In Jurkat cells, the response to LY294002 depended on BRAP2 status. At 30 μM, p-Akt fell at 24 h in mock cells and recovered toward baseline by 48 h; little reduction occurred in BRAP2-deficient cells. BRAP2 deletion also attenuated G1 accumulation and inhibition of cell accumulation, while the MTT signal declined in both genotypes. [2]
In representative RT-PCR and immunoblot panels from GLI1-overexpressing HL60 and NB4 cells, LY294002 (20 μM) was associated with weaker p-AKT bands while GLI1 transcript and protein bands remained visible. These qualitative observations do not establish unchanged GLI1 expression, a total-AKT-normalized phosphorylation response, or chemotherapy sensitization. [3]
PI3K and CK2 inhibition in recombinant kinase assays
Biochemical assay · Recombinant p110α and human CK2 [1]
Table 1 reports mean IC50 values of 0.186 μM for p110α and 0.098 μM for CK2. Activity against CK2 limits interpretation as a selective PI3K probe.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response assays; input concentration range not specified in acquired main text.
- Exposure duration
- PI3K assay: 1 h at 24 °C; CK2 IC50 assay duration not specified in acquired main text.
- Method
- Recombinant kinase inhibition; PI3K radiometric assay uses 1 μM ATP.
- Reported limits
- Table 1 gives means without n or dispersion. CK2 IC50-specific ATP, substrate, vehicle and normalization control details are not explicit in acquired main text; the separate 10 μM ATP kinase-panel screen is not assigned to these IC50 values. SI not retrieved.
Methods and source locations
Compound reported in the paper: LY294002; Compound label in the paper: LY294002
Applies to the named research compound and reported experimental material; no validation of a current Adooq lot.
Table 1; Materials and methods: Reagents and PI3K activity assays; Results: Evaluation of putative targets of LY294002
Inhibition of recombinant p110α: Radiometric PI3K activity assay — Table 1, LY294002 row, p110α column; PI3K activity assays
Inhibition of recombinant human CK2: Recombinant CK2 inhibition as reported in Table 1 — Table 1, LY294002 row, CK2(h) column
BRAP2 status modifies the Akt phosphorylation response
Cell-based study · Jurkat mock-transfected and BRAP2-deficient cells [2]
At 30 μM, LY294002 lowered p-Akt in mock cells at 24 h, followed by recovery toward baseline at 48 h. BRAP2-deficient cells showed little reduction. These are representative immunoblot observations, not a target-selectivity assay.
Literature-Reported Treatment Conditions
- Treatment concentration
- 30 μM
- Exposure duration
- 24 and 48 h; 0 h baseline
- Method
- Cell treatment followed by immunoblotting for p-Akt, total Akt and β-actin.
- Vehicle
- All inhibitors dissolved in DMSO; final DMSO concentration not stated.
- Reported limits
- No independent replicate number or uncertainty is given specifically for the Figure 1E blot. A time-matched solvent-only series is not shown; comparison includes 0 h baseline.
Methods and source locations
Compound reported in the paper: LY294002; Compound label in the paper: LY294002
Applies to the named research compound and reported experimental material; no validation of a current Adooq lot.
Figure 1A,C,E and caption; Results: BRAP2 regulated the signal transduction; Methods: Reagents and SDS-PAGE and western blotting
p-Akt in Jurkat mock cells: p-Akt with total Akt and β-actin controls — Figure 1E, mock lanes
p-Akt in BRAP2-deficient Jurkat cells: Same blot assay; BRAP2 loss checked in Figure 1A — Figure 1A,E, ΔBRAP2 lanes
BRAP2 deletion alters cell-cycle and growth responses
Cell-based study · Jurkat mock-transfected and BRAP2-deficient cells [2]
LY294002 increased G1 accumulation and inhibited cell accumulation more strongly in mock cells than in BRAP2-deficient cells. The 24 h MTT dose response declined in both genotypes; BRAP2 loss did not protect the MTT endpoint. MTT alone does not establish apoptosis.
Literature-Reported Treatment Conditions
- Treatment concentration
- MTT: 0–50 μM dose series; cell-cycle and cell-count assays: 30 μM
- Exposure duration
- MTT and cell-cycle: 24 h; cell count: 48 h
- Method
- MTT reduction, propidium-iodide DNA-content flow cytometry and direct cell counting.
- Vehicle
- Inhibitors dissolved in DMSO; final solvent concentration not stated.
- Reported limits
- Controls are described as nonadditive/untreated; exact solvent matching is not explicit. No numerical bar/curve estimates are transcribed.
Methods and source locations
Compound reported in the paper: LY294002; Compound label in the paper: LY294002
Applies to the named research compound and reported experimental material; no validation of a current Adooq lot.
Figure 6A-C and caption; Methods: MTT assay, SubG1 and cell cycle analysis, Measurement of cell proliferation rate and Statistical analysis; Figure 1A-B baseline controls
MTT viability signal in both genotypes: MTT; Figure 6 caption: n=3, error bars SD — Figure 6A and Results: Loss of BRAP2 suppressed cell cycle arrest
G1-phase proportion: PI flow cytometry; caption n=3, SD; **P<0.01 vs. mock cells — Figure 6B and caption
Cell accumulation at 48 h: Direct cell count; caption n=3, SD; ***P<0.001 vs. mock cells — Figure 6C and caption
Qualitative p-AKT and GLI1 band patterns after LY294002
Cell-based study · Wild-type and GLI1-overexpressing HL60 and NB4 leukemia cell lines [3]
Figure 3 shows weaker p-AKT bands in LY294002-treated GLI1-overexpressing HL60 and NB4 cells than in the untreated overexpressing lanes; GLI1 transcript and protein bands remain visible. Wild-type and untreated overexpressing lanes provide baseline comparisons, and GANT61 is a parallel comparator. No total-AKT-normalized effect, quantitative absence of GLI1 suppression, pathway directionality, or chemotherapy sensitization is established by these representative panels.
Literature-Reported Treatment Conditions
- Treatment concentration
- 20 μM
- Exposure duration
- The Methods drug-response paragraph reports 72 h before subsequent measurements; Figure 3 itself does not restate exposure duration.
- Method
- Drug treatment followed by transcript RT-PCR and p-AKT/GLI1 immunoblotting.
- Co-treatment
- GANT61 (20 μM) tested in parallel, not combined with LY294002 in Figure 3.
- Reported limits
- Drug solvent and final vehicle concentration not specified. Untreated lane labels do not establish a matched solvent control. Figure 3 has no numerical densitometry, total AKT panel or panel-specific n; general statistics state at least three independent repeats.
Methods and source locations
Compound reported in the paper: LY294002; Compound label in the paper: LY294002
Applies to the named research compound and reported experimental material; no validation of a current Adooq lot.
Figure 3a-d and caption; Results: Overexpression of GLI1 upregulated p-AKT; Methods: Cell culture and transfection (drug-response paragraph), RT-PCR and Western blot analysis
Displayed p-AKT band signal in GLI1-overexpressing HL60 and NB4 cells: Representative p-AKT (Ser473) immunoblot with GAPDH loading control; total AKT is not displayed in Figure 3c-d, so no p-AKT/total-AKT ratio is available. — Figure 3c-d; Western blot analysis
Persistence of displayed GLI1 transcript and protein bands: Representative RT-PCR panels with β-actin and immunoblots with GAPDH; wild-type and untreated overexpressing lanes shown. No numerical densitometry or equivalence test is provided for these panels. — Figure 3a-d and corresponding Results paragraph
