Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: ( A ) Western blotting of the indicated proteins in H358 cells incubated with 100 μM Carboplatin ( n = 2), 25 μM irinotecan ( n = 2), or γ-irradiation (4 Gy) ( n = 1) for the indicated times. p-ATM, phosphorylated (activated) ATM. ( B ) Immunofluorescence analysis of γH2AX (in red) and 53BP1 (in green) expression in H358 cells after 24 or 48 h of incubation with 100 μM Carboplatin ( n = 1). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. ( C ) Western blotting of the indicated proteins after cellular fractionation of H358 cells incubated (+) or not (−) with 100 μM Carboplatin for 48 h ( n = 3). S1 cytoplasmic fraction, S2 nucleoplasmic fraction, P2 chromatin fraction, TUBULIN loading control for S1, TBP loading control for P2. ( D ) Western blotting of the indicated proteins in H358 cells incubated with 100 μM Carboplatin for the indicated times ( n = 1). ( E ) Western blotting of the indicated proteins in H358 cells incubated or not with 100 μM Carboplatin for 48 h and with 50 μg/μl Cycloheximide (CHX) for the indicated times before the end of Carboplatin treatment ( n = 3). Upper panel: Schematic representation of the experiment design. ( F ) Graph showing the mean ± SEM of three different experiments performed as described in ( E ) and the statistics between curves was determined using a modified Chi-square-based method. NICD level was normalized to TUBULIN at each time point and then to untreated sample (lane 0) that was set at 100%. Hours correspond to Cycloheximide treatment. In the legend, n indicates the number of biological replicates. .

Article Snippet: Phosphorylated ATM , Cell Signaling Technology , 13050.

Techniques: Western Blot, Incubation, Irradiation, Immunofluorescence, Expressing, Staining, Cell Fractionation, Control, Modification

Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: ( A ) Western blotting of the indicated proteins in A549 cells exposed to 100 μM Carboplatin ( n = 2), 25 μM irinotecan ( n = 2), or γ-irradiation (4 Gy) ( n = 1) for the indicated time. P-ATM, phosphorylated (activated) ATM. ( B ) Immunofluorescence analysis of γH2AX (in red) and 53BP1 (in green) in A549 cells after 24 or 48 h of incubation with 100 μM Carboplatin ( n = 1). Nuclei were stained with DAPI (in blue). Scale bar: 10 μm. ( C ) Western blotting of the indicated proteins after cellular fractionation of A549 cells incubated (+) or not (−) with 100 μM Carboplatin for 48 h ( n = 2). S1, cytoplasmic fraction; S2, nucleoplasmic fraction; P2, chromatin fraction; TUBULIN, loading control for S1; TBP, loading control for P2. ( D ) Western blotting of the indicated proteins in A549 cells incubated with 100 μM Carboplatin for the indicated time ( n = 1). ( E ) Western blotting of the indicated proteins in A549 cells incubated or not with 100 μM Carboplatin for 48 h and with 50 μg/μl Cycloheximide (CHX) for the indicated time before the end of Carboplatin treatment ( n = 2). Upper panel: Schematic representation of the experimental design. In the legend, n indicates the number of biological replicates. .

Article Snippet: Phosphorylated ATM , Cell Signaling Technology , 13050.

Techniques: Western Blot, Irradiation, Immunofluorescence, Incubation, Staining, Cell Fractionation, Control

Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: ( A ) H358 cells were incubated with 100 μM Carboplatin and with/without 10 μM KU-55933 (ATM inhibitor) or 5 μM Ceralasertib (ATR inhibitor) for the indicated time followed by immunoblotting to detect the expression of the indicated proteins ( n = 2). P phosphorylated. ( B ) H358 cells were incubated with 100 μM Carboplatin for 48 h and with or without 10 μM KU-55933 (ATM inhibitor). 50 μg/μl Cycloheximide (CHX) was added for the indicated time before the end of Carboplatin treatment ( n = 3). Inhibitors were added 2 h before Carboplatin addition. Upper panel: Schematic representation of the experimental design. ( C ) Graph showing the mean ± SEM of three different experiments performed as described in ( B ) and the statistics between curves was determined using a modified Chi-square-based method. NICD level was normalized to TUBULIN at each time point and then to untreated sample (lane 0) that was set at 100%. Hours correspond to Cycloheximide treatment. In the legend, n indicates the number of biological replicates. .

Article Snippet: Phosphorylated ATM , Cell Signaling Technology , 13050.

Techniques: Incubation, Western Blot, Expressing, Modification

Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: ( A ) A549 cells were incubated with 100 μM Carboplatin and with/without 10 μM KU-55933 (ATM inhibitor) or Ceralasertib (ATR inhibitor) for the indicated time followed by immunoblotting to detect the expression of the indicated proteins ( n = 2). p, phosphorylated. ( B ) A549 cells were incubated with 100 μM Carboplatin for 48 h and with or without 10 μM KU-55933 (ATM inhibitor; ATMi). 50 μg/μl Cycloheximide (CHX) was added for the indicated time before the end of Carboplatin treatment ( n = 2). Upper panel: Schematic representation of the experimental design. ( C ) Western blotting of the indicated proteins in A549 cells incubated with 100 μM Carboplatin for the indicated time, with or without 10 μM KU-55933 (ATM inhibitor; ATMi) ( n = 2). ( D ) Western blotting of the indicated proteins in A549 cells transfected with a non-targeting siRNA ( siNT ) or with a siRNA against MDM2 ( siMDM2 ) for 6 h, followed by addition of 100 μM Carboplatin for the indicated time ( n = 3). ( E ) At 6 h post-transfection, A549 cells transfected with siMDM2 or siNT were incubated with 100 μM Carboplatin for 48 h and with 50 μg/μl of Cycloheximide (CHX) for the indicated time before the end of Carboplatin treatment ( n = 2). Upper panel: schematic representation of the experimental design. In the legend, n indicates the number of biological replicates. .

Article Snippet: Phosphorylated ATM , Cell Signaling Technology , 13050.

Techniques: Incubation, Western Blot, Expressing, Transfection

Journal: Frontiers in Pharmacology

Article Title: CS-6-induced p62 accumulation exacerbates DNA damage in colorectal cancer

doi: 10.3389/fphar.2025.1568339

Figure Lengend Snippet: CS-6 induced DNA damage in SW620 and DLD1 cells. SW620 and DLD1 cells were treated with 80 nM CS-6 for 24 h. The untreated cells were used as Control group. (a–d) Western blot analysis was conducted to assess the levels of phosphorylated-ataxia telangiectasia mutated kinase (p-ATM), ATM, and γH2AX levels in both Control and CS-6-treated SW620 and DLD1 cells. (e–h) Confocal microscopy images were captured to visualize γH2AX and nuclear staining in Control and CS-6-treated SW620 and DLD1 cells, presented as single-channel and merged images. Scale bar = 5 µm. (i,j) The Comet assay was performed to illustrate CS-6-induced DNA damage in CRC cells. The presented values indicate the mean ± standard deviation obtained from three independent experiments. Statistical significance is established at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Anti-phosphorylated-ataxia telangiectasia mutated kinase (P-ATM), Anti-ataxia telangiectasia mutated kinase (ATM), Anti-LC3A/B and Anti-autophagy-related protein 5 (ATG 5) were purchased from Cell Signaling Technology (#13050, #2873, #12741, #12994, Boston, America).

Techniques: Control, Western Blot, Confocal Microscopy, Staining, Single Cell Gel Electrophoresis, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: CS-6-induced p62 accumulation exacerbates DNA damage in colorectal cancer

doi: 10.3389/fphar.2025.1568339

Figure Lengend Snippet: CS-6-induced autophagy defects exacerbated DNA damage in SW620 and DLD1 cells. SW620 and DLD1 cells were treated with 80 nM CS-6 and/or 10 μM chloroquine (CQ) for 24 h (a,b) Cell viability of CS-6-treated and CS-6/CQ-treated SW620 and DLD1 cells was measured using the CCK8 assay. (c,d) Western blot assay was conducted to measure the levels of phosphorylated-ataxia telangiectasia mutated kinase (p-ATM), ATM, and γH2AX in CS-6-treated and CS-6/CQ-treated SW620 and DLD1 cells. The presented values indicate the mean ± standard deviation obtained from three independent experiments. Significance levels are indicated as * p < 0.05 and ** p < 0.01.

Article Snippet: Anti-phosphorylated-ataxia telangiectasia mutated kinase (P-ATM), Anti-ataxia telangiectasia mutated kinase (ATM), Anti-LC3A/B and Anti-autophagy-related protein 5 (ATG 5) were purchased from Cell Signaling Technology (#13050, #2873, #12741, #12994, Boston, America).

Techniques: CCK-8 Assay, Western Blot, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: CS-6-induced p62 accumulation exacerbates DNA damage in colorectal cancer

doi: 10.3389/fphar.2025.1568339

Figure Lengend Snippet: Inhibition of CS-6-induced autophagy reversed DNA damage in SW620 and DLD1 cells. SW620 and DLD1 cells were transfected with scrambled siRNA (Control) and si-ATG5 for 48 h, followed by treatment with 80 nM CS-6 for 24 h (a,b) Cell viability of Control and si-ATG5-transfected/CS-6-treated SW620 and DLD1 cells was measured using the CCK8 assay. (c,d) Western blot assay was conducted to measure the levels of phosphorylated-ataxia telangiectasia mutated kinase (p-ATM), ATM, and γH2AX in Control and si-ATG5-transfected/CS-6-treated SW620 and DLD1 cells. The values presented indicate the mean ± standard deviation obtained from three independent experiments. Significance levels are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Anti-phosphorylated-ataxia telangiectasia mutated kinase (P-ATM), Anti-ataxia telangiectasia mutated kinase (ATM), Anti-LC3A/B and Anti-autophagy-related protein 5 (ATG 5) were purchased from Cell Signaling Technology (#13050, #2873, #12741, #12994, Boston, America).

Techniques: Inhibition, Transfection, Control, CCK-8 Assay, Western Blot, Standard Deviation