Journal: Journal for immunotherapy of cancer

Article Title: Cantharidin overcomes IL-2Rα signaling-mediated vorinostat resistance in cutaneous T-cell lymphoma through reactive oxygen species.

doi: 10.1136/jitc-2024-009099

Figure Lengend Snippet: Figure 4 Attenuated ROS levels promote protein synthesis of IL-2Rα and strengthen its down-stream pathways in SAHA- resistant cutaneous T-cell lymphoma cells. (A, B) The Flow cytometry analysis and fluorescence microscope analysis of ROS production in parental H9 and HH cells, SAHA-resistant H9 and HH cells in the absence or presence of H2O2 (1 mM, pretreated for 2 hours). Scale bar, 20 μm. (C) Western blotting analysis of IL-2Rα expression in parental H9 and HH cells, SAHA-resistant H9 and HH cells in the absence or presence of H2O2 (1 mM, pretreated for 2 hours). (D) Flow cytometry analysis of IL-2Rα expression in SAHA-resistant H9 and HH cells in the absence or presence of H2O2 (1 mM, pretreated for 2 hours). (E) Western blotting analysis of IL-2Rα expression in H9SR and HHSR cells stimulated with H2O2 (1 mM) for 2 hours and then treated with MG132 (5 μM) or NH4Cl (5 mM) for the indicated time. (F) H9SR cells were treated with shIL-2Rα or H2O2 (1 mM, 2 hours). Cell lysates were immunoprecipitated with an anti-IL-2Rβ, or IgG antibodies. p-JAK1, JAK1, and p-STAT5, STAT5, p-STAT3, STAT3 expression was assessed using western blotting. The 10% input shows results obtained from cell extracts without immunoprecipitation. (G) Western blotting analysis of MEK, p-MEK, Erk, p-Erk, JAK1, p-JAK1, STAT3, p-STAT3, STAT5, p- STAT5, AKT, p-AKT, mTOR and p-mTOR expression in parental H9 and HH cells, SAHA-resistant H9 and HH cells in the absence or presence of H2O2 (1 mM, pretreated for 2 hours). AKT, protein kinase B; DCFH-DA, 2′,7′-Dichlorodihydrofluorescein diacetate; Erk, extracellular regulated protein kinases; FITC, fluorescein isothiocyanate; H9SR, H9‐SAHA resistant cells; HHSR, HH‐SAHA resistant cells; JAK, Janus kinase; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; p-AKT, phosphorylated protein kinase B; p-Erk, phosphorylated extracellular regulated protein kinases; p-JAK, phosphorylated Janus kinase; p-MEK, phosphorylated mitogen-activated protein kinase kinase; p-mTOR, phosphorylated mammalian target of rapamycin; p-STAT, phosphorylated signal transducer and activator of transcription; ROS, reactive oxygen species; SAHA, vorinostat; SR, SAHA resistant; STAT, signal transducer and activator of transcription.

Article Snippet: Rabbit monoclonal antibodies targeting phosphorylated Janus kinase 1 (p- JAK1), phosphorylated mitogenactivated protein kinase kinase (p- MEK), phosphorylated extracellular regulated protein kinases (p- Erk), phosphorylated signal transducer and activator of transcription 3 (p- STAT3), phosphorylated signal transducer and activator of transcription 5 (p- STAT5), phosphorylated protein kinase B (p- AKT), phosphorylated mammalian target of rapamycin (p- mTOR) were obtained from Cell Signaling Technology (Boston, Massachusetts, USA).

Techniques: Flow Cytometry, Fluorescence, Microscopy, Western Blot, Expressing, Immunoprecipitation

Journal: Journal for immunotherapy of cancer

Article Title: Cantharidin overcomes IL-2Rα signaling-mediated vorinostat resistance in cutaneous T-cell lymphoma through reactive oxygen species.

doi: 10.1136/jitc-2024-009099

Figure Lengend Snippet: Figure 6 Cantharidin restrains IL-2Rα and its downstream signaling pathway in a reactive oxygen species dependent manner. (A) Effects of cantharidin on IL-2Rα protein levels in H9SR and HHSR cells. (B, C) Effects of cantharidin on IL-2Rα protein levels in H9SR and HHSR cells in the absence or presence of NAC (10 mM, 24 hours). Cells were treated by 3.13 μM of cantharidin for 48 hours. (D) H9SR cells were treated with 3.13 μM of cantharidin for 48 hours in the absence or presence of NAC (10 mM, 24 hours). Cell lysates were immunoprecipitated with an anti-IL-2Rβ, or IgG antibodies. p-JAK1, JAK1, and p-STAT5, STAT5, p- STAT3, and STAT3 expressions were assessed using western blotting. The 10% input shows results obtained from cell extracts without immunoprecipitation. (E) Effects of cantharidin on MEK, p-MEK, Erk, p-Erk, JAK1, p-JAK1, STAT3, p-STAT3, STAT5, p-STAT5, AKT, p-AKT, mTOR and p-mTOR expressions in H9SR and HHSR cells in the absence or presence of NAC (10 mM, 24 hours). Cells were treated by 3.13 μM of cantharidin for 48 hours. AKT, protein kinase B; CTD, cantharidin; Erk, extracellular regulated protein kinases; FITC, fluorescein isothiocyanate; H9SR, H9‐SAHA resistant cells; HHSR, HH‐SAHA resistant cells; IL, interleukin; JAK, Janus kinase; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; NAC, N-acetyl cysteine; p-AKT, phosphorylated protein kinase B; p-Erk, phosphorylated extracellular regulated protein kinases; p- JAK, phosphorylated Janus kinase; p-MEK, phosphorylated mitogen-activated protein kinase kinase; p-mTOR, phosphorylated mammalian target of rapamycin; p-STAT, phosphorylated signal transducer and activator of transcription; STAT, signal transducer and activator of transcriptiona.

Article Snippet: Rabbit monoclonal antibodies targeting phosphorylated Janus kinase 1 (p- JAK1), phosphorylated mitogenactivated protein kinase kinase (p- MEK), phosphorylated extracellular regulated protein kinases (p- Erk), phosphorylated signal transducer and activator of transcription 3 (p- STAT3), phosphorylated signal transducer and activator of transcription 5 (p- STAT5), phosphorylated protein kinase B (p- AKT), phosphorylated mammalian target of rapamycin (p- mTOR) were obtained from Cell Signaling Technology (Boston, Massachusetts, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Cantharidin overcomes IL-2Rα signaling-mediated vorinostat resistance in cutaneous T-cell lymphoma through reactive oxygen species.

doi: 10.1136/jitc-2024-009099

Figure Lengend Snippet: Figure 7 Cantharidin synergizes with SAHA to trigger SAHA-resistant cell death via IL-2R signaling. (A) Fa-CI plot in which fa and CI indicate fraction affected and combination index of SAHA and cantharidin in H9SR or HHSR cells. The fixed ratio combination of SAHA (0.20–12.5 μM) and cantharidin (0.39–25 μM) for 48 hours against H9SR or HHSR cells is 1:2. (B) Effects of SAHA alone, cantharidin alone and combination for 48 hours on reactive oxygen species production in H9SR or HHSR cells. (C) Effects of SAHA alone, cantharidin alone and combination for 48 hours on cell apoptosis in H9SR or HHSR cells. Values are presented as mean±SD (n=3), **p<0.01 and ***p<0.001 versus the control group; #p<0.05 and ###p<0.001 versus the indicated group. (D) Effects of SAHA alone, cantharidin alone and combination for 48 hours on IL-2Rα protein levels in H9SR or HHSR cells. (E) Effects of SAHA alone, cantharidin alone and combination for 48 hours on MEK, p-MEK, JAK1, p-JAK1, STAT3, p- STAT3, mTOR and p-mTOR expression in H9SR and HHSR cells. (F) The image of the tumor with different treatments in HHSR tumor model at day 14. (G) Tumor sizes were plotted as tumor volumes at different time point in HHSR tumor model. Values are presented as mean±SD (n=4). (H) The tumor weight of each group at day 14 in HHSR tumor model. Values are presented as mean±SD (n=4), *p<0.05, **p<0.01 and ***p<0.001 versus the control group; #p<0.05 and ##p<0.01 versus the indicated group. (I) Immunochemistry assay of Ki67 in tumor tissues. Scale bar, 20 μm. (J) Effects of SAHA alone, cantharidin alone and combination for 48 hours on MEK, p-MEK, JAK1, p-JAK1, STAT3, p-STAT3, mTOR and p-mTOR expressions in HHSR tumor tissues. CTD, cantharidin; HHSR, HH‐SAHA resistant cells; H9SR, H9‐SAHA resistant cells; IL, interleukin; JAK, Janus kinase; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; p-JAK1, phosphorylated Janus kinase 1; p-MEK, phosphorylated mitogen-activated protein kinase kinase; p-mTOR, phosphorylated mammalian target of rapamycin; p-STAT3, phosphorylated signal transducer and activator of transcription 3. SAHA, vorinostat; STAT, signal transducer and activator of transcription.

Article Snippet: Rabbit monoclonal antibodies targeting phosphorylated Janus kinase 1 (p- JAK1), phosphorylated mitogenactivated protein kinase kinase (p- MEK), phosphorylated extracellular regulated protein kinases (p- Erk), phosphorylated signal transducer and activator of transcription 3 (p- STAT3), phosphorylated signal transducer and activator of transcription 5 (p- STAT5), phosphorylated protein kinase B (p- AKT), phosphorylated mammalian target of rapamycin (p- mTOR) were obtained from Cell Signaling Technology (Boston, Massachusetts, USA).

Techniques: Control, Expressing

Journal: Cell biochemistry and biophysics

Article Title: sPLA2-IB and PLA2R Mediate Aberrant Glucose Metabolism in Podocytes via Hyperactivation of the mTOR/HIF-1α Pathway.

doi: 10.1007/s12013-025-01714-5

Figure Lengend Snippet: Fig. 8 sPLA2- IB activated PLA2R in human podocytes, causing podocyte injury via aberrant energy alteration, evidenced as the enhanced glycolytic enzymes and the inhibited TCA cycle-related enzymes, which is regulated by mTOR/ HIF- 1α pathway. TCA, tricarboxylic acid

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against HK (1:2000, Proteintech Cat# 22029-1- AP, RRID: AB_11182717, China), PKM2 (1:1000, Proteintech Cat# 15822-1-AP, RRID: AB_1851537, China), LDHA (1:1000,Cell Signaling Technology Cat# 2012, RRID: AB_2137173, USA), SDHD (1:1000, Abcam Cat# ab189945, RRID: AB_3665949, USA), FH (1:1000,Proteintech Cat# 11375-1-AP, RRID: AB_763171, China), mTOR (1:1000, Cell Signaling Technology Cat# 2983, RRID: AB_2105622, USA), HIF-1α (1:1000, Cell Signaling Technology Cat# 36169, RRID: AB_2799095, USA), and β-actin (1:5000, Proteintech Cat# 66009-1-Ig, RRID: AB_2687938, China).

Techniques:

Journal: bioRxiv

Article Title: Chronic exercise protects against the progression of renal cyst growth and dysfunction in rats with polycystic kidney disease

doi: 10.1101/2021.03.11.434857

Figure Lengend Snippet: Representative images of kidney specimens immunostained for (A) p-ERK and (B) p-mTOR in the Con-SD, Sed-PCK, and Ex-PCK groups. Western blotting analysis of (C) p-ERK, (D) p-mTOR, and (E) p-S6 expression in the Con-SD (rectangle dots), Sed-PCK (closed dots), and Ex-PCK (round dots) groups ( n =8 in each group). Top panels show representative immunoblotting. Each lane was loaded with a protein sample prepared from four different rats per group. Ratios of the relative band intensity of the phosphorylated protein to that of the total protein were calculated. The ratio in the Con-SD group was assigned a value of 1. Data are presented as the mean ± SEM. ** P <0.01 compared with the Con-SD group; ## P <0.01 compared with the Sed-PCK group; ns: no significant difference.

Article Snippet: Antibodies against Raf-B (#5284; Santa Cruz), ERK (#4695; Cell Signaling Technology), p-ERK (#4376; Cell Signaling Technology), mTOR (#2983; Cell Signaling Technology), p-mTOR (#2971; Cell Signaling Technology), S6 (#2217; Cell Signaling Technology), and p-S6 (#2211; Cell Signaling Technology) were used.

Techniques: Western Blot, Expressing