In stimulated Jurkat T cells, SP600125 inhibited c-Jun phosphorylation with a reported cellular IC50 of 5–10 μM. ERK1/2 phosphorylation and IκBα degradation were not blocked at 50 μM, whereas partial inhibition of the p38/ATF2 pathway was observed at that concentration. [1]
In rabbit chondrocyte scaffold cultures, SP600125-containing schedules increased DNA content but could reduce cartilage matrix-related measures. Continuous combination with chondrogenic medium performed best among the six tested schedules; the benefit depended on culture context and did not establish selective JNK causation. [3]
ATP-competitive inhibition of recombinant JNK2
Biochemical assay · Recombinant human JNK2 with c-Jun phosphoacceptor [1]
Varying ATP and inhibitor concentrations supported ATP-competitive inhibition. Reversibility was reported in Results as data not shown. This experiment establishes JNK inhibition, not kinome-wide selectivity.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0, 50, 100 and 300 nM SP600125 in Figure 2a
- Method
- Cell-free kinetic assay; ATP varied
- Reported limits
- Assay duration and replicate number not given in the acquired Figure 2a caption; full kinetic method is referenced to prior work. ± is not identified as SD or SEM.
Methods and source locations
Compound reported in the paper: SP600125
Compound-level research evidence; no salt conversion or validation of the current Adooq product lot.
Figure 1a; Figure 2a and caption; Results opening paragraphs; Biochemical Characterization of JNK Enzyme Activity
JNK2 ATP-competitive inhibition constant: Nonlinear least-squares fit to competitive inhibition kinetics; JNK2 9 nM and c-Jun 2 μM; ATP varied. Meaning of ± and replicate number not specified in caption. — Figure 2a and caption
c-Jun phosphorylation in stimulated Jurkat cells
Cell-based study · Human Jurkat T cells stimulated with PMA, anti-CD3 and anti-CD28 [1]
SP600125 reduced c-Jun phosphorylation with a reported cellular IC50 of 5–10 μM. At 50 μM, phospho-ERK1/2 and IκBα degradation were not blocked, but partial inhibition of phospho-p38 and ATF2 was observed. These pathway comparisons do not establish target-exclusive action.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0, 1, 5, 10, 25 and 50 μM
- Exposure duration
- 10-min pretreatment followed by 30-min stimulation
- Method
- PMA 50 ng/ml, anti-CD3 0.5 μg/ml and anti-CD28 2 μg/ml; immunoblot readout
- Vehicle
- DMSO stock described in shared methods; exact final DMSO percentage for Figure 3a not specified
- Reported limits
- Replicate count and exact final vehicle percentage not stated for Figure 3a.
Methods and source locations
Compound reported in the paper: SP600125
Compound-level research evidence; no salt conversion or validation of the current Adooq product lot.
Figure 3a and caption; Results paragraph beginning On the basis; Use of SP600125 in Cell Culture
Inhibition of stimulated c-Jun phosphorylation: Immunoblot analysis of the same lysates; nonphosphorylated IKK-1 used as loading indicator. IC50 is the authors’ textual estimate, not a new curve fit. — Figure 3a; adjacent Results
Comparator pathway readouts: Immunoblot comparison with unstimulated and stimulated no-inhibitor lanes. — Figure 3a and adjacent Results
Broad kinase inhibition in an independent profiling panel
Biochemical assay · Purified protein kinase panel; inhibitor-free incubations as reference [2]
At 1 μM, SP600125 inhibited JNK isoforms and several other kinases. The authors identified PKD1, CHK2, Aurora B/C, MELK, CK1, DYRK2/3 and HIPK3 among off-targets and advised against interpreting SP600125 as a specific JNK probe in cells.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 μM
- Exposure duration
- 30 min
- Method
- Radiometric phosphorylation assay at 21°C; ATP at or below Km. JNK1/2 and Aurora B: 20 μM ATP; JNK3 and MELK: 50 μM ATP; Aurora C: 5 μM ATP.
- Reported limits
- Final inhibitor solvent percentage and meaning of table ± not specified in selected main-text methods/caption.
Methods and source locations
Compound reported in the paper: SP600125
Compound-level research evidence; no salt conversion or validation of the current Adooq product lot.
Table 7 SP600125 column and caption; Protein kinase inhibitors and other materials; Protein kinase assays; JNK inhibitors subsection
Kinase activity relative to inhibitor-omitted controls: Radiometric kinase assays. Caption: averages of duplicate determinations; each experiment repeated two or three times with similar results. Table does not identify ± as SD or SEM. — Table 7, SP600125 (1 μM) column and caption
Culture schedule affects engineered cartilage matrix and cell content
Cell-based study · Passage-two rabbit auricular chondrocytes on PGA/PLA scaffolds; six culture modes at high or low initial cell-seeding density [3]
Continuous chondrogenic medium plus SP600125 (LCM+SP) gave the highest DNA content and stronger cartilage matrix/mechanical measures among the six schedules. SP600125 first followed by regular medium produced more DNA but less collagen/GAG and lower stiffness than low-density regular medium. Thus greater cell content did not consistently mean greater cartilage matrix production.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.02 mg/ml in SP-containing culture groups
- Exposure duration
- 8 weeks total; phased groups: first 10 days, then 46 days
- Method
- Six schedules defined in Table 1; high density 6.0 × 10^7 cells/ml and low density 3.0 × 10^7 cells/ml
- Co-treatment
- Continuous LCM+SP combines SP600125 with serum-free chondrogenic medium containing TGFβ1, dexamethasone, IGF-1 and other supplements. Comparators: HRM, LRM, LRM+SP-RM, LRM-CM and LRM+SP-CM.
- Reported limits
- SP600125 stock solvent/final vehicle and catalog/lot not reported in acquired main text. Donor/biological versus technical replicate count not clearly resolved.
Methods and source locations
Compound reported in the paper: SP600125
Compound-level research evidence; no salt conversion or validation of the current Adooq product lot.
Table 1; Materials and methods Group design; Figures 3–5 and captions; Tissue-engineered cartilage regeneration in vitro
Cartilage matrix and mechanical properties after culture: Histology; hydroxyproline and GAG assays; mechanical testing. No numerical values digitized from bars. — Figures 3–4; Table 1; Results quantitative determinations
DNA content and cartilage-related gene expression: PicoGreen DNA content is a proxy for cell content; qRT-PCR normalized to β-actin. Quantitative data described as at least three replicates, mean ± SD; one-way ANOVA with Student–Newman–Keuls post hoc testing. — Figures 4F and 5; Results characteristic gene expression; Statistical analysis
