Journal: bioRxiv

Article Title: Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells

doi: 10.1101/2024.08.02.606284

Figure Lengend Snippet: (A) Pyruvate sits at a branch point between fermentation through lactate dehydrogenase (Ldh) or oxidative metabolism starting with the pyruvate dehydrogenase (Pdh) complex. Pyruvate dehydrogenase kinase isoforms (Pdks) are regulated by the products and substrates of the Pdh reaction, forming a negative feedback loop that reinforces an increase in lactate secretion when glycolytic flux is high. (B) Gene expression data from major CHO cell lines shows that Pdh complex subunits are all expressed, as are all four Pdk isoforms. Ldhb shows near zero expression (0 expression in CHO-S and DG44, below 0.1 FPKM in CHO-K1 and DXB11) while Ldha expression is high. All gene expression values are given in fragments per kilobase per million reads (FPKM), data sources are provided in Supplementary Table 4. (C) Simultaneously targeting Ldha and the Pdk genes for knockout using CRISPR/Cas9 leads to no detectable Ldha expression, as verified by chemiluminescent Western blot. (D) No phosphorylation of Pdh at S293 is detected in knockout lines. (E) Total Pdh remains stable in knockout lines. (F) Enzymatic assay verifies a loss of lactate dehydrogenase activity in knockout lines (shown here as percent of wildtype Ldh activity, n=3 technical replicates per cell line). (G) Measurement of maximum lactate levels during growth in batch culture ( , clones with 4 Pdk knockouts) shows that lactate cannot be detected (n=2 shake flasks per cell line). Data shown as mean ± standard deviation (uncertainty due to variance in background in (F) accounted for via propagation of error). Gene abbreviations are as follows: Pdk1-pyruvate dehydrogenase kinase 1, Pdk2-pyruvate dehydrogenase kinase 2, Pdk3-pyruvate dehydrogenase kinase 3, Pdk4-pyruvate dehydrogenase kinase 4, Pdha1-pyruvate dehydrogenase E1 alpha 1 subunit, Pdhb-pyruvate dehydrogenase E1 beta subunit, Dlat-dihydrolipoamide S-acetyltransferase, Dld-dihydrolipoamide dehydrogenase, Ldha-lactate dehydrogenase A, Ldhb-lactate dehydrogenase B.

Article Snippet: CHO-S TM cells (Gibco Cat. # A11557-01) were transfected using either FreeStyle MAX reagent (Gibco Cat. # 16447100) or FuGENE HD reagent (Promega Cat. # E2311).

Techniques: Expressing, Knock-Out, CRISPR, Western Blot, Enzymatic Assay, Activity Assay, Clone Assay, Standard Deviation

Journal: bioRxiv

Article Title: Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells

doi: 10.1101/2024.08.02.606284

Figure Lengend Snippet: Warburg-null cell lines (n=2 shake flasks for each clone) remain proliferative with (A) a growth rate comparable to WT while (B) drastically reducing their glucose uptake rate. This effect is observed whether in-frame mutations are counted as knockouts (left) or wildtype (right). The growth of wildtype CHO-S is shown in blue. Long-term passaging over approximately 87 days shows that Warburg-null clones (C) have higher residual glucose in culture while (D) not producing lactate and (E) maintain a comparable growth rate to WT cells (n=1 shake flask for each cell line in long-term passaging experiment).

Article Snippet: CHO-S TM cells (Gibco Cat. # A11557-01) were transfected using either FreeStyle MAX reagent (Gibco Cat. # 16447100) or FuGENE HD reagent (Promega Cat. # E2311).

Techniques: Passaging, Clone Assay

Journal: Cell reports

Article Title: BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis

doi: 10.1016/j.celrep.2022.111826

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: NVP-TAE 226 , Adooq Bioscience , A11507.

Techniques: Virus, Recombinant, Modification, Staining, Membrane, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Transfection, Saline, Protein Array, Control, Software