EMBO/soda-nel huggingface dataset:
Aryl Hydrocarbon Receptor is required for optimal B cell proliferation (2016)
Figure 2

Figure 2. AhR translocates to the nucleus and induces Cyp1a1 expression upon BCR engagement and in presence of FICZ in vitro.

A. Western blot of nuclear and cytoplasmic protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured with anti-IgM (α-IgM) for 4h, followed by the indicated treatment for 2h. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to Sam68 or Tubulin and compared to the DMSO-treated sample. Representative data of n = 3 independent experiments.

B. qPCR analysis of Cyp1a1 expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 24h as indicated. Cyp1a1 expression was normalized to Hprt1. Cyp1a1 expression among groups was normalized to Medium DMSO. n = 2 independent experiments; mean ± range.

C. Flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in splenic CD19+ cells isolated from Cyp1a1Cre R26R eYFP mice and cultured for 72h as indicated. Representative data of n = 3 independent experiments.

D. qPCR analysis of Cyp1a1 expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 24h. Cyp1a1 expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.
Aryl Hydrocarbon Receptor is required for optimal B cell proliferation (2016)
Figure 3

Figure 3. B cell activation in presence of 3-MC promotes Cyp1a1 transcription in vivo.

A. Host C57Bl/6 mice were injected with CD45.1+ splenocytes, isolated from SWHEL x Cyp1a1Cre R26R eYFP mice together with SRBC-HEL or SBRC-mock. The transferred mice were treated with 3-MC or vehicle. Readout at d7 post-challenge was eYFP expression in transferred CD45.1+ cells.

B. Representative flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in CD45.1+ cells harvested from mice challenged as in A. Gating is indicated above the plots. Representative data of n = 3 independent experiments. GC cells: CD95+ GL-7+; non-GC cells: CD95- GL-7-.

C, D. Summary of percentage of GC (C) and non-GC eYFP+ (D) cells from flow cytometry analysis. n = 3 independent experiments; lines indicate mean; one-way ANOVA, Tukey’s multiple comparison test.
Aryl Hydrocarbon Receptor is required for optimal B cell proliferation (2016)
Figure 4

Figure 4. AhR deficiency impairs B cell proliferation in vitro.

A. Flow cytometry analysis of CTV dilution in splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, solid grey) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, black) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. Representative data of n = 4-5 independent experiments.

B-D. Flow cytometry analysis of expansion index (B), % of divided cells (C) and replication index (D) of splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. n = 4-5 independent experiments; mean ± SEM; 2-way ANOVA, Sidak’s multiple comparison test.

E. Flow cytometry analysis of cell cycle distribution of splenic CD19+ cells purified from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 48h with 5 µg/ml α-IgM. Data representative of n = 2 independent experiments; lines indicate mean; 2-way ANOVA, Sidak’s multiple comparison test.
Aryl Hydrocarbon Receptor is required for optimal B cell proliferation (2016)
Figure 5

Figure 5. AhR deficiency impairs BCR-dependent B cell proliferation in vivo.

A, B. Flow cytometry analysis of distribution of CD45.1+ and CD45.2+ cells in indicated cell subsets purified from bone marrow (A) and lymph node (B) of sub lethally-irradiated Rag1-/- mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr+/+ (CD45.1+) and Ahr-/- (CD45.2+) mice. Dashed lines indicate 50% level. Representative data of n = 2 independent experiments; two-tailed paired t test.

C. Host CD45.1 mice were co-transferred with a 1:1 mixture of HEL-specific Ahr+/+ CD45.1+CD45.2+ splenocytes isolated from SWHEL AhR+/+ mice and HEL-specific Ahr-/- CD45.2+ splenocytes isolated from SWHEL AhR-/- mice in the presence of SRBC-HEL or SRBC-mock. Readout at d7 post-challenge was distribution of Ahr+/+ CD45.1+CD45.2+ vs Ahr-/- CD45.2+ cells.

D. Flow cytometry analysis of distribution of CD45.1+CD45.2+ and CD45.2+ cells harvested from host mice challenged as indicated above the dot plots. Representative data of n = 3 independent experiments.

E. Flow cytometry analysis of distribution of Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Indicated distribution is quantified relative to transferred cells. X symbols indicate SRBC-mock-treated control. Dashed line indicates 50% threshold. Representative data of n = 3 independent experiments; two-tailed paired t test.

F, G. Flow cytometry analysis of distribution (F) and cell numbers (G) of IgG1+ and IgG1- HEL3x-binding Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Representative data of n = 2 independent experiments; two-tailed paired t test.
Aryl Hydrocarbon Receptor is required for optimal B cell proliferation (2016)
Figure 6

Figure 6. Ahr-deficient B cells fail to up-regulate Ccno expression.

A, B. Tables showing top 20 down-regulated (A) and up-regulated (B) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.

C. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, black) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, white) mice, stimulated with 10 µg/ml anti-IgM for the indicated time points. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; two-way ANOVA, Sidak’s multiple comparison test.

D. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 8h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey’s multiple comparison test.

E. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM in presence or absence of 250 nM FICZ or 1 μM 3-MC. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey’s multiple comparison test.

F. Chromatin Immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter and an irrelevant region (-3.3 Kb from Ccno transcription starting site) in Ahr+/+ (black) and Ahr-/- (white) B cells 5h after activation with 10 µg/ml α-IgM + 20 ng/ml IL-4. 250 nM FICZ was added in the last hour of culture. Representative data of n = 2 independent experiments; mean ± SEM; two-way ANOVA, Sidak’s multiple comparison test.
Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interactions (2021)
Figure 1

Figure 1. Licochalcone B (LicoB) inhibits NLRP3 inflammasome activation.

(A) Licochalcone B (LicoB) structure.

(B) Cell Counting Kit 8 (CCK-8) was used to assess the viability of BMDMs treated with different doses of LicoB for 24 h.

(C-I) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control.

Data information: Error bars, mean ± SEM from three biological replicates. **P<0.01, ***P<0.001, and n.s.: not significant (One-way ANOVA with Dunnett's post-hoc test).
Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interactions (2021)
Figure 2

Figure 2. Licochalcone B is a specific inhibitor of the NLRP3 inflammasome. (A-C) BMDMs were primed with LPS and then treated with LicoB (20 μΜ) for 1 h, followed by stimulation with nigericin, ATP, poly (I:C), or MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then transfected with LPS. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs. (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured.

(D-F) LPS-primed BMDMs were treated with LicoB (20 μM) for 1 h and then stimulated with nigericin for 45 min, or poly(dA:dT)/Salmonella for 6 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (D). Caspase-1 activity (E) and IL-1β secretion (F) in the SN were measured.

Data information: Error bars, mean ± SEM from three biological replicates. ***P<0.001, and n.s.: not significant (unpaired Student's t-test).
Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interactions (2021)
Figure 3

Figure 3. Licochalcone B inhibits ASC oligomerisation during NLRP3 inflammasome activation.

(A-C) LPS-primed BMDMs were pre-treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h. Western blot analysis was used to detect cross-linked ASC in the Triton X-insoluble pellet, the arrow indicates ASC (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured again to verify NLRP3 activation.

(D) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, ATP, poly(I:C), and MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then stimulated with LPS.

(E) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, poly (dA:dT), or Salmonella.

Data information: Error bars, mean ± SEM from three biological replicates. ***P<0.001, and n.s.: not significant (One-way ANOVA with Dunnett's post-hoc test).
Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interactions (2021)
Figure 5

Figure 5. Licochalcone B inhibits NLRP3 inflammasome activation in vivo and ameliorates LPS-induced septic shock.

(A) Survival of WT mice pre-treated with vehicle, LicoB (20 mg/kg), LicoB (40 mg/kg), MCC950 (40 mg/kg), or LicoB (40 mg/kg) + MCC950 (40 mg/kg), followed by i.p. injection with LPS (20 mg/kg); mice survival was monitored for 72 h (n=10 mice per group).

(B-G) Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with LPS (20 mg/kg) and treated for 3 h. The levels of IL-1β (B, C) and TNF-α (D, E) in the serum and peritoneal lavage fluid were measured using ELISA. Quantification of peritoneal exudate cells (PECs) (F) and macrophages (F4/80+ cells) (G) using flow cytometry (n=8 mice per group).

(H-K) C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of ALT (H), AST (I), and CRE (J) were measured, along with the body weight (K) (n=8 mice per group).

Data information: Error bars, mean ± SEM from biological replicates. The significance of the differences was analysed using log-rank test (A), one-way ANOVA with Sidak's post-hoc test (B-G), unpaired Student's t-test (H-K). *P<0.05, **P<0.01, ***P<0.001, and n.s.: not significant vs. control group.
Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interactions (2021)
Figure 6

Figure 6. LicoB suppresses NLRP3-dependent peritonitis.

(A-D) Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with MSU (50 mg/kg) and treated for 6 h (n=6 mice per group). The levels of IL-1β in the peritoneal lavage fluid (A) and serum (B) were measured using ELISA. Quantification of peritoneal exudate cells (PECs) (C) and neutrophils (Ly6G and CD11b) (D) using flow cytometry.

Data information: Error bars, mean ± SEM from biological replicates. ***P<0.001, and n.s.: not significant vs. control group (One-way ANOVA with Sidak's post-hoc test).
Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interactions (2021)
Figure 7

Figure 7. Licochalcone B exhibits therapeutic effect in the non-alcoholic steatohepatitis (NASH) model.

(A-H) Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950, or a combination of LicoB and MCC950 (n=8 mice per group (B, C and I) or n=6 mice per group (E-H)). (A) Representative liver section images stained with H&E, Sirius Red, and Masson's trichrome stains. Scale bar: 0.5 cm (top row) and 200 μm (3 bottom ows). (B, C) The serum levels of ALT and AST (n=8 mice per group) were measured using ELISA. (D) Western blot analyses of pro-caspase-1, cleaved caspase-1, α-SMA, and ASC in the mice livers, as described in (A). (E-H) Real-time quantitative PCR was used to detect the mRNA levels of Col1a1 (E), TNF-α (F), IL-1β (G), and IL-18 (H) in the mice livers, as described in (A) (n=6 mice per group).

(I) The levels of IL-18 (I) in the serum was measured using ELISA (n=8 mice per group).

Data information: Error bars, mean ± SEM from biological replicates. *P<0.05, **P<0.01, ***P<0.001, and n.s.: not significant vs. control group (One-way ANOVA with Dunnett's post-hoc test).

Expanded View Figure Legends
Targeting the Warburg Effect via LDHA Inhibition Engages ATF4 Signaling 
for Cancer Cell Survival (2018)
Figure 1

Figure 1. LDHA is dispensable for melanoma cell proliferation under normoxic conditions.

A Proliferation of melanoma cell lines treated with LDHA inhibitor GSK-2837808A (10 μM) for the indicated duration under hypoxic (1% O2) and normoxic conditions.

B (left) Immunoblotting of LDHA in melanoma cell lines transfected with non-targeting siRNA (NT-siRNA) or LDHA-specific siRNA for 72 hr. (right) Proliferation of melanoma cell lines transfected with NT-siRNA or LDHA-specific siRNA for the indicated timepoints.

C-F Immunoblotting of LDHA (C) and proliferation under normoxic (D) and hypoxic (E and F) conditions of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs.

G Anchorage-independent growth of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs.

Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than 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. In (A) and (B), statistical comparison for the treatments that showed a significant change. In (B and F), statistical significance is denoted by blue (for si/sh-LDHA#1, hypoxia) and black (for sish-LDHA#2, hypoxia) stars respectively.
Targeting the Warburg Effect via LDHA Inhibition Engages ATF4 Signaling 
for Cancer Cell Survival (2018)
Figure 2

Figure 2. Loss of LDHA function increases glutamine dependence.

A Schematic representation of the regulation of glucose levels and glycolysis by LDHA activity. Suppressed NAD+ regeneration consequent to LDHA inhibition compromises the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an enzyme required for the conversion of glyceraldehyde 3-phosphate to 1,3-biphosphoglycerate. This in turn triggers a build-up of the glycolytic intermediates in the first few steps of glycolysis; increase in cellular levels of unused glucose; and a suppression in glucose uptake.

B, C Glucose (B) and glutamine (Gln, C) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr.

D Medium Gln levels after transfection of melanoma cells with non-targeting (NT) or LDHA-specific siRNA for 96 hr.

E GC-MS-based quantification of intracellular Gln levels in cells treated as in (B).

F qRT-PCR analysis of expression of the indicated SLC family transporters in MeWo (left) and A375 (right) melanoma cells after treatment with LDHAi for 48 hr.

G Immunoblotting of SLC1A5 in melanoma cells treated with LDHAi for 96 hr.

H Immunoblotting of LDHA and SLC1A5 in melanoma cell lines transfected with NT-siRNA or indicated si-LDHA for 96 hr.

I Immunoblotting of SLC1A5 and LDHA in MeWo cells stably transduced with empty vector (EV) or four different LDHA-­specific shRNAs.

J Immunoblotting of SLC1A5 in MeWo cells mock (NT-siRNA + DMSO) or si-SLC1A5-treated for 24 hr and then treated with LDHAi for 48 hr.

K Medium Gln levels (left), intracellular Gln levels (right) of MeWo cells treated as described for (J).

L Proliferation of melanoma cell lines for the indicated timepoints following treatment with LDHA-i, si-SLC1A5 or both.

M (left) Long-term viability of melanoma cells treated as indicated for 8 days. (right) corresponding quantification showing change in viability.

N Immunoblotting of SLC1A5 and cleaved caspase-3 in melanoma cells subjected to mock (NT-siRNA + DMSO) or si-SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr.

Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. N.D., not determined. 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 3

Figure 3. LDHA targeting induces intracellular essential amino acid levels and mTORC1 activity.

A GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells treated for 72 hr with LDHAi .

B GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells mock or si-SLC1A5-transfected for 24 hr and then treated with LDHAi for 48 hr.

C (left) Immunoblotting of SLC7A5 in MeWo cells transfected with non-targeting (NT) or SLC7A5-specific siRNAs for 48 hr. (right) GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells mock or si-SLC7A5-transfected for 24 hr and then treated with LDHAi for 48 hr.

D-F Immunoblotting of total and phosphorylated S6, ULK1, and eIF4EBP1 in melanoma cells (D) treated with LDHAi for 96 hr, (E) transfected with non-targeting (NT) or LDHA-specific siRNAs for 96 hr, and (F) si-SLC1A5-transfected for 24 hr followed by treatment with LDHAi for 72 hr.

G Model summarizing Gln uptake-mediated cellular build-up of EAA and the consequent mTORC1 activation.

H Proliferation of melanoma cells treated with LDHAi, m-TORC1 inhibitor (Rapamycin, 100 nM), or a combination of the two.

I (top) Long-term viability of melanoma cells treated as indicated for 10 days. (bottom) corresponding quantification showing change in viability.

J Immunoblotting of cleaved caspase-3 and p-(S780)-Rb in melanoma cells treated as indicated for 72 hr.

Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. 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 4

Figure 4. ATF4 orchestrates the survival response to LDHA targeting.

A, B Immunoblotting of ATF4 in melanoma cell lines (A) treated with the LDHA inhibitor LDHAi for 96 hr, (B) transfected with non-targeting (NT) or LDHA-specific siRNAs for 96 hr.

C Immunoblotting of ATF4 in MeWo cells stably transduced with empty vector (EV) or indicated sh-LDHAs.

D Immunoblotting of ATF4, c-MYC and SLC1A5 in melanoma cell lines treated with LDHAi for 2, 24, or 48 hr.

E Immunoblotting of ATF4 and SLC1A5 in melanoma cell lines mock- or si-ATF4-treated for 24 hr and then incubated with LDHAi for 48 hr.

F qRT-PCR analysis of SLC1A5 transcript levels in melanoma cell lines transfected with si-ATF4 for 24 hr and then incubated with LDHAi for 24 hr.

G GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells treated as indicated. Figures 3B and 4G were part of the same experiment and share Mock and LDHA-i controls.

H Proliferation of melanoma cell lines mock- or si-ATF4-transfected and incubated with LDHAi as indicated for up to 96 hr.

I qRT-PCR analysis of asparagine synthetase (ASNS) and glutamine-pyruvate transaminase 2 (GPT2) mRNA levels in the indicated cell lines after 24 hr treatment with LDHAi.

J GC-MS-based quantification of intracellular asparagine (Asn) and alanine (Ala) levels in MeWo and 1205Lu cells treated with LDHAi for 72 hr.

K, L GC-MS-based quantification of intracellular Asn and Ala levels in MeWo cells mock-transfected or transfected with si-ATF4 (K) or si-SLC1A5 (L) for 24 hr and then treated with LDHAi for 48 hr. (K) and (L) were part of the same experiment and shared Mock and LDHA-i controls.

M (left) Immunoblotting of ASNS in MeWo cells mock- or si-ASNS-transfected for 24 hr and then incubated with LDHAi for 48 hr. (right) Proliferation of MeWo cells mock- or si-ASNS-transfected for 24 hr and then incubated with LDHAi for the indicated times.

N Immunoblotting of GCN2, ATF4 and SLC1A5 in MeWo cells mock- or si-GCN2-treated for 24 hr and then incubated with LDHAi for 48 hr.

O 3H-Glutamine uptake in MeWo cells transfected with si-GCN2 and treated 48 hr later with LDHAi for 6 hr.

P Gln levels in the medium after treatment of MeWo cells with mock- or si-GCN2 for 24 hr and subsequent incubation with LDHA-i for 48 hr.

Q GC-MS-based quantification of intracellular Gln and essential amino acid levels in MeWo cells treated as in (N).

Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001