In DU145 prostate cancer cells, SB 202190 at 50 µM for 24 h caused cytoplasmic vacuolation and reduced PI(3,5)P2 immunofluorescent staining relative to DMSO. These cellular observations accompany the biochemical evidence for PIKfyve inhibition. [2]
SB 202190 produced opposite growth responses across colorectal cancer cell lines: SRB readouts decreased in RKO, CACO2 and SW480 but increased in HCT116, SW1116 and SW620. Its effect on cancer-cell growth therefore depended on the tested model. [3]
In HepG2 and PLC5 cells, pretreatment with 20 µM SB 202190 for 1 h reduced the mature SREBP2 immunoblot signal associated with subsequent 20 µM GSK343 exposure for 6 h. This combination experiment supports an effect on SREBP2 processing under those conditions. [4]
p38-pathway inhibition and vacuolation controls in HT29 cells
Cell-based study · Human HT29 colorectal adenocarcinoma cells [1]
SB202190 suppressed stimulated downstream p38-pathway phosphorylation. SB202190 and SB203580 caused vacuolation, whereas BIRB-796 and VX-745 inhibited the pathway without comparable vacuolation. These controls show that p38 inhibition alone was insufficient for this phenotype in HT29 cells.
Literature-Reported Treatment Conditions
- Treatment concentration
- SB202190 5 µM; comparators: SB203580 5 µM, SB202474 5 µM, BIRB-796 1 µM, VX-745 2 µM
- Exposure duration
- Vacuolation: 24 h. Pathway control: inhibitor pretreatment 1 h followed by anisomycin for 30 min.
- Method
- Microscopy and immunoblotting
- Vehicle
- DMSO
- Co-treatment
- Anisomycin 10 µg/mL only in the stimulated pathway-control experiment
- Reported limits
- Final DMSO fraction and biological replicate count for the microscopy comparison are not specified in the read caption.
Methods and source locations
Compound reported in the paper: SB 202190; Compound label in the paper: SB202190
Evidence concerns the material used by these authors; no validation of an Adooq lot or salt conversion is implied.
Figure 5A,B,E and caption; Results inhibitor comparison; Materials and Methods
Anisomycin-stimulated phosphorylation of MK2 Thr334, Hsp27 Ser82 and keratin 20 Ser13: Western blot; total Hsp27 and GAPDH controls — Figure 5E and caption; Results: p38 MAPK inhibition is not sufficient; Materials; Immuno blotting
Vacuolated HT29 cells after inhibitor treatment: Crystal-violet microscopy; caption describes means from three independent images, not three biological experiments — Figure 5A,B and caption
Direct inhibition of recombinant PIKfyve
Biochemical assay · Recombinant full-length GST-PIKfyve (Carna Biosciences #11-118) [2]
SB202190 inhibited PIKfyve-dependent ATP hydrolysis in an ADP-Glo assay, establishing an additional biochemical target under these assay conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- SB202190 inhibitor series 0–2000 nM; this range is an assay input, not an IC50
- Exposure duration
- 30 min inhibitor preincubation at room temperature; kinase reaction 2 h
- Method
- Cell-free ADP-Glo kinase assay
- Vehicle
- 0.02% DMSO
- Reported limits
- Figure 5C and shared statistics do not identify the error type for the reported ±36.2 nM or a panel-specific n. Shared statistics state experiments were performed at least three times unless otherwise indicated.
Methods and source locations
Compound reported in the paper: SB 202190; Compound label in the paper: SB202190
Evidence concerns the material used by these authors; no validation of an Adooq lot or salt conversion is implied.
Figure 5C and caption; Results PIKfyve subsection; Reagents; PIKfyve inhibition assay; Statistical analyses
Inhibition of recombinant PIKfyve ATP hydrolysis: ADP-Glo luminescence; PI(3)P 50 µM, phosphatidylserine 400 µM and ATP 50 µM; comparison with SB203580 and YM201636 — Figure 5C; Results: SB203580 and SB202190 stimulate vacuolation through off-target inhibition of PIKfyve; Methods: PIKfyve inhibition assay
Vacuolation and reduced PI(3,5)P2 staining in DU145 cells
Cell-based study · Human DU145 prostate cancer cells [2]
SB202190 produced visible vacuolation and reduced PI(3,5)P2 immunofluorescence relative to DMSO. The cellular concentration greatly exceeds the cell-free PIKfyve IC50; these endpoints do not measure intracellular target selectivity.
Literature-Reported Treatment Conditions
- Treatment concentration
- SB202190 50 µM; separate comparator groups: SB203580 50 µM and YM201636 1 µM
- Exposure duration
- 24 h
- Method
- Cell exposure followed by phase-contrast or immunofluorescence microscopy
- Vehicle
- DMSO control
- Reported limits
- Panel-specific replicate count, error-bar definition and final cellular DMSO fraction are not specified in the read caption/shared statistics. Immunostaining is a relative signal rather than an absolute lipid mass assay.
Methods and source locations
Compound reported in the paper: SB 202190; Compound label in the paper: SB202190
Evidence concerns the material used by these authors; no validation of an Adooq lot or salt conversion is implied.
Figure 5A,D and caption; Results PIKfyve subsection; Reagents; Cell culture and transfections; Immunofluorescence and western blotting
Cytoplasmic vacuolation: Phase-contrast microscopy — Figure 5A and caption
PI(3,5)P2 immunofluorescent dots per cell: Immunofluorescence microscopy; anti-PI(3,5)P2 reagent Z-P035 listed in Reagents — Figure 5D and caption; Reagents; Immunofluorescence and western blotting
Opposite growth responses across colorectal cancer cell lines
Cell-based study · Human RKO, CACO2, SW480, HCT116, SW1116 and SW620 colorectal cancer cells [3]
SB202190 reduced SRB growth readouts in RKO, CACO2 and SW480 but increased them in HCT116, SW1116 and SW620. Response depended on the cell model.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response: plotted SB202190 0, 1, 5 and 10 µM; time course: 10 µM
- Exposure duration
- 48 h for concentration response; plotted time course through 72 h
- Method
- SRB growth assay
- Vehicle
- DMSO (Figure 1 vehicle comparator)
- Reported limits
- Final DMSO fraction is not specified in the read methods. SRB reflects cellular biomass; it is not a direct count of viable cells.
Methods and source locations
Compound reported in the paper: SB 202190; Compound label in the paper: SB202190
Evidence concerns the material used by these authors; no validation of an Adooq lot or salt conversion is implied.
Figure 1A and caption; Results 2.1; Methods 4.1,4.5,4.10
Cell growth relative to vehicle control: Sulforhodamine B (SRB) assay; Figure 1A reports means ± s.d. and uses the wording “n = 3 in triplet”; biological versus technical replication is not further resolved here — Figure 1A and caption; Results 2.1; Methods 4.5
Suppression of GSK343-associated SREBP2 processing
Cell-based study · Human HepG2 and PLC/PRF/5 (PLC5) hepatic cancer cell lines [4]
SB202190 pretreatment reduced the GSK343-associated mature SREBP2 immunoblot signal in both cell lines. This is a pharmacological combination observation and alone cannot establish exclusive p38 dependence.
Literature-Reported Treatment Conditions
- Treatment concentration
- SB202190 20 µM; GSK343 20 µM (Figure 10B lane labels)
- Exposure duration
- SB202190 pretreatment 1 h followed by GSK343 exposure for 6 h
- Method
- Immunoblot of whole-cell lysates
- Vehicle
- DMSO comparator shown in Figure 10B
- Co-treatment
- GSK343; separate inhibitor-only and GSK343-only conditions are shown
- Reported limits
- Final DMSO concentration and a panel-specific biological replicate count are not specified. Figure 10C viability data are excluded because its caption says MTS while shared methods describe MTT.
Methods and source locations
Compound reported in the paper: SB 202190; Compound label in the paper: SB202190
Evidence concerns the material used by these authors; no validation of an Adooq lot or salt conversion is implied.
Figure 10B image and caption; Results p38α subsection; Materials; Cell culture
Mature SREBP2 protein signal after GSK343 exposure: Western blot of whole-cell lysates; precursor/mature SREBP2 and GAPDH loading control; PD98059 and SP600125 comparator conditions also shown — Figure 10B and caption; Results: Activation of p38α is involved in GSK343-induced SREBP2 activation
