anti pp38  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Starvation of leukemic cells enhances DNA damage-induced apoptosis in vitro via ROS/p38 MAPK and prevents leukemia progression in fasting xenograft mice

doi: 10.1016/j.jbc.2026.111143

Figure Lengend Snippet: Starvation-induced killing of ALL cells involves ROS-mediated activation of p38 MAPK. A , REH cells (0.8 × 10 6 cells/ml) were cultured in CM or DM in the presence or absence of forskolin (Forsk, 70 μM) for 45 min prior to IR, 7 Gy. The cells were harvested 1 hour after IR, and total cell lysates were subjected to immunoblot analyses with antibodies against p38 MAPK (p38), phosphorylated p38 MAPK (pp38). Antibodies against vinculin were used as a control of equal loading. Left panel ( upper ) shows one representative Western blot of six independent experiments. Right panel shows a quantified overview of pp38 signal intensity relative to the vinculin signal. The data represent the mean ± SEM, n = 6, ∗ P < 0.05 (paired t-test). Left panel (lower) shows a representative Western blot from one of one experiment using antibodies against p38 and vinculin. B , REH cells (0.8 × 10 6 cells/ml) were cultured in DM in the presence or absence of 40 μM of the p38 MAPK inhibitor (SB 202190) for 30 min, followed by incubation with or without forskolin (Forsk, 70 μM) for 45 min prior to IR, 7 Gy. The cells were harvested 1 hour after IR, and total cell lysates were subjected to immunoblot analyses with antibodies against p38, pp38 and vinculin. Left panel (upper) shows one representative Western blot of four independent experiments. Right panel shows a quantification of the pp38 signal intensity relative to vinculin signal. The data represent the mean ± SEM, n = 3, ∗ P < 0.05 (paired t-test). Left panel ( lower ) shows a representative Western blot from one of one experiment using antibodies against p38 and vinculin. C , REH cells (0.8 × 10 6 cells/ml) were cultured in DM in the presence or absence of 10 mM NAC for 30 min, followed by incubation with or without forskolin (Forsk, 70 μM) for 45 min prior to IR, 7 Gy. The cells were harvested 1 hour after IR, and total cell lysates were subjected to immunoblot analyses with antibodies against pp38 and vinculin. Left panel shows one representative Western blot of three independent experiments. Right panel shows quantification of the pp38 signal intensity relative to the vinculin signal. The data represent the mean ± SEM, n = 4, ∗ P < 0.05 (paired t-test). In all panels, ‘control’ represents cells cultured in the relevant medium alone. CM, complete medium; DM, depleted medium.

Article Snippet: The phospho-p38 MAPK (pp38) rabbit monoclonal antibody (#4511), the p38 MAPK rabbit polyclonal antibody (#9212), the ULK1 (A705, #4776) rabbit polyclonal antibody and the LC3B (# 2775) rabbit polyclonal antibody were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Cell Culture, Western Blot, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Starvation of leukemic cells enhances DNA damage-induced apoptosis in vitro via ROS/p38 MAPK and prevents leukemia progression in fasting xenograft mice

doi: 10.1016/j.jbc.2026.111143

Figure Lengend Snippet: Apoptosis induced by forskolin and IR in starving ALL cells involves the activation of p38 MAPK. A and D , REH cells (0.4 × 106 cells/ml) were cultured in DM in the presence or absence of 10 μM of the p38 MAPK inhibitor SB 202190 for 30 min, followed by incubation with forskolin (Forsk, 70 μM) for 45 min prior to IR, 10 Gy. A , the percentages of dead cells were monitored by flow cytometry of cells stained with PI 2 hours after IR. The data represent the mean ± SEM, n = 4, ∗ P < 0.05 (paired t-test). B , the loss of mitochondrial membrane potential was assessed by flow cytometry of cells stained with TMRM 2 hours after IR and shown as percentage of TMRM-low cells, representing apoptotic cells. The data represent the mean ± SEM, n = 3, ∗ P < 0.05 (paired t-test). C , total ROS levels were analyzed by staining the cells with CellROX green 1 hour after IR, and the MFI was analyzed by flow cytometry. The data represent the mean ± SEM, n = 4, ∗ P < 0.05 (paired t-test). D , the cells were subjected to CYTO-ID staining for detection of autophagy 24 hours after IR, and the MFI was analyzed by flow cytometry. The data represent the mean ± SEM, n = 3, ∗ P < 0.05 (paired t-test). E , model explaining how DNA damage-induced killing of ALL cells is enhanced under starving culture conditions in the presence of cAMP signaling. Our model suggests that starvation promotes cAMP-mediated ( via forskolin, Forsk) enhancement of DNA damage-induced ( via IR) killing of ALL cells by enhancing the ROS levels. Increased ROS levels activate p38 MAPK, which subsequently leads to apoptosis. Additionally, ROS is involved in starvation-induced autophagy, which is prompted by cAMP signaling in the presence of a DNA damaging agent. Notably, activation of p38 MAPK appears to function both upstream and downstream of ROS. According to our model, the cells die with – but not due to the high levels of autophagy. In all panels, ‘control’ represents cells cultured in the relevant medium alone. DM, depleted medium; MFI, mean fluorescence intensity.

Article Snippet: The phospho-p38 MAPK (pp38) rabbit monoclonal antibody (#4511), the p38 MAPK rabbit polyclonal antibody (#9212), the ULK1 (A705, #4776) rabbit polyclonal antibody and the LC3B (# 2775) rabbit polyclonal antibody were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Cell Culture, Incubation, Flow Cytometry, Staining, Membrane, Control, Fluorescence