EMBO/SourceData huggingface dataset:
Figure 3 qRT-PCR analysis of INH1 mRNA levels. Data are mean ± SD from three biological replicates. Western blot analysis of Inh1 protein levels. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot.Deletion of INH1 benefits the proliferation of ρ0 cells at 37 ℃. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ and 37 ℃ for 2 days. Western blot analysis of copper-inducible overexpression of Inh1. A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus. Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours.  Overexpression of Inh1 decreases ∆ψm in ρ0 cells. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Cells were then stained with 125 nM TMRM for FACS analysis.  Overexpression of Inh1 represses the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30℃ for 2 days.  Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins (Nfs1 and Yah1) and nuclear ISC-containing protein Pol3. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Nfs1, Yah1 were endogenously tagged with FLAG, and Pol3 was endogenously tagged with HA.
Figure 5 Puf3 is phosphorylated in WT cells and is hyper-phosphorylated in ρ0 cells. Puf3-FLAG was immunoprecipitated from the indicated strains and treated with/without λ-phosphatase, and analyzed by regular and Phos-tag SDS-PAGE.  Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Treatments and strains with Puf3 hyper-phosphorylation was highlighted in red. Glucose depletion was used as a positive control. Asterisk indicates a non-specific band.  Puf3-15A mutation inhibits Puf3 hyper-phosphorylation in ρ0 cells. A cassette expressing WT or mutant Puf3-FLAG (11A or 15A) under the control of PUF3 promoter was inserted into the HO site in puf3∆ cells. The 15A mutant that blocks Puf3 hyper-phosphorylation is highlighted in red. Puf3-FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos-tag SDS-PAGE.  Puf3-15A mutation inhibits Puf3 hyper-phosphorylation upon oligomycin treatment and in ∆V and ρ0 cells. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Asterisk indicates a non-specific band.  Puf3-15A mutation downregulates Puf3-associated mitochondrial transcripts in ρ0 cells. ρ0 and ρ0 puf3-15A cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV7). All the transcripts and the Puf3-associated transcripts encoding mitochondrial proteins (gene lists in Table EV1D) are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated. Puf3-15A mutation inhibits the upregulation of Mia40 in ∆V and ρ0 cells. Mia40 was endogenously tagged with FLAG. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Puf3-15A mutation decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.  Puf3-15A mutation has no effect on the proliferation of WT cells.   Puf3-15A mutation inhibits the proliferation of ρ0 cells.   Mia40 overexpression rescues the proliferation defect of ρ0 puf3-15A cells. Single copy plasmid expressing Mia40 driven by MIA40 promoter was transformed into the indicated strains.
Figure 6 Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells, and represses ribosome and Ribi gene expression in ρ0 cells. ρ0 snf1∆ cells were prepared by acute EtBr (25 µg/ml) treatment of snf1∆ cells in SCD for 2 days. RNAseq was performed with three biological replicates per genotype (Dataset EV8). All the transcripts and two categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated.  Snf1 represses ribosome biogenesis in WT and ρ0 cells. Polysomes of the indicated strains were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.  Deletion of SNF1 decreases ∆ψm in both ∆V and ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis  Deletion of SNF1 decreases the proliferation of both ∆V and ρ0 cells. Serial dilutions of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.  Deletion of MIG1 in ρ0 snf1∆ cells rescues ∆ψm. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis  Deletion of MIG1 in ρ0 snf1∆ cells partially rescues cell proliferation. A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.  Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1∆ cells fully rescues cell proliferation. A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.
Figure 7 Heat map plots of the mRNA level changes of chaperones, ubiquitin-proteasome pathway, and autophagy genes (gene list in Table EV1F) in the indicated strains (data from Dataset EV1 and Dataset EV10). Selected genes for growth analysis in B are highlighted in red.  Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3∆ cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Data are mean ± SD from three biological replicates.  Deletion of HSC82 selectively decreases ∆ψm in ρ0 puf3∆ cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.
Figure 4A Histochemical stained roots from composite plants transformed with proMtNPF6.5:GUS (left column), proMtNPF6.7:GUS (middle column), proMtNPF6.6:GUS (right column).B Expression of MtNPFs in roots after transfer to media containing different levels of NO3- for 30 minutes. C=KCl control, N=KNO3 (Biological replicates: n = 3 (3 pooled root organs in each replicate), asterisk denotes significance p<0.05, Student's t-test). The means of all samples were compared to that of their concentration-matched KCl controls. Expression is shown relative to Ubiquitin.
Figure 5A, B I-V responses in oocytes (subtracted of the water injected controls) exposed to 40 mM Cl at pH5.8 (A) and pH7.4 (B). The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration Data information: Value = mean ± SEM, biological replicates: n = 4C, D I-V responses in the (C) water and (D) MtNPF6.5 (subtracted of the water injected controls) injected oocytes exposed to different concentrations of Cl- at pH5.8. The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration. Data information: Value = mean ± SEM, biological replicates: n = 6
Figure 6C Oocytes were exposed to 1.17 mM 36Cl- for 2h with 1 and 10 mM NO3-. Biological replicates: n=6 (4 oocytes in each sample). D The same data as in C is shown without AtNPF6.3 to allow for better comparison between MtNPFs. Biological replicates: n=6 (4 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, and black cross represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05.
Figure 7A, B Relative expression of NPFs in M. truncatula roots grown in FP and exposed to different NaCl treatments. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEMD Relative expression of NPFs in M. truncatula roots grown in FP and exposed to 100mM KCl treatments for different times. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM
Figure 8A Seedling Cl- content determined by ion chromatography 2 h after treatment with 50mM NaCl. *p<0.05, Student's t-test; biological replicates: n=4. B Cl- content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3. *p<0.05, Student's t-test; biological replicates: n=4. C On the left, relative Cl- content determined by XRF of NO3- starved seedlings after KNO3 supplementation. *p<0.05, Student's t-test; biological replicates: n=3-5. On the right, a representative XRF image of Cl- signal across WT and mutant roots at the 0h timepoint. Five roots per genotype are shown. The outlines indicate the measurement areas. The color legend indicates relative Cl- concentration.
Figure 9A Biomass (fresh weight) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl. *p<0.05, Student's t-test; biological replicates: n=3 (2-5 pooled organs in each replicate).B Image of seedlings grown on FP or 50 mM NaCl. Data information: roots were re-arranged to better display their length, they did not exhibit curling/agravitropic growth.
Figure 10B Changes in NO3- and Cl- content (mol/g dry weight) of NO3- starved plants after NO3- provision Data information: , *p< 0.05, Student's t-test; biological replicates: n=4 ±SEM.E Relative MtNPF expression following treatment with 5mM KNO3 +100mM NaCl Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM.
Figure 11A The induction of MtNPF6.7 and repression of MtNPF6.5 by NO3- requires MtNLP1. Plants were grown on FP plates and then treated with 5 mM KNO3. expression shown is relative to UBQ; *p<0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate).B The switch from Cl- to NO3- as the major anion upon NO3- addition is lost in mtnlp1. Plants were grown on FP plates and then treated with 5 mM KNO3 treatment. *p< 0.05, Student's t-test; biological replicates: n=4 (3 pooled root organs in each replicate).C
Figure 1(C) The Hippo WW domain-containing proteins show binding specificity to the known Hippo PY motif-containing proteins. TAP-MS analysis of a series of WW domain-containing proteins were performed and their binding with the indicated Hippo PY motif-containing proteins was summarized in a heatmap.(E) Validation of the binding specificity for the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.(F) Validation of the binding specificity for the derived WW domains from the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.
Figure 2(C-G) Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was respectively examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.
Figure 3(B) STXBP4 forms a complex with several Hippo PY motif-containing proteins. Immunoprecipitation was performed with STXBP4 antibody.(C) The identified 9-amino acid sequence is required for the association between STXBP4 and AMOT. HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay.(E) The YAP-WW1/SMAD7-PY and STXBP4-WW/SMAD7-PY complexes show similar cumulative average trend and average binding free energy (∆G) within standard deviation (the shaded region) of one another.(F) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation. Western blotting was performed with the indicated antibodies.Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP nuclear translocation (G)Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP downstream gene transcription (mean ± s.d., n=3 biological replicates) (H). Scale bar, 20 μm.*** p < 0.001 (Student's t-test).(I) WW domain is required for the STXBP4-mediated YAP cytoplasmic translocation. STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed. HA-positive cells (arrows) from ~30 different views (~200 cells in total) were randomly selected and quantified for YAP localization. Percentage of HA-positive cells with nuclear YAP enrichment is shown. Scale bar, 20 μm.(J) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition. The indicated cells were subjected to serum starvation (treatment with no-serum medium for 12 hours), glucose starvation (treatment with no-glucose medium for 6 hours) or actin inhibition (treatment with 0.5 μg/mL latrunculin B or 5 μM blebbistatin for 30 min). YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.
Figure 4(B) STXBP4 forms a protein complex with α-catenin and a group of Hippo pathway proteins.(C) STXBP4 promotes the association of α-catenin with AMOT and LATS1. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.(D) Loss of STXBP4 diminishes the association of α-catenin with AMOT, LATS1 and YAP. HEK293A and STXBP4 KO cells were transfected with the SFB-tagged α-catenin construct and subjected to the pulldown assay.(E) STXBP4 induces the co-localization between α-catenin and AMOT as well as LATS1 and YAP. HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed. Scale bar, 20 μm.Identification of several STXBP4 missense mutations that disrupt its interaction with α-catenin and AMOT. The identified missense mutations respectively disrupted the STXBP4- α-catenin (G) and STXBP4-AMOT (H) complex formation.(I) Inhibition of actin cytoskeleton promotes the STXBP4-associated protein complex formation. HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre-immune serum and anti-STXBP4 serum under the indicated treatments.(J) The missense mutations of STXBP4 (F) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension. YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.
Figure 5STXBP4 is downregulated in human kidney cancer. The mRNA level of STXBP4 is analyzed in the Firebrowse web database (A), where 14,729 tumor sample data generated by TCGA were included. The first quartile, median and third quartile values were indicated as the boxplots. Outliers were plotted as individual points. Error bars indicated the standard deviation above and below the mean of the data.STXBP4 is downregulated in human kidney cancer. The expression of STXBP4 was also examined using kidney tissue microarray, where percentage of the indicated tissue samples with downregulated STXBP4 was shown (B). The p value was calculated by using the paired Student's t-test.(C) Kaplan-Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level. Clinical data of STXBP4 were analyzed in TCGA-KIRC project containing total 611 patient samples. The p value was calculated by using the Log-rank (Mantel-Cox) test.(D) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray, where the indicated regions in the box were shown three times enlarged. Brown staining indicates positive immunoreactivity. Scale bar, 100 μm.(E) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables. Statistical significance was determined by chi-square test. R, correlation coefficient. N, nuclear localization. C, cytoplasmic localization.(F) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting.(G and H) Both the association with α-catenin and the functional WW domain are required for the STXBP4's tumor suppressive function in 786-O cells. Overexpression of STXBP4, but not the indicated STXBP4 missense mutants, significantly suppressed the 786-O cell xenograft tumor formation. Xenograft tumors are shown in (G), and the tumor weight is quantified in (H) (n = 5 mice, mean ± s.d.). ** p < 0.01 (Student's t-test). Scale bar, 1 cm.
Figure 1(B) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. unparasitized hosts. Day-4 of last instar larvae were used for this assay. Test larvae were parasitized by the wasp on day 0 of last instar.(C) Survival of armyworm larvae injected with PBS, unparasitized, and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. PBS. Test larvae were exposed to the heat stress 4 h after injection of each sample.(D) Time-dependent survival of armyworm larvae injected with 0.8 µmol/larva N-acetyl-L-tyrosine (NAT): post NAT-injection 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. zero time.(E) Dose-dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min (data are means ± SEM; n=12). Injection of more than 10 nmol NAT/larva caused a significant difference compared with zero (log rank test using R version 3.2.2).(F) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. PBS. Each chemical (0.5 µmol/larva) was dissolved in PBS; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine.
Figure 2(A) HPLC chromatogram and L-MS spectrogram of N-acetyltyrosine purified from pooled human serum (#12181201, Cosmo Bio Co., Japan). The peak fraction purified by HPLC was confirmed to be N-acetyltyrosine by LC-MS (inserted spectrogram).(B) NAT concentrations in the serum of mice (Slc:ddY, 18 wks old, male) exposed to heat (40oC ) and restraint stress (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. zero time.(C) Effects of NAT on corticosterone concentrations in the serum of mice (Slc:ddY, 9-10 wks old, male) exposed to restraint stress for 60 min (data are means ± SEM; n=6). ** P<0.01 vs. without stress. Test mice drank 1.5 mg/ml NAT solution for 24 h before exposure to stress.(D) Effect of NAT on stress-induced peroxidized lipid in the serum of mice (Slc:ddY, 9 wks old, male) forced to swim for 60 min (data are means ± SEM; n=10). *P<0.05 vs. PBS.
Figure 3(A) Survival of Drosophila melanogaster (W1118) larvae fed with 2 µmol NAT/g-diet 2 d after heat stress at 38.5oC for 60 min (data are means ± SEM; n=10). **P<0.01 vs. zero time.(B) Effects of NAT feeding on expression of antioxidant enzymes, catalase, SOD1, and SOD2 (data are means ± SEM; n=10). *P<0.05 vs. zero time.(C) Representative images of nuclei (left) and FoxO (middle) in the fat body of Drosophila larvae before and after NAT feeding for 12 h. White signals in the merged image (right) show the overlap of both signals. Scale bar: 50 µm. (D) Relative whiteness intensities before and after NAT feeding (data are means ± SEM; n=15). **P<0.01 vs. zero time. (E) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae (data are means ± SEM; n=8). *P<0.05, **P<0.01 vs. zero time.(F) Effects of N-acetyltransferase (Dat) knockdown in gut enteroendocrine cells (TK-Gut-Gal4>UAS-dsDat) on survival of Drosophila larvae (data are means ± SEM; n=15). **P<0.01 vs. without NAT.
Figure 4(A) Survival of Drosophila S2 cells treated with preheating, 100 µM NAT, and 100 µM NAT with 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, those were removed by changing the medium. *P<0.05, **P<0.01 vs. PBS.(B) Survival of Drosophila S2 cells pretreated with 100 µM NAT, 100 µM NAT with 30 µg/ml antimycin A, and preheating with or without 30 µg/ml antimycin A 48 h after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, they were removed by changing the medium. *P<0.05 vs. PBS.(C) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells. Upper: representative images of time-dependent changes in MitoRed localization in mitochondria after adding NAT. Scale bar: 20 µm. Lower: relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader. Different letters above bars represent significant differences [P <0.05 (data are means ± SEM, n=12)].(D) ROS concentrations in S2 cells after adding 100 µM NAT (data are means ± SEM; n=12). *P<0.05 vs. zero time.(F) Effects of dsRNA targeting of FoxO on NAT-dependent expression of Keap1 (data are means ± SEM; n=12). *P<0.05, **P<0.01 vs. without NAT. Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies.(G) Effect of NAT on growth of HCT116 colon cancer cells in mice (BALB/cSlc-nu/nu, 6 wks old, female) (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. without NAT. Test mice began to freely drink 5 mg/ml NAT solution for 7 d before transplantation of the tumor cells on day 0: the average amount of NAT consumed by each mouse was about 8.8 mg/day (0.4 - 0.5 g/kg weight/day). HCT116 cells (5.0 x 106 cells) were transplanted into each test mouse using a 1 ml syringe with a 27G needle.
f1a, Percentage of ciliated cells in control (Ctrl) and IFT20− MEFs (*P = 0.030, n = 3) and wild-type (WT) and Ift88−/− KECs (**P = 0.003, 25 cells each, n = 3) after 24 h of serum removal. Arrows, cilia.b, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl and IFT20− MEFs (top, **P = 0.0003, n = 7), and in WT and Ift88−/− KECs (bottom, *P = 0.035, n = 9). PI, protease inhibitors; S, serum. cc, Autophagic flux in WT and Ift88−/−KECs by mCherry-GFP-LC3 puncta quantification. Puncta: yellow, autophagosomes; red only, autophagolysosomes; total, both together (**P = 0.008, *P = 0.025).. d, LC3 immunoblot (left) for quantification of autophagosome formation (right). (*P = 0.048, n = 4).e, Electron microscopy images (left) and morphometric quantification of autophagic vacuoles (AV, right) in Ctrl and IFT20− MEFs (top, **P = 0.001, 10 fields in 2 experiments) and KECs WT and Ift88−/− (bottom, *P = 0.037, 5 fields). Arrows, autophagic vacuoles (AV, black) and endosomal compartments (yellow).f, Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml−1 platelet-derived growth factor (PDGF) treatment. (**P = 0.001, ***P = 0.0009, 121, 230 and 162 cells, n = 5).g, LC3immunoblot (top) for autophagic flux quantification (bottom) upon PDGF treatment (*P = 0.005, n = 3). Scale bars in a, 10 µm. n.s., statistically non-significant. Mean ± s.d. in b, d and mean ± s.e.m. in other panels.