-SY5Y cells were grown in RPMI+5% FCS and the amount of nt and PINK1 knockdown (kd) cells in S-phase was determined by BrdU incorporation. No differences in cell proliferation were visible between cells without (nt) and with PINK1 kd; n = 3.
Loss of PINK1 impairs stress-induced autophagy and cell survival. (2014)
Figure 5

Increased apoptosis in cells with PINK1 knockdown.

5A) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS or starved with HBSS for 12 h. DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to the protein content. Cells with PINK1 knockdown demonstrated a significantly increased caspase-3 activity under both conditions; n = 4; RPMI+5% FCS: p<0.05; HBSS: p<0.01. 5B) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing. 5C) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH. Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation; n = 4; 24 h: p<0.005. 5D) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP-Parkin or PINK1-GFP or PINK1G309D-GFP. After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined. The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1. Starvation-mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D-GFP but not by GFP or Parkin; GFP: n = 5, p<0.005; Parkin: n = 3, p<0.05; PINK1: n = 5, ns; PINK1G309D-GFP: n = 3, ns. 5E) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA. After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to 1,000,000 cells. PINK1 knockdown increased caspase-3 activity significantly, which was prevented by LC3 overexpression; n = 3; p<0.05. 5F) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA. 48 h post transfection cells were either left untreated (+ medium) or starved (+HBSS) for additional 24 h and afterwards the amount of adherent cells was determined. The amount of non-starved cells was set as 1. PINK1 knockdown resulted in elevated cell loss, which was prevented by LC3 overexpression; HeLa: n = 6, p<0.05; HeLa LC3: n = 5.
Parkin-mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes (2019)
Figure 1

Figure 1.

MITOL redistributes to peroxisomes in response to a loss in the mitochondrial membrane potential.

(A-C) MITOL-HA formed small dot-like structures following CCCP treatment. HeLa cells transiently expressing Flag-Parkin and MITOL-HA were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry. MITOL and Tom20 (mitochondrial marker; A) signals co-localized well without CCCP treatment, whereas the MITOL-positive small dots were not coincident with Tom20, Sec61β (ER marker; B), or LAMP1 (lysosomal marker; C) after CCCP treatment. Scale bars, 10 µm.

(D) MITOL-HA co-localized with catalase (peroxisome marker) following CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-HA co-localization with catalase.

(E) Correlation statistics for the localization of MITOL-HA and Tom20, Sec61β, LAMP1, or catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.

(F) The line-graph shows a line scan of fluorescence through three MITOL-positive peroxisomes (red bar in Fig 1D) that clearly indicates co-localization of MITOL (green line) and catalase (magenta line).

(G) Peroxisomal membrane protein (PMP) 34-FusionRed also co-localized with MITOL-GFP in Parkin-expressing HeLa cells after 3 hours of CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.

(H) Correlation statistics for the localization of MITOL-GFP and PMP34-FusionRed. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.
Parkin-mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes (2019)
Figure 2

Figure 2.

Parkin is required for MITOL redistribution to peroxisomes.

(A) MITOL-HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild-type HeLa cells or HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.

(B) Correlation statistics for the localization of MITOL-HA and Tom20 in the absence or presence of GFP-Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.

(C) MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag-MITOL were transfected with a GFP-Parkin plasmid or the pEGFP-C1 vector, treated with 15 µM CCCP +/- 10 µM MG132 for 3 hours, and then immunoblotted with anti-Flag, anti-ubiquitin, and anti-tubulin antibodies. Black arrowheads indicate 3Flag-MITOL in the upper panel and mono-ubiquitin in the middle panel, respectively.

(D) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that Mitofusin2 (MFN2) underwent rapid degradation within 3 hours of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria-rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 hours 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor ZVAD-FMK (10 μM) was added to cells along with valinomycin to prevent cell death.

(E) Quantification of 3Flag-MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 µM valinomycin + ZVAD-FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m relative to untreated samples in three biological replicates.

(F) Pre-existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 hours to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 hours, cells were immunostained using anti-Flag, anti-Pex14 (peroxisomal membrane protein), and anti-Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.