a304 309a t  (Bethyl)

Journal: Frontiers in Pharmacology

Article Title: Dexmedetomidine alleviates lung ischemia-reperfusion injury by inhibiting cuproptosis: an in vivo study

doi: 10.3389/fphar.2025.1562535

Figure Lengend Snippet: WB results of cuproptosis-related protein expression. (A) Western blots for FDX 1, SLC 31 A1, LIAS, DLST, DLD, and SDHB in rat lung tissue. (B–G) relative levels of FDX 1, SLC 31 A1, LIAS, DLST, DLD, and SDHB in rat lung tissue (n = 3). ## p < 0.05 vs. control group; ** p < 0.05 vs. I/R group.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against: lipoic acid (Abcam, 1:1,000), FDX1 (Abcam, 1:1,000), LIAS (Proteintech, 1:1,000), SDHB (Proteintech, 1:1,000), DLAT (Proteintech, 1:1,000), DLD (Proteintech, 1:1,000), SLC31A1 (MCE, 1:1,000), DLST (MCE, 1:1,000), and β-actin (Sevier, 1:5,000).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Dexmedetomidine alleviates lung ischemia-reperfusion injury by inhibiting cuproptosis: an in vivo study

doi: 10.3389/fphar.2025.1562535

Figure Lengend Snippet: Immunohistochemical and WB results of lipoacylated proteins. (A–C) representative images of lipoacylated protein immunohistochemical staining in rat lung tissue. (D) Western blots for lip-DLAT and lip-DLST. (E, F) relative levels of lip-DLAT and lip-DLST in rats lung tissue (n = 3). ## p < 0.05 vs. control group; ** p < 0.05 vs. I/R group.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against: lipoic acid (Abcam, 1:1,000), FDX1 (Abcam, 1:1,000), LIAS (Proteintech, 1:1,000), SDHB (Proteintech, 1:1,000), DLAT (Proteintech, 1:1,000), DLD (Proteintech, 1:1,000), SLC31A1 (MCE, 1:1,000), DLST (MCE, 1:1,000), and β-actin (Sevier, 1:5,000).

Techniques: Immunohistochemical staining, Staining, Western Blot, Control

Journal: Cell Reports

Article Title: A lncRNA-mediated metabolic rewiring of cell senescence

doi: 10.1016/j.celrep.2025.115747

Figure Lengend Snippet: sin-lncRNA interacts with DLST and controls its subcellular localization (A) RNA-FISH based on locked nucleic acid (LNA) technology on fixed IMR90 ER:RAS cells depleted or not of sin-lncRNA (siRNA_sin-lncRNA#1/3) and treated for 5 days with 4OHT. Two different LNAs were used. Incubation without LNA probe (“No_probe”) was used as control. Scale bar: 10 μm. (B) RT-qPCR analysis of a set of genes after mitochondria purification from proliferating (−4OHT) or senescent cells (+4OHT) using Mitotracker (green, 488) staining and FACS sorter. The graph shows a representative experiment. (C) Schematic representation of the TCA cycle that takes place in the mitochondria. DLST enzyme is highlighted in red. (D) RNA immunoprecipitation analysis monitoring DLST binding to sin-lncRNA, MALAT1 , and GAPDH RNA in control and sin-lncRNA -depleted (siRNA#1/3) senescent IMR90 ER:RAS, using anti-DLST antibody or IgG as a negative control. The graph represents the mean ± SD ( n = 2). (E) Left, western blot analysis of DLST cellular fractionation in control and sin-lncRNA -depleted (siRNA#1/3) senescent IMR90 ER:RAS. GAPDH and H3 were used as cytoplasmic and nuclear controls, respectively. Right, percentage of nuclear and cytoplasmic distribution of DLST normalized to GAPDH (cyto) or H3 (nucl). The graph represents the mean ± SD ( n = 3). Two-tailed Student’s t test, ∗ ∗ p < 0.01.

Article Snippet: After centrifugation, the samples were precleared with Dynabeads Protein A (Invitrogen) for 1 h. One per cent of the sample was used as the input control and the remaining extracts were incubated with 10 μg DLST antibody (Bethyl) or IgG at 4 °C overnight.

Techniques: Incubation, Control, Quantitative RT-PCR, Purification, Staining, RNA Immunoprecipitation, Binding Assay, Negative Control, Western Blot, Cell Fractionation, Two Tailed Test

Journal: Science advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair.

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: Fig. 1. Metabolic CRISPR screen identifies lipoylation as metabolic vulnerability for radiation response in NSCLC. (A) Schematic illustration of CRISPR-Cas9 screen workflow. (B) Top 10 negatively selected genes ranked by robust rank aggregation (RRA) score in 10 Gy–irradiated group compared to the control. (C) Manhattan plot of the entire 2981 metabolic genes by log10 P value. The top 10 negatively selected genes are highlighted. (D) Schematic illustration of the function of LIPT1, LIAS, DLD, and PDHX, the four top hits involved in lipoylation. (E) Pathway analysis of the 10 most significantly depleted metabolic pathways by Gene Ontology classification in 10 Gy–irradiated cells compared to the nonirradiated cells. (F) Immunoblots of total and lipoylated PDH and α-KGDH subunits (DLAT and DLST, respectively). Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH), DLAT, and DLST were blotted as loading controls. (G) Clonogenic assay of indicated cell lines after 2, 4, 6, and 8 Gy IR. The surviving fraction was normalized to the corresponding sham control, and survival curves were fitted using the linear-quadratic model.

Article Snippet: We used the following primary antibodies: anti- LA (437695, RRID:AB_212120, Millipore), anti- DLAT (12362, RRID:AB_2797893, Cell Signaling Technology), anti- DLST (5556, RRID:AB_10695157, Cell Signaling Technology), anti- tubulin (T5168, RRID:AB_477579, Sigma- Aldrich), anti–glyceraldehyde- 3- phosphate dehydrogenase (8884, RRID:AB_11129865, Cell Signaling Technology), anti- ATM (2873, RRID:AB_2062659, Cell Signaling Technology), anti–p- ATM (S1981) (Ab81292, Abcam), anti- Chk2 (6334, RRID:AB_11178526, Cell Signaling Technology), anti–p- Chk2 (Thr68) (2197, RRID: AB_2080501, Cell Signaling Technology), anti- γH2AX (05- 636, MilliporeSigma), anti- Ku70 (sc- 17789, Santa Cruz Biotechnology), anti– histone H3 (4499, RRID:AB_10544537, Cell Signaling Technology), anti- L2HGDH (15707- 1- AP, RRID:AB_2133202 Proteintech), antiKDM4A (5328, RRID:AB_10828595, Cell Signaling Technology), anti- KDM4B (8639, RRID:AB_11140642, Cell Signaling Technology), anti–Myc tag (2276, RRID:AB_331783, Cell Signaling Technology), and anti–acetyl- lysine Antibody (06- 933, RRID:AB_310304, MilliporeSigma).

Techniques: CRISPR, Irradiation, Control, Western Blot, Clonogenic Assay

Journal: Science advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair.

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: Fig. 5. LIPT1−/− cells have impaired activation of ATM and its downstream DNA damage repair signaling cascade. (A) Representative images and quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT, LIPT1−/− H460, and LIPT1−/− H460 reconstituted Myc-LIPT1 cells. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. (B) Quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT and LIPT1−/− H157 cells. (C and D) Immunoblotting analysis of ATM-pS1981, Ku70, γH2AX, and histone 3 in the soluble nuclear and chromatin fractions of WT and LIPT1−/− in H460 (C) and in H157 (D) cells, with or without IR (0.5 hours post 10 Gy). Histone H3 and γH2AX served as chromatin markers. LE, long exposure; SE, short exposure. (E and F) Immuno- fluorescence images and quantification of colocalized ATM-pS1981 (red) and γH2AX (green) foci at 0.5 hours post-4 Gy in WT, LIPT1−/− H460, and Myc-LIPT1–reconstituted H460 cells (E), as well as WT and LIPT1−/− H157 cells (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. (G and H) Immunoblot analysis of ATM-pS1981, total ATM, Chk2-pT68, total Chk2, lipoyl-DLAT/DLST, and γH2AX in H460 (G) and H157 (H) cells at 0.5 hour post-10Gy. (A), (B), (E), and (F), quantification was performed on >100 cells per treatment. Two-way ANOVA was used for (A) and (B), one-way ANOVA was used for (E), and unpaired t test was used for (F). ****P < 0.0001.

Article Snippet: We used the following primary antibodies: anti- LA (437695, RRID:AB_212120, Millipore), anti- DLAT (12362, RRID:AB_2797893, Cell Signaling Technology), anti- DLST (5556, RRID:AB_10695157, Cell Signaling Technology), anti- tubulin (T5168, RRID:AB_477579, Sigma- Aldrich), anti–glyceraldehyde- 3- phosphate dehydrogenase (8884, RRID:AB_11129865, Cell Signaling Technology), anti- ATM (2873, RRID:AB_2062659, Cell Signaling Technology), anti–p- ATM (S1981) (Ab81292, Abcam), anti- Chk2 (6334, RRID:AB_11178526, Cell Signaling Technology), anti–p- Chk2 (Thr68) (2197, RRID: AB_2080501, Cell Signaling Technology), anti- γH2AX (05- 636, MilliporeSigma), anti- Ku70 (sc- 17789, Santa Cruz Biotechnology), anti– histone H3 (4499, RRID:AB_10544537, Cell Signaling Technology), anti- L2HGDH (15707- 1- AP, RRID:AB_2133202 Proteintech), antiKDM4A (5328, RRID:AB_10828595, Cell Signaling Technology), anti- KDM4B (8639, RRID:AB_11140642, Cell Signaling Technology), anti–Myc tag (2276, RRID:AB_331783, Cell Signaling Technology), and anti–acetyl- lysine Antibody (06- 933, RRID:AB_310304, MilliporeSigma).

Techniques: Activation Assay, In Situ, Control, Staining, Western Blot, Fluorescence

Journal: Science advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair.

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: Fig. 6. LIPT1−/− cells are functionally deficient in HR repair. (A) Recruitment of EXO1–yellow fluorescent protein to laser-generated DSBs in single living WT and LIPT1−/− H157 cells. Relative fluorescence intensity of the local accumulation of EXO1 at laser-induced DNA damage sites was quantified for over 10 min and displayed as mean ± SD in WT and LIPT1−/− H157 cells. Simple linear regression was used for the statistical analyses. ****P < 0.0001. Scale bar, 10 μm. (B) Representative images and quantification of RAD51 foci by immunofluorescence staining in nonirradiated control and at 6 hours after 4 Gy in WT, LIPT1−/− H460, and LIPT1−/− H460 stably expressing Myc-LIPT1 cells. Nuclei were stained with DAPI. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. Two-way ANOVA was used for the statistical analyses. ****P < 0.0001. (C) Representative immunoblots demonstrate knockout of LIPT1 in U2OS and HEK-293 DR-GFP HR cells using the CRISPR-Cas9 gene editing system. Lipoylation was detected on DLAT and DLST, with GAPDH serving as the internal control. (D) Schematic illustration of the DR-GFP HR reporter assay. (E) HR activity assay by quantifying the population of cells positive for both DsRed and GFP in WT and LIPT1−/− U2OS (top) and WT and LIPT1−/− HEK-293 DR-GFP HR cells (bottom). Data were represented as means ± SD, unpaired t tests were used for the statistical analyses. **P < 0.01, ****P < 0.0001.

Article Snippet: We used the following primary antibodies: anti- LA (437695, RRID:AB_212120, Millipore), anti- DLAT (12362, RRID:AB_2797893, Cell Signaling Technology), anti- DLST (5556, RRID:AB_10695157, Cell Signaling Technology), anti- tubulin (T5168, RRID:AB_477579, Sigma- Aldrich), anti–glyceraldehyde- 3- phosphate dehydrogenase (8884, RRID:AB_11129865, Cell Signaling Technology), anti- ATM (2873, RRID:AB_2062659, Cell Signaling Technology), anti–p- ATM (S1981) (Ab81292, Abcam), anti- Chk2 (6334, RRID:AB_11178526, Cell Signaling Technology), anti–p- Chk2 (Thr68) (2197, RRID: AB_2080501, Cell Signaling Technology), anti- γH2AX (05- 636, MilliporeSigma), anti- Ku70 (sc- 17789, Santa Cruz Biotechnology), anti– histone H3 (4499, RRID:AB_10544537, Cell Signaling Technology), anti- L2HGDH (15707- 1- AP, RRID:AB_2133202 Proteintech), antiKDM4A (5328, RRID:AB_10828595, Cell Signaling Technology), anti- KDM4B (8639, RRID:AB_11140642, Cell Signaling Technology), anti–Myc tag (2276, RRID:AB_331783, Cell Signaling Technology), and anti–acetyl- lysine Antibody (06- 933, RRID:AB_310304, MilliporeSigma).

Techniques: Generated, Fluorescence, Immunofluorescence, Staining, Control, Stable Transfection, Expressing, Imaging, Western Blot, Knock-Out, CRISPR, Reporter Assay, Activity Assay

Journal: Science advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair.

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: Fig. 10. Inhibition of lipoylation enhances NSCLC’s radiation response in vivo. (A) Schematic illustrating timeline of WT and LIPT1−/− H460 tumor inoculation and treatments. (B) Tumor growth rate of WT and LIPT1−/− H460 xenografts in athymic nude mice with or without 10 Gy IR. n = 8 to 10 tumors. (C and D) Representative im- ages (C) and quantification (D) of immunoblotting analysis of lipoylated-DLAT and DLST using pooled tumors from H460 tumor–bearing athymic nude mice that either received vehicle or CPI-613 (20 mg/kg) for 14 doses. Intensity was quantified by ImageJ and normalized by GAPDH, n = 4. Data were represented as means ± SD, and two-way ANOVA was used for the statistical analyses. *P < 0.05, ****P < 0.0001. (E) Schematic illustrating timeline of tumor inoculation and treatments. Mice received daily treatment with either CPI-613 (20 mg/kg) or vehicle daily for 14 days. In the irradiated group, 10-Gy IR was administered to tumors after the first two doses of CPI-613. (F) Tumor growth rate of WT H460 xenografts in athymic nude mice treated with vehicle, CPI-613, 10-Gy, or combined treatment. n = 7 to 10 tumors. (G and H) Represen- tative images (G) and quantification (H) of Ki67 and γH2AX-positive nuclei by immunohistochemistry staining. Scale bar, 100 μm. Data were represented as means ± SD, and two-way ANOVA was used for the statistical analyses. *P < 0.05, ****P < 0.0001. (I) Schematic illustrating timeline of KP9-3 tumor inoculation and treatments. C57B/ L6 mice received daily treatment with either CPI-613 (20 mg/kg) or vehicle every other day for a total of eight doses. In irradiated group, 10-Gy IR was administered to tumor between the first two doses of CPI-613. (J) Tumor growth rate of KP9-3 xenografts in C57B/L6 mice treated with vehicle, CPI-613, 10-Gy, or combined treatment. n = 8 to 12 mice. For (A), (C), and (G), error bars represent the SEM.

Article Snippet: We used the following primary antibodies: anti- LA (437695, RRID:AB_212120, Millipore), anti- DLAT (12362, RRID:AB_2797893, Cell Signaling Technology), anti- DLST (5556, RRID:AB_10695157, Cell Signaling Technology), anti- tubulin (T5168, RRID:AB_477579, Sigma- Aldrich), anti–glyceraldehyde- 3- phosphate dehydrogenase (8884, RRID:AB_11129865, Cell Signaling Technology), anti- ATM (2873, RRID:AB_2062659, Cell Signaling Technology), anti–p- ATM (S1981) (Ab81292, Abcam), anti- Chk2 (6334, RRID:AB_11178526, Cell Signaling Technology), anti–p- Chk2 (Thr68) (2197, RRID: AB_2080501, Cell Signaling Technology), anti- γH2AX (05- 636, MilliporeSigma), anti- Ku70 (sc- 17789, Santa Cruz Biotechnology), anti– histone H3 (4499, RRID:AB_10544537, Cell Signaling Technology), anti- L2HGDH (15707- 1- AP, RRID:AB_2133202 Proteintech), antiKDM4A (5328, RRID:AB_10828595, Cell Signaling Technology), anti- KDM4B (8639, RRID:AB_11140642, Cell Signaling Technology), anti–Myc tag (2276, RRID:AB_331783, Cell Signaling Technology), and anti–acetyl- lysine Antibody (06- 933, RRID:AB_310304, MilliporeSigma).

Techniques: Inhibition, In Vivo, Western Blot, Irradiation, Immunohistochemistry, Staining