EMBO/soda-nel huggingface dataset:
Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel (2021)
Figure 5

Figure 5. The reporter assay enables quantification of serum-derived sICs from SLE patients.

Serum derived sIC from systemic lupus erythematosus (SLE) patients activate human FcγR reporter cells. 25 patients and 4 healthy control individuals were separated into three groups for measurement.

A) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals. Dose-dependent reactivity of FcγRs IIIA and IIB/C was observed only for SLE patient sera and not for sera from healthy individuals. Each titration represents one donor measured in technical duplicates.

B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right).

C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.
Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel (2021)
Figure 6

Figure 6. The reporter assay can be applied to mouse models of autoimmune disease.

A) Reporter cells expressing mFcγRIII, mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF-α and mouse-anti-hTNF-α at a 1:1 ratio by mass. One experiment performed in technical duplicates. Error bars indicate reproducibility as range.

B) Titrations of three mouse sera per group (C57BL/6, K/BxN or NZB/WF1) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above. Sera from BL/6 mice served as negative control. Each symbol represents mean values of one mouse measured in technical duplicates. Error bars = Mean with SD.

Expanded view tables and figure legends
A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis (2022)
Figure 1

Figure 1. ATX (ENPP2) expression is upregulated in human and mouse livers.

A ATX gene expression from publicly available datasets in NASH patients (n=9) compared to healthy controls (n=7) (*P < 0.05); cirrhosis patients (n=40) compared to healthy controls (n=19) (***P < 0.001). Bars represent the mean ± SEM, statistical analysis was performed by two-tailed student's t-test. *P < 0.05, ***P < 0.001 denotes significance versus respective healthy controls.

B Relative ATX gene expression in the MCD-diet-fed (n=9) compared to control mice (n=9) (***P < 0.001); CCl4 (n=8) compared to control mice (n=5) (*P < 0.05). Bars represent the mean ± SEM, statistical analysis was performed by two-tailed student's t-test. *P < 0.05, ***P < 0.001 denotes significance versus respective healthy controls.

C Representative images (scale=50μm) of ATX stained liver sections from human NASH (n=5) compared to healthy controls (n=4); human cirrhosis (n=5) compared to healthy controls (n=4).

D Representative images (scale=50μm) of ATX stained liver sections (using AEC chromogen denoted by red color staining or DAB chromogen denoted by brown color staining; nuclei were stained blue with hematoxylin) from MCD-diet-fed NASH (n=9) and CCl4-induced liver fibrosis (n=8) mouse models compared to respective controls (n=9 or n=5).
A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis (2022)
Figure 2

Figure 2. ATX inhibitors PF8380 and Cpd17 decrease LPA production.

A Structural binding of type I ATX inhibitor PF8380 to ATX; Potency (IC50) of PF8380 for inhibiting the catalysis of LPC to LPA. Bars represent the mean + SEM, n=3.

B Structural binding of type IV ATX inhibitor Cpd17 to ATX; Potency (IC50) of Cpd17 for inhibiting the catalysis of LPC to LPA. Bars represent the mean + SEM, n=3.
A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis (2022)
Figure 3

Figure 3. ATX inhibitors decrease steatosis in hepatocytes, and migration and expression of inflammation-related genes in macrophages.

A Representative images (scale=10µm) and quantification of HepG2 cells stained with oil-red-O after 48 hours of incubation with or without 200μM palmitate with or without 1µM of PF8380 or Cpd17. Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. ##P < 0.01 denotes significance versus control (non-palmitate) and *P < 0.05 denotes significance versus vehicle (palmitate); ns=non-significant.

B Representative images (at 0h and 24h) (scale=50µm), and quantitative analysis (after 24h) of migration by control (M0) and LPS- and IFNγ-induced M1 RAW264.7 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus M0 (non-stimulated macrophages) and *P < 0.05 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.

C Relative gene expression (normalized with GAPDH) for CCL2 and iNOS in control (M0) and LPS- and IFNγ-induced M1 RAW 264.7 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus M0 (non-stimulated macrophages) and *P < 0.05, **P < 0.01 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.

D Relative gene expression (normalized with 18s RNA) for CCL2 and iNOS in control (M0) and LPS-induced PMA-differentiated human THP1 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ##P < 0.01, ###P < 0.001 denotes significance versus M0 (non-stimulated macrophages) and **P < 0.01, ***P < 0.001 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.
A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis (2022)
Figure 4

Figure 4. ATX inhibition decreases TGFβ-induced HSCs activation, migration, and contraction.

A Collagen I and α-SMA stained images (scale=50µm) of control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM) (n=3).

B Relative Collagen I, α-SMA and PDGFβR gene expression (normalized with GAPDH) in control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by two-tailed student's t-test. #P < 0.05, ###P < 0.001 denotes significance versus control (non-TGFβ cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.

C,D Representative images (at 0h and 24h) (scale=100µm) (C) and quantitative analysis (after 24h) (D) of migration by control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.

E,F Representative images (E) and quantitative analysis (F) showing 3D collagen gel contractility of control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ cells) and **P < 0.01, ****P < 0.0001 denotes significance versus vehicle (TGFβ-activated cells).
A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis (2022)
Figure 5

Figure 5. Inhibition of ATX results in blockade of LPAR-driven activation of stellate cells and macrophages.

A Schematic overview of LPA-induced signaling pathways.

B Top panel shows time-course stimulation to LPA and uninhibited or inhibited LPC-treated ATX followed by RhoA activation, measured as a YFP/CFP fluorescent ratio. Bottom panel depicts quantitation of the burst response to the stimulants. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05, ***P <0.001 denotes significance versus ATX+LPC treated cells.

C Representative images and quantification showing Western-blot analysis for p-Akt and p-ERK normalized with β-actin in control, LPC, LPA, ATX, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17 stimulated LX2 cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC treated cells.

D Representative images and quantification showing Western-blot analysis for LPAR1 on the cell surface and in lysate, normalized with β-actin in lysate, in LPC, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17 stimulated LX2 cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC treated cells.
A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis (2022)
Figure 6

Figure 6. Cpd17 ameliorated CCl4 induced acute liver injury.

A Schematic showing CCl4-mediated liver disease induction and Cpd17 treatment regimen.

B Liver weight to total body weight ratio from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (healthy mice); ***P < 0.001 denotes significance versus CCl4 mice.

C Serum ALT levels from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ###P < 0.001 denotes significance versus control (healthy mice); P = 0.26 denotes significance versus CCl4 mice.

D,E Representative images (scale=100μm) (D) and quantitative analysis (E) of liver sections from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice and stained with H&E, collagen-I and F4/80. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); P = 0.098 or P = 0.08 denotes significance versus CCl4 mice.
A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis (2022)
Figure 7

Figure 7. Cpd17 attenuated steatosis, inflammation and fibrosis in MCD-diet induced NASH mice.

A Schematic showing NASH induction and Cpd17 treatment regimen.

B Representative macroscopic images of livers from control (n=5), MCD (n=6) and MCD+Cpd17 (n=6).

C Serum ALT, serum cholesterol and serum triglyceride levels in control (n=5), MCD (n=6) and MCD+Cpd17 (n=6) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ##P < 0.01, ###P < 0.001 denotes significance versus control (healthy, chow-diet fed mice); *P < 0.05, **P < 0.01, ***P < 0.001 denotes significance versus MCD-diet fed mice.

D,E Representative images (scale=100μm) (D) and quantitative analysis (E) of liver sections from control (n=5), MCD (n=6) and MCD+Cpd17 (n=6) mice and stained with H&E, oil-red-O and Collagen I. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); *P < 0.05, **P < 0.01, ***P < 0.001 denotes significance versus MCD-diet fed mice.

F Liver hydroxyproline levels as assessed in livers from control (n=5), MCD (n=6) and MCD+Cpd17 (n=6) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (healthy mice); *P < 0.05 denotes significance versus MCD-diet fed mice.
Mitotic WNT signalling orchestrates neurogenesis in the developing neocortex (2021)
Figure 6

Figure 6. Sox4/11 overexpression and GSK3 inhibition both rescue differentiation defects of DKO NPCs.

(A) Schematic illustrating strategy to rescue DKO NPC differentiation defect by simultaneous lentiviral overexpression of Flag-Sox4 and Flag-Sox11 with two lentiviruses. Created with Biorender.com.

(B) IF for Tuj1 and Map2 (not shown) to identify newly formed neurons in NPCs after seven-day culture in differentiation medium. Empty CAG-IRES-EGFP vector was used as control in cells not transduced with Sox4/11. Transduced cells were identified by IF for GFP (Co and DKO) or FLAG (Co + Sox4/11 and DKO + Sox4/11). Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm.

(C) Quantification of transduced Tuj1+ cells from (B). Note the decrease in neuron number in DKO cultures compared to control, and the rescue in DKO cultures upon Sox4/11 expression (experiment performed twice in triplicates, representative experiment shown). Data are means ± SEM.

(D) Schematic illustrating the rescue DKO NPC differentiation defect with the GSK3 inhibitor CHIR99021 (chemical name: 6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile). Created with Biorender.com.

(E) IF for Tuj1 to identify newly formed neurons in NPCs after seven-day culture in differentiation medium. Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm.

(F) Quantification of Tuj1+ cells in control, control + CHIR (1 µM, 3 µM), DKO, and DKO + CHIR NPC cultures. Data are means ± SEM (experiment performed twice in triplicates, representative experiment is shown). Numbers below asterisks show fold change.

Data information: One way ANOVA test for all statistical analyses: ns, not significant; * p < 0.05, ** p < 0.01 *** p < 0.001. p = 0.97 (C, yellow bar); p = 0.00010 (C, red bar); p = 0.0023 (C, purple bar); p = 0.031 (F, yellow bar); p = 5 × 10-6 (F, grey bar); p = 2 × 10-5 (F, purple bar); p = 1 × 10-6 (F, green bar).
The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism (2020)
Figure 1

Figure 1. IKKε induces remodelling of cellular carbon metabolism by activating the serine biosynthesis pathway (SBP) and suppressing pyruvate oxidation

(A) Top panel: Scheme illustrating the tetracycline inducible Flp-In 293 system that controls the expression of HA-IKKε or HA-GFP. Bottom panel: Representative western blot showing induced expression of HA-IKKε in Flp-In 293 cells treated with doxycycline (Dox, 50 ng/ml) for 16 hours compared to endogenous IKKε in T47D, MDA-MB-231 and MDA-MB-468 breast cancer cell lines.

(B) Heatmap and hierarchical clustering of metabolite concentrations in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (Dox, 50 ng/ml, 16 hours) (n=5 technical replicates).

(C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates).

(D) Schematic representation of the 13C6-glucose and 15N2-glutamine labelling strategy to assess the effect of HA-IKKε induction on glycolysis, the TCA cycle and serine metabolism.

(E) Contribution of pyruvate and glucose-derived carbon to TCA cyle metabolites in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (n=5 technical replicates).

(F) Fractional enrichment of serine, malate and citrate 13C-isotopologues in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates).

(G) Fractional enrichment of the serine 15-N-isotopologue in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). m+1 shows the naturally occurring 13C isotopologue (n=5 technical replicates).

Data Information: In (A-G), Flp-In 293 HA-IKKε or GFP cells were treated with doxycycline (Dox) 50ng/ml, 16 hours. In (C, E-G), metabolite levels were normalised to the internal standard HEPES. In (C) and (E-G), data are presented as mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed Student's t-test or Mann-Whitney test).
The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism (2020)
Figure 2

Figure 2. The effect of IKKε silencing on metabolism of breast cancer cell lines

(A-B) Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (A) T47D and (B) MDA-MB-468 breast cancer cell lines.

(C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates).

(E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates).

(F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells. (n=5 technical replicates).

Data Information: In (C, E), metabolite levels were normalised to the internal standard HEPES. In (D, F), metabolite levels were normalised to total ion count. In (C-D) metabolite levels were scaled to maximum and minimum levels of each metabolite. In (E-F), data are presented as mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed Student's t-test).
The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism (2020)
Figure 3

Figure 3. IKKε inhibits mitochondrial metabolism via reducing PDH activity

(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells.

(B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells.

(C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines, measured using Seahorse XF96e or XF24 analysis.

(D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry.

(E) Principal component analysis of differentially phosphorylated substrates in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (100 ng/ml, 16 hours). The phosphoproteomes in the three clones were analysed by mass spectrometry as described in material and methods.

(F) Representative western blot showing l­evel of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours).

(G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours).

(H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells.

(I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry.

(J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry.

Data Information: All data are n≥3 biological replicates, with the exception of (E) which is n=3 technical replicates. Data are presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. In (C, G), data were normalised to total sample protein concentration. The following statistical tests were applied; in (A, B) two-way ANOVA with Fisher's LSD post-hoc tests, in (C, D, G) two-tailed paired Student's t-tests, in (H, I) one-way ANOVA with Fisher's LSD post-hoc tests, and in (J) one-way ANOVA with Tukey's multiple comparison tests.
The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism (2020)
Figure 4

Figure 4. IKKε stimulates SBP enzyme gene transcription via ATF4

(A) Representative western blot showing level of ATF4 in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours).

(B) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4 biological replicates)

(C) Representative western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.

(D) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in a non-silenced, non-treated control, and normalised to β-Actin (n=5 biological replicates).

(E) Representative western blot showing levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.

Data Information: In (B, D), data are presented as mean ± SEM, statistics were performed using log transformed fold change values. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, measured using two way ANOVA with Bonferroni post-hoc tests.