Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: ATF4 promotes DDIT4 transcription leads to EC necroptosis and BBB injury in tMCAO mice. A, Western blot analysis of ATF4 protein levels in brain microvascular tissues isolated from 8-week-old and 20-month-old tMCAO mice, with quantification of protein levels (right panel, n = 6 per group). B, Real-time quantitative PCR analysis of ATF4 mRNA levels in brain microvascular tissues isolated from 8-week-old and 20-month-old tMCAO mice (n = 4 per group). C, Heat map illustrates the mRNA expression levels of key target genes associated with ATF4 transcriptional activation in the microvascular tissues of aged mice. Values were determined by qRT-PCR and plotted as fold change relative to control. D-E, qChIP analysis of the DDIT4 promoters was performed using antibodies against ATF4 in microvascular tissues from aged mice after ischemic-reperfusion (n = 3 per group). F, Changes in the mRNA levels of the downstream target gene DDIT4 were observed following ATF4 overexpression (n = 5 per group). G, Changes in the mRNA levels of the downstream target gene DDIT4 were observed following simultaneous ATF4 knockdown during in vitro oxygen-glucose deprivation and reperfusion (n = 5 per group). H. Schematic diagram of the AAV used for ATF4 knockdown in vivo . I, Experimental design and timeline of ischemic stroke, ATF4 knockdown, and analysis in aged mice. J, Representative images results of immunofluorescence staining for TUNEL (green, indicating apoptosis) and PI (red, indicating necrosis) in ECs within the ischemia-reperfusion cortical region following ATF4 knockdown. K, Representative images illustrating the co-staining of p-MLKL with BMECs (CD31) of the ischemia-reperfusion in cortical region following ATF4 knockdown. L, Illustration of TEER permeability assay and FITC-dextran permeability assay. M-N, TEER and FITC permeability of vehicle-treated (AdV-shNT) versus AdV-shDDIT4 treated ECs under ATF4 overexpression. (n = 5 independent experiments). Data are mean ± SEM.

Article Snippet: The following primary antibody was utilized: Pan Kla (PTM-1401, PTM BIO), H3K9la (PTM-1419, PTM BIO), H3K14la (PTM-1414, PTM BIO), H3K18la (PTM-1406, PTM BIO), H4K5la (PTM-1407, PTM BIO), H4K8la (PTM-1415, PTM BIO), H4K12la (PTM-1411, PTM BIO), H4K16la (PTM-1417, PTM BIO), H3 (PTM-1001, PTM BIO), H4 (PTM-1015, PTM BIO), LDHA (19987-1-AP, Proteintech), β-actin (Abclonal), DDIT4 (10638-1-AP, Proteintech), p-MLKL (ab196436, Abcam), MLKL (37705, Cell Signaling Technology), p-RIP1 (65746S, Cell Signaling Technology), RIP1 (7519-1-AP, Proteintech), p-mTOR (5536S, Cell Signaling Technology), mTOR (66888-1-Ig, Proteintech), NDUFS3 (15066-1-AP, Proteintech), Ubiquitin (43124S, Cell Signaling Technology), ATF4 (60035-1-Ig, Proteintech).

Techniques: Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Activation Assay, Quantitative RT-PCR, Control, Over Expression, Knockdown, In Vitro, In Vivo, Immunofluorescence, Staining, TUNEL Assay, Permeability, FITC-Dextran Permeability Assay

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: Genetic knockdown of DDIT4 ameliorates EC necroptosis and BBB injury in tMCAO mice. A, Representative images of reconstructed Z-stack showing colocalization of DDIT4 and endothelial cell marker CD31 in the cortex of tMCAO mice. B, Representative images co-stained with fibrin, DDIT4 and cortical microvascular ECs (CD31) in the ischemia-reperfusion area. C, Quantitative analysis of the co-localization of DDIT4 and ECs (n = 8 per group). D, Analysis of correlation between co-localization ratio of DDIT4 and CD31 (represented by DDIT4/CD31) and Fibrinogen area. E, Schematic diagram of the AAV used for DDIT4 knockdown in vivo . F, Experimental design and timeline of ischemic stroke, DDIT4 knockdown, and analysis in aged mice. G, Representative images co-stained with fibrin and cortical microvascular ECs (CD31) in the ischemia-reperfusion area from DDIT4 conditional knockdown mice. H, Representative images results of immunofluorescence staining for TUNEL (green, indicating apoptosis) and PI (red, indicating necrosis) in ECs within the ischemia-reperfusion cortical region following DDIT4 knockdown. I, Representative images illustrating the co-staining of p-MLKL with BMECs (CD31) of the ischemia-reperfusion in cortical region following DDIT4 knockdown. J-K, Statistical analysis results of immunofluorescence staining for TUNEL (green, indicating apoptosis) and PI (red, indicating necrosis) in ECs within the ischemia-reperfusion cortical region following DDIT4 knockdown (n = 6 per group). L, Quantitative analysis of the co-localization of p-MLKL and ECs (n = 6 per group). M, CCK-8 results of cell viability of primary microvascular ECs (n = 4 independent experiments). N-O, Representative flow cytometry images and statistical analysis results of ECs stained with PI/annexin V. The necroptosis activation was represented by ratio of PI + /annexin V + (n = 5 per group). Data are mean ± SEM.

Article Snippet: The following primary antibody was utilized: Pan Kla (PTM-1401, PTM BIO), H3K9la (PTM-1419, PTM BIO), H3K14la (PTM-1414, PTM BIO), H3K18la (PTM-1406, PTM BIO), H4K5la (PTM-1407, PTM BIO), H4K8la (PTM-1415, PTM BIO), H4K12la (PTM-1411, PTM BIO), H4K16la (PTM-1417, PTM BIO), H3 (PTM-1001, PTM BIO), H4 (PTM-1015, PTM BIO), LDHA (19987-1-AP, Proteintech), β-actin (Abclonal), DDIT4 (10638-1-AP, Proteintech), p-MLKL (ab196436, Abcam), MLKL (37705, Cell Signaling Technology), p-RIP1 (65746S, Cell Signaling Technology), RIP1 (7519-1-AP, Proteintech), p-mTOR (5536S, Cell Signaling Technology), mTOR (66888-1-Ig, Proteintech), NDUFS3 (15066-1-AP, Proteintech), Ubiquitin (43124S, Cell Signaling Technology), ATF4 (60035-1-Ig, Proteintech).

Techniques: Knockdown, Marker, Staining, In Vivo, Immunofluorescence, TUNEL Assay, CCK-8 Assay, Flow Cytometry, Activation Assay

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: Knockdown of DDIT4 in BMECs ameliorates mitochondrial dysfunction and reduces mtROS release. A, Mitochondrial morphology in primary ECs with overexpression of DDIT4. B, Mitochondrial morphology in OGD/R-treated primary ECs after DDIT4 shRNA. C-D, Mean branch length (µm), mean perimeter (µm) and mean form factor (au) of mitochondria (n = 5 independent experiments). E, Representative images of LC3 co-stained with Tomm20 in primary BMECs after OGDR. F-G, The number of LC3 punch and co-location of LC3 and TOMM20 were presented (right). H-I, Mitochondrial membrane potential measured by flow cytometry of JC-1. JC-1 aggregation = normal mitochondrial membrane potential; JC-1 monomers = low membrane potential x-axis. J, Histograms display y-axis. K, Quantification of JC-1 flow cytometry (n = 5 per group). L, Representative images of staining with mitoSOX (red) and live cell nuclear stain hoechst33342 (blue). M, IntDen/cell measured using ImageJ (n = 5 per group). Data are mean ± SEM.

Article Snippet: The following primary antibody was utilized: Pan Kla (PTM-1401, PTM BIO), H3K9la (PTM-1419, PTM BIO), H3K14la (PTM-1414, PTM BIO), H3K18la (PTM-1406, PTM BIO), H4K5la (PTM-1407, PTM BIO), H4K8la (PTM-1415, PTM BIO), H4K12la (PTM-1411, PTM BIO), H4K16la (PTM-1417, PTM BIO), H3 (PTM-1001, PTM BIO), H4 (PTM-1015, PTM BIO), LDHA (19987-1-AP, Proteintech), β-actin (Abclonal), DDIT4 (10638-1-AP, Proteintech), p-MLKL (ab196436, Abcam), MLKL (37705, Cell Signaling Technology), p-RIP1 (65746S, Cell Signaling Technology), RIP1 (7519-1-AP, Proteintech), p-mTOR (5536S, Cell Signaling Technology), mTOR (66888-1-Ig, Proteintech), NDUFS3 (15066-1-AP, Proteintech), Ubiquitin (43124S, Cell Signaling Technology), ATF4 (60035-1-Ig, Proteintech).

Techniques: Knockdown, Over Expression, shRNA, Staining, Membrane, Flow Cytometry

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: DDIT4 facilitates the ubiquitination of NDUFS3 resulted in mitochondrial dysfunction. A, Mass spectrometry analysis of proteins pulled down by anti-DDIT4 antibodies identified potential DDIT4-interacting proteins. B, Volcano plot of differentially expressed proteins between mitochondria isolated from cerebral microvascular tissues before and after ischemia-reperfusion. C, Overlapping analysis of differential proteins in mitochondrial proteomic and interacting proteins of DDIT4. D, Heat map showed the expression of 11 overlapping proteins in mitochondrial proteomic. E, Co-immunoprecipitation of NDUFS3 (left) or DDIT4 (right) in ECs (IgG as a control antibody). F, Western blotting of NDUFS3 in indicated cells. Endothelial cell was treated with MG132 6h for harvest. And the quantification of protein was shown in below (n = 6 per group). G, Western blotting of NDUFS3 in cerebral microvascular tissues from conditional DDIT4 knockdown mice. The quantification of protein was shown in below (n = 6 per group). H, Co-IP of NDUFS3 and then western blotted with anti-ubiquitin in DDIT4 overexpression ECs. I, Representative images of staining with mitoSOX (red) and live cell nuclear stain hoechst33342 (blue). J, Phospho-MLKL aggregation in indicated cells. K, IntDen/cell measured using ImageJ (n = 6 per group). L, p-MLKL aggregates/nucleus quantified using Fiji (n = 6 per group). M, The OCR was measured in indicated cells. Data are mean ± SEM.

Article Snippet: The following primary antibody was utilized: Pan Kla (PTM-1401, PTM BIO), H3K9la (PTM-1419, PTM BIO), H3K14la (PTM-1414, PTM BIO), H3K18la (PTM-1406, PTM BIO), H4K5la (PTM-1407, PTM BIO), H4K8la (PTM-1415, PTM BIO), H4K12la (PTM-1411, PTM BIO), H4K16la (PTM-1417, PTM BIO), H3 (PTM-1001, PTM BIO), H4 (PTM-1015, PTM BIO), LDHA (19987-1-AP, Proteintech), β-actin (Abclonal), DDIT4 (10638-1-AP, Proteintech), p-MLKL (ab196436, Abcam), MLKL (37705, Cell Signaling Technology), p-RIP1 (65746S, Cell Signaling Technology), RIP1 (7519-1-AP, Proteintech), p-mTOR (5536S, Cell Signaling Technology), mTOR (66888-1-Ig, Proteintech), NDUFS3 (15066-1-AP, Proteintech), Ubiquitin (43124S, Cell Signaling Technology), ATF4 (60035-1-Ig, Proteintech).

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Isolation, Expressing, Immunoprecipitation, Control, Western Blot, Knockdown, Co-Immunoprecipitation Assay, Over Expression, Staining

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: Interruption of glycolysis/H3K18la/ATF4-DDIT4 feedback loop of BMECs alleviate edema and brain injury in tMCAO mice. A, Western blotting of DDIT4 in primary BMECs treated with varying concentrations of lactate. The quantification of protein was shown in the right panel (n = 5 per group). B, Western blotting of DDIT4 in primary BMECs treated with lactate for different durations at the same concentration. The quantification of protein was shown in the right panel (n = 5 per group). C, Western blotting of DDIT4 in primary BMECs with or without LDHA overexpression. The quantification of protein was shown in the right panel (n = 6 per group). D, Western blotting of DDIT4 in OGDR treated primary BMECs with or without LDHA knockdown. The quantification of protein was shown in the right panel (n = 3 per group). E, Colorimetric assay for lactate levels in primary BMECs with or without DDIT4 overexpression (n = 5 per group). F, Colorimetric assay for lactate levels in OGDR treated primary BMECs with or without DDIT4 konckdown (n = 5 per group). G, Western blotting of Pan- and site-specific histone lactylation in primary BMECs with or without DDIT4 overexpression. The quantification of protein was shown in the right panel (n = 3-4 per group). H, Experimental design and timeline of ischemic stroke, DDIT4 knockdown, and analysis in tMCAO mice. I-J, Evans blue leakage of mice brains in coronal sections and extravasation (fold change relative to sham) from Sham, AAV-shControl Tie2-cre tMCAO, and AAV-shDDIT4 Tie2-cre tMCAO groups (n = 6 per group). K-L, Representative images and statistical results of TTC (n = 6 per group). M, Experimental design and timeline of ischemic stroke and analysis in LDHA flox/flox mice and LDHA flox/flox ; CDH5-CreERT mice. N-O, Representative images and statistical results of TTC (n = 5 per group). P, Statistical results of brain water content (n = 10 per group). Q, Modified neurological severity score at 3 days after tMCAO in different groups (n = 6 per group). Data are mean ± SEM.

Article Snippet: The following primary antibody was utilized: Pan Kla (PTM-1401, PTM BIO), H3K9la (PTM-1419, PTM BIO), H3K14la (PTM-1414, PTM BIO), H3K18la (PTM-1406, PTM BIO), H4K5la (PTM-1407, PTM BIO), H4K8la (PTM-1415, PTM BIO), H4K12la (PTM-1411, PTM BIO), H4K16la (PTM-1417, PTM BIO), H3 (PTM-1001, PTM BIO), H4 (PTM-1015, PTM BIO), LDHA (19987-1-AP, Proteintech), β-actin (Abclonal), DDIT4 (10638-1-AP, Proteintech), p-MLKL (ab196436, Abcam), MLKL (37705, Cell Signaling Technology), p-RIP1 (65746S, Cell Signaling Technology), RIP1 (7519-1-AP, Proteintech), p-mTOR (5536S, Cell Signaling Technology), mTOR (66888-1-Ig, Proteintech), NDUFS3 (15066-1-AP, Proteintech), Ubiquitin (43124S, Cell Signaling Technology), ATF4 (60035-1-Ig, Proteintech).

Techniques: Western Blot, Concentration Assay, Over Expression, Knockdown, Colorimetric Assay, Modification

Journal: Journal of translational medicine

Article Title: Establishment of a prognostic model based on ER stress-related cell death genes and proposing a novel combination therapy in acute myeloid leukemia.

doi: 10.1186/s12967-025-06615-y

Figure Lengend Snippet: Fig. 9 DDIT4 promotes AML cell proliferation in vitro. A Knockdown of DDIT4 in U937 and HL60 cell lines. B‒H Analysis of cell viability (B), cell counting (C), colony formation (D‒E), cell apoptosis (F), the cell cycle (G), and migration (H) in control and DDIT4-knockdown cells. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The primary antibodies used for the western blotting assays were rabbit antibodies against DDIT4 (1:1000, 10638-1- AP; Proteintech, Wuhan, Beijing) and mouse antibodies against beta-actin (1:1000, AC004; ABclonal Technology, Wuhan, China).

Techniques: In Vitro, Knockdown, Cell Counting, Migration, Control