EMBO/soda-nel huggingface dataset:
Aneuploid senescent cells activate NF-κB to promote their immune clearance by NK cells (2021)
Figure 4

Figure 4. NF-κB pathway is activated in ArCK cells.

Significantly differentially expressed hallmarks in ArCK cells compared to euploid control cells are shown in the first column in the heatmap (FDR q value0.05). Normalized enrichment scores are plotted. The corresponding NES for these hallmarks in doxorubicin (Doxo), palbociclib (Palbo), nutlin3 (Nutlin) and torin1 (Torin) treated cells are also plotted. Hallmarks did not reach statistical significance (FDR q value0.05) are shown in grey.

Enrichment plots for "Interferon alpha response", "TNFalpha signaling via NFKB" and "Interferon gamma response" hallmark signatures in ArCK cells compared to the euploid control cells.

Significantly differentially expressed RELA and RELB target genes in ArCK cells compared to euploid control cells were identified by ingenuity pathway analysis based on RNA sequencing data (Log2 fold change, p value0.05).

RT-qPCR quantifying NF-κB downstream target gene expression in ArCK and euploid control (Ctrl) cells. n=6 biological replicates; mean± SEM. IL-1b, *p= 0.029; IL-6, *p= 0.032; IL-8, **p= 0.008; unpaired t-test.

Measurement of NF-κB activity with NF-κB alkaline phosphatase (SEAP) reporter assay. The reporter was expressed in RPE1-hTERT cells by transient transfection. 10 hours after transfection, cells were treated with either DMSO (Ctrl) or reversine (500 nM; Aneuploid senescent) for 96 hours and the secretion of alkaline phosphatase in the culture supernatant was measured. The secretion level was normalized to cell number for each condition. n=3 biological replicates; mean± SEM; unpaired t-test, ****p<0.0001.
Aneuploid senescent cells activate NF-κB to promote their immune clearance by NK cells (2021)
Figure 5

Figure 5. Both canonical and non-canonical NF-κB pathway are required for NK cell-mediated killing of ArCK cells

NK cell-mediated killing of RELA ArCK cells was compared to clones harboring an empty vector. ArCK RELA knock out cells were generated and the NK cell-mediated cytotoxicity was measured as described in the method section. Dot plot of individual data points and mean were presented. n=2 biological replicates; ArCK-c1 vs ArCK RELA-c1, p= 0.70, n.s.; KS test.

Measurement of RelA protein levels in RELA KO single cell clones generated in RPE1-hTERT cells.

The effect of inactivating RELB on NK cell-mediated cytotoxicity in ArCK cells. The same experimental methods were used as described in (A). n=3 biological replicates; ArCK-c1 vs ArCK RELB-c1, p= 0.47, n.s.; KS test.

Measurement of RelB protein levels in RELB KO single cell clones generated in RPE1-hTERT cells.

The effect of inactivating both RELA and RELB on NK cell-mediated cytotoxicity in ArCK cells. n=2 biological replicates; ArCK-c1 vs ArCK RELA RELB- c1, p= 0.0014; KS test.

Measurement of RelA and RelB protein levels in RELA RELB double KO single cell clones generated in RPE1-hTERT cells.

ArCK or euploid proliferating control cells were treated with either DMSO or the NF-κB inhibitor BMS-345541 (5 μM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean± SEM. ArCK vs ArCK NF-κB inhibitor, p< 0.0001; KS test.

(H, I) The effect of inactivating both RELA and RELB on NK cell-mediated cytotoxicity in 7-day doxorubicin (H) and nutlin3-treated (I) cells. n2 biological replicates; Doxo-c1 vs. Doxo RELA RELB-c1, p = 0.02. Nutlin-c1 vs. Nutlin RELA RELB-c1, p = 0.44, n.s.; KS test.
Aneuploid senescent cells activate NF-κB to promote their immune clearance by NK cells (2021)
Figure 6

Figure 6. NF-κB is active in highly aneuploid cancer cell lines

Comparison of the Hallmark gene expression signature "TNFA_signaling via NFKB" in near-diploid (low aneuploidy) and highly-aneuploid (high aneuploidy) human cancer cell lines from the CCLE. *, p= 5e-08, empirical Bayes-moderated t-statistics. The y-axis represents the ssGSEA expression score. The grey line marks an ssGSEA score of 0, indicating the genes in the hallmark "TNFA_signaling via NFKB" geneset is not differentially regulated.

Correlation plot between ssGSEA expression score of "TNFA_signaling via NFKB" and the aneuploid score for human cancer cell lines from the CCLE. The trend line for Spearman's correlation plot is indicated in red. Spearman's ρ= 0.181, p-value = 2.2e-08.

HCT116 (left) or DLD1 cells (right) were treated with either DMSO or the Mps1 inhibitor reversine (500 nM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean±SEM. HCT116 vs. HCT116 Mps1 inhibitor, p= 0.44, n.s.; DLD1 vs. DLD1 Mps1 inhibitor, p= 0.44, n.s.; KS test.

Expanded View Figure Legends
Structure of the Frequency-Interacting RNA Helicase: a protein interaction hub for the circadian clock (2016)
Figure 1

Figure 1: Function and structure of FRH. (A) In the Neurospora crassa circadian core clock oscillator FRH associates with FRQ and CK1a to form a repressor complex (FFC) that inhibits the positive-acting white-collar transcription factors (WC-1 and WC-2) which form the white-collar complex (WCC). Stoichiometries of the components are not represented in the schematic. FFC composition has been estimated as 1 FRH molecule: 2 FRQ molecules (Cheng et al, 2005; Hurley et al., 2013)), or 2 FRH molecules: 2 FRQ molecules (Lauinger et al, 2014). (B) Structure of FRH from the large crystallographic cell with an accordingly colored domain map below.
Structure of the Frequency-Interacting RNA Helicase: a protein interaction hub for the circadian clock (2016)
Figure 2

Figure 2: Comparison of FRH and Mtr4 structures. (A) FRH large cell (cyan), Mtr4 (pdb 2XGJ, green, RNA backbone in orange), and FRH small alternate cell (blue) differ primarily in the orientation of the arch domain. The FRH RNA-bound structure also crystallizes in the small-cell configuration. The two different FRH structures have an overall RMSD of 3.9 Å and the RMSD between Mtr4 and FRH is 2.9 Å (over 734 residues, for large cell FRH). (B) SAXS data indicates that FRH-Δ114 is monomeric and flexible. The crystal structure of the large cell structure predicts the solution scattering curve (left, χ2 = 0.92) and fits well to the calculated molecular envelope (right).
Structure of the Frequency-Interacting RNA Helicase: a protein interaction hub for the circadian clock (2016)
Figure 3

Figure 3: The N-terminal region of FRH is unstructured in the absence of FRQ. (A) Comparison of the FRH N-terminus (large cell structure, magenta) to that of Mtr4 (green) aligned on their RecA domains. Only the additional elements of FRH are shown (cyan). The FRH N-terminal extension has little ordered secondary structure, although regions 143-147 and 129-135 form small turns that interact with the helicase core. (B) The very N-terminus of FRH extends away from the RecA-2 domain to interact with a symmetry related molecule in the crystal (below), whereas the Mtr4 N-terminus turns back to form an outer β‐strand. Center panel shows the crystal contacts, with close-ups for each region highlighted as insets on the left and right.
Structure of the Frequency-Interacting RNA Helicase: a protein interaction hub for the circadian clock (2016)
Figure 4

Figure 4: The RecA and arch-elbow domains of FRH compared to Mtr4. (A) The two RecA domains are rotated relative to one another in FRH (cyan, large cell) compared to Mtr4 (green, RNA backbone in orange). The change in positioning of RecA-2 with respect to RecA-1 results in an overall displacement from the Mtr4 domain juxtaposition that ranges from 1.1 – 4.0 Å throughout RecA-2 with the largest differences at the external helices. (B) Comparison of arch domains conformations from Mtr4 (green) and FRH (large cell in cyan, small cell in marine) after superposition of the most N-terminal helical region. The isolated KOW modules of FRH, Mtr4 (with FRH superimposed), and Ski2 have considerably different loop configurations.
Structure of the Frequency-Interacting RNA Helicase: a protein interaction hub for the circadian clock (2016)
Figure 5

Figure 5: Interactions of FRH with RNA and ADP. (A) 3.8 Å resolution mFo-DFc omit map density (green, calculated with sharpened B-factors and contoured at 2.0) reveals the presence of ADP and a short segment of RNA bound to FRH. (B) 3.8 Å resolution 2mFo-DFc calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6 orange density shown for surrounding protein (0.6. (C) Comparison of Mtr4 and FRH ADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds). (c) Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains. (d) Hel308 DNA helicase DNA (pdb 2PR6; backbone in orange, bases in gray bonds) after aligning the RecA domains of Hel308 with those of FRH (cyan, RNA in yellow); see also Appendix Fig S4. The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains.
Structure of the Frequency-Interacting RNA Helicase: a protein interaction hub for the circadian clock (2016)
Figure 6

Figure 6: Features of FRH critical for helicase and clock function. (A) Key residues for FRH function shown on the large-cell structure: K208 and S324 compose the ATP binding site, E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712 and K766 contribute to a positively charged surface on the KOW domain. R806H disrupts the clock. Q131, G132 and V142 in the N-terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig1, ADP and RNA are in orange bonds). (B) Same as (A) after rotation about a horizontal axis. (C) Close-up of the ADP-binding pocket and N-terminal interaction for the large-cell structure (cyan ribbons) and the small-cell structure (blue ribbons). (D) Electrostatic potential surface of FRH (blue positive, red negative) shown with the same orientation as in (B). (E) Close-up of the KOW domain showing the large number of basic (aqua sticks) and acidic side chains (yellow sticks) on the surface that also includes the mutational sites R806, R712, K811 and K766 (grey sticks)
Structure of the Frequency-Interacting RNA Helicase: a protein interaction hub for the circadian clock (2016)
Figure 7

Figure 7: FRH mutant interactions with the core clock components examined by race tube and immunoprecipitation assays. The assays probe the interactions between the core clock components in strains with a wild type copy of FRH and FRH with several different amino acid mutations. (A) Race tube assays with FRH mutants. Every circadian cycle N. crassa lays down aerial hyphae that appear as fluffy yellow/orange conidial bands. The distance between the bands reflects the clock period. The strains were grown in constant darkness and growth fronts were labeled every 24 h (black vertical lines), rhythmicity on right (τ= period in hours, σ = standard deviation, n = number of race tubes, ARR, arrhythmic). The race tubes shown here are representative samples from three replicate tubes. (B) Pull-down assays evaluating interactions among FRH, FRQ, WC-1 and WC-2. Ratio of FRQ, WC-1 and WC-2 binding to the V5 tagged FRH mutants as compared to the inter-strain variations in FRH levels, error bars represent standard deviation for the assay run in triplicate. *P < 0.05 (unpaired two-tailed t-test with Gaussian distribution assumption). Representative immunoblots are shown in Appendix Fig S5.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 1

Mfn2 ablation promotes abnormal ER expansion during ER stress conditions. (A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm. (B) EM images of Tg‐treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm. (C) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β‐GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm. (D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A‐bodipy to stain ER and Golgi. Representative flow‐cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P0.05 versus WT group.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 2

Mfn2 ablation prevents caspase activation during ER stress and promotes paraptosis‐like cell death. (A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (D) Flow‐cytometry analysis of the Sub G1 DNA fragmentation in methanol‐fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group. (E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3‐L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z‐VAD‐fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P0.05 versus WT+Tg group. (H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm. (I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 3

Mfn2 loss‐of‐function decreases autophagy in response to ER stress. (A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P0.05 versus WT group; #P0.05 versus WT non‐Tg‐treated group. (B, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b‐I and LC3b‐II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b‐II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P0.05 versus WT+Baf+Tg group. (C) WT or Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm. (D) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus WT group. (E) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group. (F) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions. (G) Expression of Beclin‐1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 4

Mfn2 modulates the UPR response. (A) Scheme on UPR branches. (B) Immunodetection of p‐PERK,PERK, GADD34, p‐eIF2α, eIF2α, ATF4, CHOP, and XBP‐1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated. (C) Densitometric quantifications. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (D) Immunodetection of p‐eIF2α, eIF2α, and ATF4 in WT and Mfn2 KO cells treated with 0.5 μg/ml Tunicamycin (Tm) for the times indicated. (E) Transcriptional activity driven by ATF6. WT and Mfn2 KO cells were co‐transfected with the 5xATF6‐GL3 and TK‐Renilla plasmids and treated with 1 μM Tg for 24 h after transfection. Data are mean±s.e.m. (n=4). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 5

PERK deficiency improves apoptosis during ER stress in Mfn2‐ablated cells. (A) Immunodetection of PERK in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed against PERK (PERK KD). (B) Caspase activity was detected by measurement of DEVD‐AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group. (C, D) Total and cleaved caspase 3 levels were detected by western blot. Densitometric quantification is shown in D. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group. (E, F) Immunodetection of LC3b‐I and ‐II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 6

XBP‐1 loss‐of‐function enhances autophagy during ER stress in Mfn2‐ablated cells. (A) Immunodetection of XBP‐1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed to XBP‐1 (XBP‐1 KD), and treated with Tg for 6 h. (B) Caspase activity was detected by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. #P0.05 versus Scr group. (C, D) Total and cleaved caspase 3 levels were detected by western blot. WT and Mfn2 KO cells subjected or not to XBP‐1 silencing (Scr, and KD) and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (E, F) Immunodetection of LC3b‐I and ‐II in Scr and XBP‐1 KD WT or Mfn2 KO cells treated as indicated (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). #P0.05 versus Scr group. (G) Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b and subjected or not to XBP‐1 silencing (Scr or KD) were treated with 1 μM Tg, 100 ng/ml Bref, or 0.5 μg/ml Tm for 24 h and examined by confocal microscopy. Scale bar: 10 μm. (H) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 7

Mfn2 regulates PERK activity. (A) Immunodetection of p‐PERK and p‐eIF2α in Mfn2 KO MEFs, Mfn2 knockdown 3T3‐L1 fibroblasts, and Mfn2 knockdown C2C12 myoblasts. (B) Densitometric quantification of p‐PERK and p‐eIF2α in MEFs. Data are mean±s.e.m. (n=3). *P0.05 versus WT cells. (C, D) Immunodetection of p‐eIF2α, eIF2α, CHOP, XBP‐1s, LC3b, and p62 in Mfn2‐deficient liver (C) or skeletal muscle (D) from tissue‐specific KO mice. (E) Co‐immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK‐myc or an empty vector (pBABE‐myc; negative control). (F) Co‐immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates. PERK was immunoprecipitated with a C‐terminal antibody and co‐immunoprecipitation of Mfn2 was detected by western blot.Source data for this figure is available on the online supplementary information page.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 8

PERK knockdown rescues mitochondrial morphology and excessive ROS production in Mfn2 KO cells. (A) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD Mfn2 KO cells stained with MitoTracker Green. (B) Mitochondrial morphology quantification. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus Scr group. (C, D) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD WT cells (C) or scrambled (Scr) and PERK KD Mfn1 KO cells (D) stained with MitoTracker Green. (E) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4‐phenyl butyric acid for 6 h and stained with MitoTracker Green. (F) Flow‐cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO cells (G) or Scr and PERK KD WT and Mfn1 cells using DHR1,2,3. Data are mean±s.e.m. (n=4). *P0.05 versus WT group, #P0.05 versus Scr group. Scale bars: 10 μm.
Mfn2 modulates the UPR and mitochondrial function via repression of PERK (2013)
Figure 9

PERK knockdown normalizes mitochondrial Ca2+ overload and mitochondrial respiration. (A) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells. Cells were loaded with Rhod‐2 and then treated with 2.5 mM CaCl2 (left). Calcium uptake was monitored by confocal microscopy. Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod‐2 and incubated with 2.5 mM CaCl2 for 5 min (right). Scale bars: 10 μm. (B–E) Mitochondrial oxygen consumption (OCR) was measured in Scr and PERK KD Mfn2 KO cells (B), Scr and PERK KD WT cells (C), Scr and PERK KD Mfn1 KO cells (D), and WT cells stably expressing PERK‐myc (E). Data are mean of three independent experiments. The following parameters were measured: oxygen consumption under routine conditions (DMEM with 5.5 mM glucose), maximal respiratory capacity reached after uncoupling with FCCP, and respiratory leak, measured after inhibition of ATP synthase with oligomycin. Data are mean±s.e.m. *P0.05 versus Scr group or versus the empty plasmid group. (F, G) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK‐myc stained with MitoTracker Green. Scale bars: 10 μm. Quantitative analysis of mitochondrial morphology is shown in G. Data are mean±s.e.m. (n=3). *P0.05 versus the empty plasmid group.