cervical adenocarcinoma hela  (ATCC)

Journal: bioRxiv

Article Title: Adaptive plasticity of aspartate metabolism in succinate dehydrogenase-deficient cancer cells

doi: 10.64898/2026.05.18.726122

Figure Lengend Snippet: (A) Schematic illustrating the generation of clonal SDHB-knockout 143B cells (B) Representative western blot showing levels of SDHB and tubulin loading control in wild-type parental cells (WT) and SDHB knockout clones 1-5 (C) Absolute proliferation rates (mean +/- S.D.) of SDHB KO clones 1-3 with or without 20 mM aspartate supplementation. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (D) Schematic illustrating the effects of CI/SDH co-suppression on alternative aspartate synthesis and cell fitness. (E) Relative whole-cell aspartate levels (mean +/- S.D.), measured using LCMS, in SDHB-KO clones 1-3 after 6 hours of treatment with vehicle control or 50 nM rotenone (ROT). Levels are normalized to the vehicle-treated condition in each respective clone. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (F) Absolute proliferation rates (mean +/- S.D.) of SDHB KO clones 1-3 with or without 50nM rotenone (ROT) supplementation. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (G) Average inter-passage proliferation rates (see methods) of five SDHB-knockout clones over ∼120 cumulative population doublings. See for plots highlighting individual clones. (H) Absolute proliferation rates (mean +/- S.D.) of early passage (EP) and late passage (LP) clones 1-3 with or without 20 mM aspartate supplementation. Statistical significance determined using an ordinary two-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance. (n=3) (I) Representative western blot showing levels of NDUFA8 and tubulin loading control in wild-type parental cells (WT), an previously-adapted (PA) SDHB-KO clone that adapted by suppressing complex I , and late passage (LP) SDHB knockout clones 1-3. Normalized NDUFA8 band densities are shown below each respective lane. (J) normalized oxygen consumption rate (OCR) traces (mean +/- S.D.) for wild-type parental cells (WT), parental cells treated with 5 uM Atpenin A5 (WT + AA5), a previously adapted, CI-deficient SDHB-KO clone (PA), and early passage (EP) and late passage (LP) SDHB knockout clones 1-3. Injections of rotenone (ROT) and antimycin (ANTI) are shown with arrows. (n=7-12) (K) basal mitochondrial oxygen consumption rates (OCR) (mean +/- S.D.) quantified from data in (J). Statistical significance determined using an ordinary one-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance. (n=7-12)

Article Snippet: 143B cells were obtained from ATCC, authenticated using small tandem repeat (STR) profiling (ATCC), and monitored at least twice yearly for mycoplasma contamination (MycoProbe, R&D Systems).

Techniques: Knock-Out, Western Blot, Control, Clone Assay

Journal: bioRxiv

Article Title: Adaptive plasticity of aspartate metabolism in succinate dehydrogenase-deficient cancer cells

doi: 10.64898/2026.05.18.726122

Figure Lengend Snippet: (A) Schematic illustrating the role of the mitochondrial pyruvate carrier (MPC) in alternative aspartate synthesis and its knockout using CRISPR/Cas9 (B) Representative western blot showing levels of MPC1, V5-tagged MPC1, and tubulin loading control in parental, MPC1-knockout (KO) and MPC1-addback (AB) cells for late passage SDHB-KO clone 2 (2 LP) and 2 EP NDUFA8-KO (A8KO). (C) Proliferation rates (mean +/- S.D.) of MPC KO and addback (AB) 2 LP with or without 20 mM aspartate supplementation. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (D) Identical setup as in (C), with A8KO cells. (n=3) (E) Relative aspartate levels (mean +/- S.D.) of MPC KO and addback (AB) 2 LP and A8KO 24 hours after media change. Levels are normalized to each corresponding AB. Statistical significance determined using an ordinary two-way ANOVA and an uncorrected Fisher’s LSD with a single pooled variance. (n=3) (F) Schematic illustrating the role of pyruvate carboxylase (PC) in alternative aspartate synthesis and its knockout using CRISPR/Cas9 (G) Representative western blot showing levels of PC, V5-tagged PC, and tubulin loading control in parental, MPC1-knockout (KO) and MPC1-addback (AB) 2 LP and A8KO cells (H) Proliferation rates (mean +/- S.D.) of PC KO and addback (AB) 2 LP with or without 20 mM aspartate supplementation. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (I) Identical setup as in (H), with A8KO cells. (n=3) (J) Relative aspartate levels (mean +/- S.D.) of PC KO and addback (AB) 2 LP and A8KO 24 hours after media change. Levels are normalized to each corresponding AB. Statistical significance determined using an ordinary two-way ANOVA and an uncorrected Fisher’s LSD with a single pooled variance. (n=3) (K) Schematic illustrating compartmentalized aspartate synthesis by GOT1/2 (L) Representative western blot showing levels of GOT1, GOT2, and tubulin loading control in parental, GOT1-KO and GOT2-KO 2 LP and A8KO cells (M) Proliferation rates (mean +/- S.D.) of GOT1- and GOT2-KO 2 LP and A8KO cells in all combinations of 20 mM aspartate and 50 nM rotenone treatments. Statistical significance determined using an ordinary two-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance (n=3) (N) Relative aspartate levels (mean +/- S.D.) of parental, GOT1-, and GOT2-KO 2 LP cells 24 hours after treatment with vehicle control or 50 nM rotenone. Levels are normalized to each corresponding parental cell vehicle treatment Statistical significance determined using an ordinary two-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance. (n=3) (O) Identical setup as in (N), with A8KO cells (n=3) (P) schematic depicting aspartate synthesis in wild-type and SDH-deficient 143B cells before and after adaptation along two distinct trajectories. Abbreviations: GOT1, glutamic-oxaloacetic aminotransferase 1; GOT2, glutamic-oxaloacetic aminotransferase 2; ASP, aspartate; OAA, oxaloacetate; cyto., cytosol; mito., mitochondria; GLC, glucose; GLN, glutamine; PYR, pyruvate; SDH, succinate dehydrogenase; SUC, succinate; PC, pyruvate carboxylase; CI, respiratory complex I; CIT, citrate.

Article Snippet: 143B cells were obtained from ATCC, authenticated using small tandem repeat (STR) profiling (ATCC), and monitored at least twice yearly for mycoplasma contamination (MycoProbe, R&D Systems).

Techniques: Knock-Out, CRISPR, Western Blot, Control

Journal: bioRxiv

Article Title: Adaptive plasticity of aspartate metabolism in succinate dehydrogenase-deficient cancer cells

doi: 10.64898/2026.05.18.726122

Figure Lengend Snippet: (A) Schematic illustrating the role of the mitochondrial pyruvate carrier (MPC) in alternative aspartate synthesis and its inhibition using UK-5099 (B) Proliferation rates (mean +/- S.D.) of late passage SDHB-KO clone 1 (1 LP) following treatment with the indicated doses of UK-5099, with or without 20 mM aspartate supplementation. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (C) Identical setup as in (B), with late passage SDHB-KO clone 2 (2 LP) (n=3) (D) Identical setup as in (B), with late passage SDHB-KO clone 3 (3 LP) (n=3) (E) Identical setup as in (B), with NDUFA8-KO early passage SDHB KO clone 2 (A8KO) (n=3) (F) Identical setup as in (B), with wild-type 143B parental cells (n=3) (G) Data from the same experiment as in , with proliferation rates (mean +/- S.D.) calculated between days 2-3 of the 3-day assay.

Article Snippet: 143B cells were obtained from ATCC, authenticated using small tandem repeat (STR) profiling (ATCC), and monitored at least twice yearly for mycoplasma contamination (MycoProbe, R&D Systems).

Techniques: Inhibition

Journal: bioRxiv

Article Title: Adaptive plasticity of aspartate metabolism in succinate dehydrogenase-deficient cancer cells

doi: 10.64898/2026.05.18.726122

Figure Lengend Snippet: (A) Schematic depicting U- 13 C glucose tracing to monitor aspartate synthesis via the MPC-PC-GOT2 pathway (B) Absolute isotopolog distributions (mean +/- S.D.) for aspartate in early passage (EP) or late passage (LP) SDHB-KO clone 2 following 24 hours of tracing in media containing U- 13 C glucose. (n=3) (C) Representative western blots showing levels of MPC1, PC, GOT2, and tubulin loading controls in 2 EP/LP (D) Quantification of PC protein levels from 2 EP/LP using western blotting. Statistical significance determined using Welsh’s t-test. (n=3) Quantification blot available in Source Data. (E) Representative western blots showing levels of PC, V5-tagged PC, and tubulin loading control in 2 EP or PC-overexpressing 2 EP (2 EP PC OE). (F) Absolute isotopolog distributions (mean +/- S.D.) for aspartate in 2 EP and 2 EP PC OE after 24 hours of tracing in media containing U- 13 C glucose. (n=3) (G) Proliferation rates (mean +/- S.D.) of 2 EP and 2 EP PC OE with or without 20 mM aspartate (ASP) supplementation. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (H) Representative western blots showing levels of PC and tubulin loading control in wild-type 143B cells or PC-overexpressing 143B cells (143B PC OE). (I) Proliferation rates (mean +/- S.D.) of 143B and 143B PC OE at the indicated doses of Atpenin A5 (AA5) with or without 20 mM aspartate (ASP) supplementation. Proliferation rates are normalized to the 0 μΜ ΑΑ5 treatment condition for each respective trace. P-values shown correspond to statistical testing comparing the relative proliferation rate of 143B vs. 143B PC OE at the indicated dose of AA5. Statistical significance determined using an ordinary two-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance. (n=3) (J) Schematic illustrating the approach in (K), described further in the main text. (K) Mean SDHA-D DepMap gene effect scores for the top and bottom 5% of expressors of the indicated metabolic genes. Statistical significance determined using an ordinary one-way ANOVA and Sidak’s multiple comparisons test with a single pooled variance. (n=57)

Article Snippet: 143B cells were obtained from ATCC, authenticated using small tandem repeat (STR) profiling (ATCC), and monitored at least twice yearly for mycoplasma contamination (MycoProbe, R&D Systems).

Techniques: Western Blot, Control