EMBO/soda-nel huggingface dataset:
Targeting the Warburg Effect via LDHA Inhibition Engages ATF4 Signaling 
for Cancer Cell Survival (2018)
Figure 5

Figure 5. LDHA inhibition suppresses Ser and Asp biosynthesis to engage ATF4-mediated survival response.

A GC-MS-based quantification of intracellular Asp (upper) and Ser (lower) levels in melanoma cell lines treated with LDHAi for 72 hr.

B (upper) GC-MS-based 13C6-glucose fractional isotope labeling of Ser and Asp in melanoma cells treated for 72 hr with LDHAi. (lower) GC-MS-based 13C5-glutamine fractional isotope labeling of Asp in melanoma cell lines treated for 72 hr with LDHAi.

C, D GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in DMEM (C) or MEM supplemented with Ser (400 μM; D) and treated with LDHAi for 24 hr.

E GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells treated with LDHAi for 1 hr

F GC-MS-based quantification of intracellular Asp levels in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with LDHAi for 1 hr.

G Immunoblotting of ATF4 in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with GSK-2837808A (10 μM) for 1, 2, or 24 hr.

H Immunoblotting of ATF4 in MeWo cells pre-treated with Ser (400 μM), Asp (10 mM), or Ser (400 μM) + Asp (10 mM) for 6 hr and then treated for 1 hr with LDHAi (10 μM).

Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
Targeting the Warburg Effect via LDHA Inhibition Engages ATF4 Signaling 
for Cancer Cell Survival (2018)
Figure 6

Figure 6. Diminished NAD+ and oxaloacetate transamination suppresses Ser and Asp biosynthesis respectively.

A Relative NAD+/NADH ratio in MeWo cells treated with LDHAi, nicotinamide mononucleotide (NMN, 1 mM), or a combination of the two for 24 hr. Cells were pretreated with nicotinamide mononucleotide for 6 hr and then treated with LDHAi for 24 hr in the combination setting.

B GC-MS-based quantification of total intracellular levels of Ser and Asp and their 13C6-glucose fractional isotope labeling in MeWo cells pretreated with nicotinamide mononucleotide (NMN, 1 mM) for 6 hr and then treated with LDHAi for 24 hr.

C Schematic diagram showing potential fates of cellular pyruvate. PDH, pyruvate dehydrogenase; CS, citrate synthase; GLS, glutaminase; PC, pyruvate carboxylase; GOT2, glutamic-oxaloacetic transaminase 2; GPT2, glutamic-pyruvic transaminase 2.

D GC-MS-based quantification of intracellular Ala levels in MeWo cells treated with LDHAi for 1 hr.

E GC-MS-based 13C6-Glc (M+2) and 13C5-Gln (M+4) isotopologue levels of the key TCA cycle metabolites and Asp in MeWo cells treated with LDHAi for 72 hr.

F GC-MS-based 13C6-Glc (left) and 13C5-Gln (right) isotopologue levels of Asp in MeWo cells treated with LDHAi for 72 hr.

G GC-MS-based quantification of intracellular Ala (left) and Asp (right) levels in si-GPT2-treated MeWo cells incubated with LDHAi for 1 hr.

H Immunoblotting of ATF4 and GPT2 in MeWo cells treated as in (G).

I Model depicting the survival program elicited by LDHA targeting that mounts resistance to LDHA inhibition in melanoma cells.

Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
Targeting the Warburg Effect via LDHA Inhibition Engages ATF4 Signaling 
for Cancer Cell Survival (2018)
Figure 7

Figure 7. Combined LDHA and mTORC1 inhibition suppresses tumor growth.

A (left) Immunoblotting of LDHA and ATF4 in A375 cells expressing inducible (Tet-On) sh-LDHA expression vector treated with doxycycline (Dox) for 48 hr. (right) qRT-PCR (n=3) analysis of the ATF4 target genes in A375 cells treated with doxycycline (0.5 μg/ml) for 24 hr. Statistical analysis was performed by unpaired t-test.

B Proliferation of inducible (Tet-On) sh-LDHA expression vector harboring A375 cells (n=3) treated with Dox (0.5 μg/ml).

C Athymic nude mice were injected subcutaneously with A375 melanoma cells expressing inducible (Tet-On) sh-LDHA expression vector. Starting day 5, animals were treated on alternate days with 1 g/L doxycycline, 4 mg/kg rapamycin, or with a combination of the two (n=8 in each group). The tumor growth was determined by measuring the tumor volume. Statistical analysis was performed using Welch's t-test (two-tailed).

D Immunoblotting of resected tumors (4 from each group) for the indicated proteins.

E, F Representative images of H&E and cleaved caspase-3 staining from each treatment group (E). Quantification of cleaved caspase-3 (F) (n = 4, each 4 fields). Scale bars, 50 μm. Statistical analysis was performed using two-way Anova.

G Relative mTORC1 expression in pan-tumor samples segregated based on high (LDHAhi) (n=2917) and low LDHA (LDHAlo) (n=2914) expression. The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR).

H Conditional essentiality of the indicated genes in LDHAlo and LDHAhi cell lines (n=17, each group). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR). p≤0.1 is significant.

Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively. Data are shown as the mean ± SEM in panels (A-C and F), ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Targeting the Warburg Effect via LDHA Inhibition Engages ATF4 Signaling 
for Cancer Cell Survival (2018)
Figure 8

Figure 8. ATF4-associated metabolic enzyme signature in melanoma subjected to MAPK signaling inhibitors.

A qRT-PCR analysis of LDHA mRNA levels in A375 cells treated with BRAF inhibitor (PLX-4032, 1 μM) for 24 hr.

B, C GC-MS-based quantification of (B) intracellular 3-PG and (C) lactate levels in A375 cells treated with PLX-4032 (1 μM) for 16 hr.

D Immunoblotting of the indicated proteins in A375 cells treated with PLX-4032 (1 μM) for 2, 6, or 24 hr.

E, F Heatmap for expression fold-change of ASNS, GPT2, PSAT1, and MITF between (E) post-treatment (BRAF inhibitor, BRAF-i; MEK inhibitor, MEK-i) and pre-treatment, or (F) post-treatment (BRAF-i) and pre-treatment tumor in each of the patients (y-axis). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR) are also displayed; post-treatment expression significantly greater than pre-treatment except in the case of MITF, where post-treatment expression is significantly less than pre-treatment.

Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.

Expanded View Figure Legends
Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency (2020)
Figure 4

Figure 4. Cysteine depletion alters expression of complex I and IV in RIRCD fibroblasts.

(A) Patient n= 4 (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts (n=2) were grown for 12 days in media containing 0.02 mM cysteine and 5% dialyzed FBS. Proteins belonging to mitochondrial complexes (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) were analysed via Western Blotting. VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20.

(B) Densitometry analysis of proteins from (A) showing no significant changes in OXPHOS proteins upon glutamine/ glutamic acid depletion. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Graphs show means ± s.d of duplicate samples. For statistical analysis unpaired Student's t-test was employed where *p≤ 0.05 was considered statistically significant.

(C) Patient (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5% dialyzed FBS. Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting. VCL was used as a loading control while the densitometry analysis (D) was done based on the mitochondrial proteinTOM20. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Although we could observe significant changes upon cysteine depletion in all conditions, the phenotype was exacerbated in the digenic fibroblasts compared to the controls. Graphs show means ± s.d of duplicate samples (*p≤0.05 unpaired Student's t-test was employed). All experiments were performed in triplicate n=3 and similar results were observed in all experiments
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies (2018)
Figure 1

Figure 1 The M45 RHIM is amyloidogenic and 1-90 and 1-277 regions of M45 confer protection against TNF-induced necroptosis in human cells.

A Sequence alignment of RHIMs from M45 and human and murine RIPK1, RIPK3 and ZBP1.

B Schematic representation of M45, RIPK1 and RIPK3 proteins. Grey regions indicate RHIM-encompassing fragments used in this study. Black bar represents 19-residue RHIM. RNR-like: inactive ribonucleotide reductase domain; DD: death domain; Z: double-stranded RNA-specific adenosine deaminase (DRADA) repeat.

C Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM. Representative of three samples examined. Scale bar 100 nm.

D HT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1. Error bars represent s.d., **** = p<0.0001. To determine statistical significance a two-tailed Students t-test was performed using GraphPad Prism (version 7; GraphPad software).
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies (2018)
Figure 2

Figure 2 The RHIM-containing fragment forms the amyloid fibril core.

A Thioflavin T fluorescence as a function of time, following dilution of WT or AAAA His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 from 8 M to 200 mM urea-containing buffer. Buffer sample contains ThT but no protein. Samples were tested in triplicate and solid line is the average of the triplicates and the error bars (in grey) represent +/- s.d.

B Absorbance spectra from solutions containing Congo red with WT (black lines) or AAAA mutant (dark grey lines) His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 that have been dialysed against Assembly Buffer. Congo red sample alone with no protein (light grey line) served as a control for the experiment. Experiment conducted once.

C Electron micrograph of material eluted in peak 1 from the three MBP fusion proteins, as labelled. These are representative micrographs of the material observed. Scale bar 100 nm in all panels.

D Representative micrographs of insoluble material generated after treatment with thrombin removed the MBP. Experiment repeated three times. Scale bar 100 nm in all panels
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies (2018)
Figure 3

Figure 3 Assembly of RHIMs gives rise to homomeric and heteromeric structures.

A Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown here.

B Schematic representation of fluorescence spectroscopy experiments used to detect the formation of homo- and heteromeric amyloid fibrils. Fusion proteins containing RHIMs and different fluorescent partners are mixed together under assembly-permissive conditions and examined in the confocal volume. Co-assembly of two different fluorophores within one complex results in detection of coincident signals in the two channels.

C, D Representative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of (C) mCHERRY-RIPK1497‑583 and YPet-RIPK1497‑583 and (D) mCHERRY-RIPK3387‑518 and YPet-RIPK3387‑518.

E, F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of (E) mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 (inset is expansion of region indicated by *) and (F) mCHERRY-RIPK1497‑583-AAAA and YPet-RIPK3387‑518. All mixtures examined in at least three different experiments on three separate occasions.
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies (2018)
Figure 4

Figure 4 Heteromeric amyloid formation including M45 RHIM requires the intact core tetrad.

A-H Representative fluorescence intensity traces collected over 50 s for mixtures of (A) mCHERRY-M451-90 and YPet‑only; (B) mCHERRY-M451-90 and YPet‑RIPK1497‑583; (C) mCHERRY-M451-90 and YPet-RIPK3387‑518; (D) mCHERRY-M451-90 and YPet-ZBP1170-355; (E) mCHERRY-M451-90AAAA and YPet‑only; (F) mCHERRY-M451-90AAAA and YPet-RIPK1497-583; (G) mCHERRY-M451-90AAAA and YPet-RIPK3387‑518 and (H) mCHERRY-M451-90 and YPet-ZBP1170-355. Independent replicates performed at least twice.
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies (2018)
Figure 5

Figure 5 Co-assembly of M451-90WT with RIPK1497‑583, His-Ub-RIPK3387‑518 and ZBP1170-355 results in the formation of heteromeric fibrils and dense fibrillar networks.

A Representative electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparations.

B Heterofibrils containing mCHERRY-M451-90 and YPet‑RIPK1497‑583

C Heterofibrils containing mCHERRY-M451-90 and YPet-RIPK3387‑518

D Heterofibrils containing mCHERRY-M451-90 and YPet‑ZBP1170‑355

E Heterofibrils containing mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518

F Heterofibrils containing mCHERRY- RIPK3387‑518 and YPet‑ZBP1170‑355

G Pearson's correlation coefficients measured from two confocal images of the indicated protein mixtures.

H Heterofibrils containing ECFP-M451-90 with mCHERRY- RIPK1497‑583 and YPet-RIPK3387‑518.

I STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518.

Data information: Scale bars represent 200 nm in panel (A), 10 μm in panels (B-F, H), and 2 μm in panel (I).
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies (2018)
Figure 6

Figure 6 SDS AGE analysis of RHIM-based assemblies.

A SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane.

B SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45.

C SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.

AGE experiments performed at least twice.

Size of assemblies indicated to the right of the panel, as large assemblies, soluble amyloid oligomers or monomers.
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies (2018)
Figure 7

Figure 7 Model of the heteromeric M45:RIPK3 amyloid fibril structure A Structure of the RIPK1:RIPK3 RHIM core, prepared using PyMol Graphics System from PDB 5V7Z. The RIPK3 sequence is coloured in blue and RIPK1 is coloured in green. Tetrad residue side chains are shown as sticks; Tyr, Asn/Gln, Ser and Cys side chains are shown in wheat, red, grey and yellow, respectively. Core tetrad interactions across the opposing β‑sheets are indicated by the dotted box. B Model of M45:RIPK3 RHIM core, prepared by appropriate mutation of RIPK1. The M45 sequence is coloured in cyan and RIPK3 in blue. Tetrad residue side chains are shown as sticks; Tyr, Asn/Gln, Val and Cys side chains are shown in wheat, red, grey and yellow, respectively. Core tetrad interactions across the opposing sheets are indicated by the dotted box. C Schematic representation of RHIM fibrils formed by full-length proteins with partner domains (e.g. kinase or RNR, coloured green) flanking either side of the amyloid core formed by RHIMs (coloured grey). D Confocal microscopy image of a single fibril composed of YPet-M451-90. Scale bar 5µm. Expanded View Figure Legends
SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres (2020)
Figure 1

Figure 1. SMCHD1 promotes c-NHEJ at dysfunctional telomeres.

(A) Western Blot detection of ATM pS1981, SMCHD1, TRF2 and hnRNPA1 loading control in wild-type and two SMCHD1 knockout (KO1 and KO2) HeLa cells transfected with shTRF2 plasmid or EV control.

(B) Representative metaphase spreads from wild-type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control. Telomeric signals were detected with Cy3-OO-(CCCTAA)3 and are false colored in red, DNA is stained with DAPI and is false colored in cyan. Scale bar: 5µm.

(C) Quantification of telomere fusions from experiment shown in D). Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>3,000 telomeres counted/condition/experiment). (*) P < 0.05; unpaired two-tailed Student's t-test.

(D) Representative cell cycle profiles from two biological replicates of WT and SMCHD1 KO HeLa cells transfected with shTRF2 plasmid or EV control. Cells were stained with propidium iodide and analyzed by Flow cytometry.
SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres (2020)
Figure 2

Figure 2. SMCHD1 loss slows down overhang processing at TRF2 depleted telomeres.

(A) Western Blot detection of SMCHD1, TRF2 and hnRNPA1 in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11).

(B) Quantification of telomere fusions in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11). Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>1,900 telomeres counted/condition/experiment). (***) P < 0.001, (**) P< 0.01; (*) P< 0.05; unpaired two-tailed Student's t-test.

(C) Terminal Restriction Fragment (TRF) analysis of telomeric DNA to detect 3'overhang processing of genomic DNA isolated from SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated as in the experiment in (B). (Left) Radiolabeled (CCCTAA)n probe was hybridized with a short run (upper panel) and long run (lower panel) native DNA gel to detect the signal of the telomeric 3' overhang. Samples used for the short and the long run were from the same digestion split into two. Exo I treatment (+ Exo) was used as a control that single stranded telomeric signal was terminal. (Right) The total TTAGGG signal in the same lane was deteced upon denaturation and hybridization with the same probe.

(D) Quantification of the telomeric overhang signal at d11 after doxycyclin addition to SMCHD1 wild-type and SMCHD1 knockout HeLa shTRF2 inducible cells. The bar graph represents the average overhang signal intensity from two biological replicates as percentage of the signal in the cells untreated with doxycycline with error bars representing the standard deviation (SD) of the sample.
SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres (2020)
Figure 3

Figure 3: SMCHD1 loss does not affect recombination frequency at telomeres in TRF2 depleted cells.

A Western blot detection of ATM pS1981, SMCHD, TRF2, γH2AX, and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and EV control.

B Representative images for CO-FISH on metaphase spreads in wild type (WT) Hela and U2OS cells. The leading strand telomeres were labeled by PNA-FISH with a (TTAGGG)3-Alexa488 probe (green), the lagging strand with a (CCCTAA)3-Cy5 probe (pink), and the DNA was stained with DAPI. Examples of telomere sister chromatid exchange (t-SCEs) events are marked with white arrows. Scale bar: 5µm

C Quantification of chromosome ends with t-SCEs detected by CO-FISH. Data represent means of 2 independent experiments± SD (>700 telomeres/condition/experiment) with individual average values indicated.
SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres (2020)
Figure 4

Figure 4. SMCHD1 promotes TIF formation and stimulates ATM signaling from TRF2 depleted telomeres.

(A-C) Representative images for detection of ATM pS9181, γH2AX, and 53BP1 at telomeres in wild-type (WT) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector (EV) control. Immunofluoresence (IF) for ATM pS1981 (gray), γH2AX (green) and 53BP1 (yellow) was combined with telomeric (CCCTAA)3-FISH (red) and DAPI staining total DNA. Scale bars: 5µm

(D) Quantification of the number of cells containing >5 Telomere dysfunction Induced Foci (TIFs) detected as in (A)-(C). Data represent the mean of 4 independent experiments ± SD (>200 cells/condition/experiment) for ATM pS1981 and 3 independent experiments ± SD (>200 cells/condition/experiment) for γH2AX and 53BP1. (***) P < 0.001; unpaired two-tailed Student's t-test.

(E) Quantification of the number of 53BP1 TIFs per cell detected as in (C) from three independent experiments. Data represent individual values per cell with the bar representing mean ± SD. (***) P < 0.001; unpaired two-tailed Student's t-test.
SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres (2020)
Figure 5

Figure 5. SMCHD1 is not required for genome-wide ATM signaling.

(A) Western blot detection of SMCHD1, ATMpS1981, CHK2 pT68, CHK1 pS345, RPA pS33, RPA pS4/8, γH2AX and Vinculin in wild-type or SMCHD1 KO HeLa cells γ-irradiated with 3Gy dose and harvested at the indicated times (1h, 12h, 24h) after the treatment.

(B) Representative images for Immunofluoresence (IF) detection of ATMpS1981 in wild-type HeLa cells γ-irradiated with a dose of 3Gy and harvested after the indicated times. Scale bar: 5µm

(C) Quantification of the average number of ATMpS1981 foci per cell detected as in (B) in wild-type and SMCHD1 KO γ-irradiated HeLa cells. Data represent mean of 3 independent experiments ± SD (>200 cells/condition/experiment) with individual average values indicated.