Journal: Science Advances

Article Title: Targeting astrocytic Dp71 attenuates BBB disruption after traumatic brain injury through WTAP-associated m 6 A regulation of MMP2

doi: 10.1126/sciadv.aed8653

Figure Lengend Snippet: ( A ) Real-time PCR analysis of Dp71 mRNA expression in astrocytes infected with shNC, shDp71-1, and shDp71-2 lentiviruses ( n = 3 per group; one-way ANOVA). ( B and C ) Western blot analysis of Dp71 protein expression in astrocytes infected with shNC, shDp71-1, and shDp71-2 lentiviruses ( n = 3 per group; one-way ANOVA). ( D ) Volcano plot showing decreased expression of typical inflammatory genes in the shDp71 group. FDR, false discovery rate. ( E ) Gene Ontology (GO) enrichment analysis of DEGs (molecular function module). ( F and G ) Real-time PCR analysis of MMP2 mRNA expression in astrocytes and activated astrocytes ( n = 3 per group; one-way ANOVA). ( H to K ) Western blot analysis of MMP2 protein expression in astrocytes and activated astrocytes ( n = 3 per group; one-way ANOVA). ( L and M ) Immunofluorescence analysis of GFAP and MMP2 colabeling in the injured region of mice at 3 days after TBI ( n = 6 per group; Student’s t test). Scale bars, 100 μm. ( N ) Real-time PCR analysis of MMP2 mRNA expression in the injured region of TBI mice and the corresponding cerebral cortex of sham mice ( n = 6 per group; two-way ANOVA). ( O and P ) Western blot analysis of MMP2 protein expression in the injured region of TBI mice and the corresponding cerebral cortex of sham mice ( n = 6 per group; two-way ANOVA). Results are expressed as means ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n.s., not significant.

Article Snippet: The MMP2 inhibitor (HY-146754, MCE) was dissolved in 1% dimethyl sulfoxide in PBS.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: Targeting astrocytic Dp71 attenuates BBB disruption after traumatic brain injury through WTAP-associated m 6 A regulation of MMP2

doi: 10.1126/sciadv.aed8653

Figure Lengend Snippet: ( A ) Co-IP for exogenous Dp71-HA, Dp71a-HA, and WTAP in astrocytes ( n = 3). ( B ) Schematic of truncated Dp71 mutants. ( C and D ) Co-IP for interactions between truncated Dp71 mutants and WTAP ( n = 3; one-way ANOVA). ( E and F ) Western blot analysis of WTAP protein expression levels in astrocytes ( n = 3; one-way ANOVA). ( G ) Real-time PCR analysis of WTAP mRNA expression levels in astrocytes ( n = 3; one-way ANOVA). ( H ) WTAP ubiquitination (Ub) assay in astrocytes treated with 20 μM MG-132 for 8 hours ( n = 3). ( I and J ) Western blot analysis of WTAP protein in the injured mouse brain cortex at 3 days post-TBI ( n = 6; two-way ANOVA). ( K ) Real-time PCR analysis of WTAP mRNA in the injured mouse brain cortex at 3 days post-TBI ( n = 6; two-way ANOVA). ( L and M ) m 6 A dot blot analysis for m 6 A levels ( n = 9; Student’s t test). ( N ) Prediction of m 6 A modification sites in MMP2 mRNA using the SRAMP tool. 3′UTR, 3′ untranslated region; CDS, coding sequence. ( O ) Methylated RNA immunoprecipitation (MeRIP) analysis of m 6 A modification levels on MMP2 mRNA ( n = 3; Student’s t test). ( P ) Real-time PCR analysis of MMP2 mRNA stability ( n = 3; Student’s t test). h, hours. ( Q ) RIP-qPCR analysis of the binding between WTAP protein and MMP2 mRNA ( n = 3; Student’s t test). ( R and S ) Western blot analysis of MMP2 protein expression levels ( n = 3; Student’s t test). ( T ) Real-time PCR analysis of MMP2 mRNA expression levels ( n = 3; Student’s t test). ( U ) MeRIP analysis of MMP2 mRNA m 6 A modification levels ( n = 3; Student’s t test). ( V ) Real-time PCR analysis of MMP2 mRNA stability ( n = 3; Student’s t test). Results are expressed as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n.s., not significant.

Article Snippet: The MMP2 inhibitor (HY-146754, MCE) was dissolved in 1% dimethyl sulfoxide in PBS.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Dot Blot, Modification, Sequencing, Methylation, RNA Immunoprecipitation, Binding Assay

Journal: Science Advances

Article Title: Targeting astrocytic Dp71 attenuates BBB disruption after traumatic brain injury through WTAP-associated m 6 A regulation of MMP2

doi: 10.1126/sciadv.aed8653

Figure Lengend Snippet: ( A ) Schematic diagram of TRAM@siNC and TRAM@siDp71 synthesis. TRAM was prepared by fusing liposomes with astrocyte membrane and conjugating with the RVG29 peptide. Created in BioRender. Jiheng, W. (2026); https://BioRender.com/p9xmg9o . ( B ) Western blot analysis of the protein expression of CD47 (a cell membrane–specific molecule) on the surface of TRAM@siNC and TRAM@siDp71 ( n = 3 per group). ( C ) Particle size analysis of TRAM@siNC and TRAM@siDp71 ( n = 3 per group). DLS, dynamic light scattering. ( D ) Transmission electron microscopy (TEM) observation of TRAM@siNC and TRAM@siDp71. Scale bars, 50 nm ( n = 3 per group). ( E ) Hemolytic activity assay of TRAM, TRAM@siNC, and TRAM@siDp71 ( n = 3 per group; one-way ANOVA). ( F ) Cell Counting Kit-8 (CCK-8) assay for the cytotoxicity of TRAM, TRAM@siNC, and TRAM@siDp71 ( n = 4 per group; one-way ANOVA). ( G and H ) Western blot analysis of Dp71 and MMP2 protein expression in astrocytes in vitro after uptake of TRAM@siNC and TRAM@siDp71 ( n = 3 per group; one-way ANOVA). ( I and J ) Real-time fluorescence imaging to detect the distribution of Cy5.5 in mouse brain regions after intravenous injection of TRAM@siNC and TRAM@siDp71 ( n = 4 per group; one-way ANOVA). Avg, average. ( K to M ) Immunofluorescence analysis of the colocalization of GFAP and Cy5.5. Scale bars, 100 and 50 μm (zoom) ( n = 6 per group). ( N ) Western blot analysis of Dp71, MMP2, Occludin, and ZO-1 protein expression in mice after TBI ( n = 6 per group; one-way ANOVA). ( O and P ) Evans blue staining analysis of BBB permeability in mice after TBI ( n = 6 per group; one-way ANOVA). ( Q ) Assessment of cerebral edema in mice at 3 days after TBI by quantifying brain water content ( n = 6 per group; one-way ANOVA). Results are expressed as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n.s., not significant.

Article Snippet: The MMP2 inhibitor (HY-146754, MCE) was dissolved in 1% dimethyl sulfoxide in PBS.

Techniques: Liposomes, Membrane, Western Blot, Expressing, Particle Size Analysis, Transmission Assay, Electron Microscopy, Activity Assay, Cell Counting, CCK-8 Assay, In Vitro, Fluorescence, Imaging, Injection, Immunofluorescence, Staining, Permeability

Journal: Oncology Reports

Article Title: Knockdown of CCT2 inhibits the malignant progression of hepatocellular carcinoma cells by impairing STAT3 activation

doi: 10.3892/or.2026.9086

Figure Lengend Snippet: Knockdown of CCT2 inhibits STAT3 signaling activation in hepatocellular carcinoma cells. The protein levels of STAT3, p-STAT3, MCL1, MMP2 and SOX2 in (A) Huh-7 and (B) HCCLM3 cells were measured by western blotting. The protein levels of (C) p-STAT3, (D) MCL1, (E) MMP2 and (F) SOX2 in subcutaneous tumor tissue were detected by immunohistochemical staining. *P<0.05, **P<0.01 vs. sh-NC. CCT2, chaperonin containing TCP1 subunit 2; sh, short hairpin; NC, negative control; p, phosphorylated; MCL1, myeloid cell leukemia sequence 1; SOX2, SRY-box transcription factor 2.

Article Snippet: The primary antibodies were as follows: CCT2 (cat. no. 24896-1-AP), β-actin (cat. no. 66009-1-Ig), MMP2 (cat. no. 10373-2-AP), myeloid cell leukemia sequence 1 (MCL1; cat. no. 16225-1-AP) and SRY-box transcription factor 2 (SOX2; cat. no. 11064-1-AP; all Proteintech Group, Inc.) and STAT3 (cat. no. 4904) and phosphorylated (p-)STAT3 (Tyr705; cat. no. 4113; both Cell Signaling Technology, Inc.) The membranes were washed three times in TBST (0.1% Tween-20) for 5 min each at room temperature.

Techniques: Knockdown, Activation Assay, Western Blot, Immunohistochemical staining, Staining, Negative Control, Sequencing

Journal: iScience

Article Title: Changes in the microglial phenotype drive neuroinflammation independent of systemic inflammation in the acute stage of heatstroke

doi: 10.1016/j.isci.2026.115254

Figure Lengend Snippet: Time course of functional damage to the intestinal barrier and blood-brain barrier during the acute stage of heatstroke The mice were exposed to 41.2 ± 0.5°C ambient temperature until their rectal temperature reached 42.4°C, and then they were allowed to recover at an ambient temperature of 25 ± 0.5°C for the indicated times. (A) The concentration of FD-4 in serum was measured after its oral administration. (B) Serum endotoxin levels. (C) EB was injected into the mice via the tail vein after the onset of heatstroke, and representative images of EB leakage was shown. (D) Quantitative analysis of EB concentrations in brain tissues. (E) Expression of MMP-2 in the cerebral cortex at 1 h, 6 h, and 24 h post heatstroke. (F) Expression of MMP-9 in the cerebral cortex at 1 h, 6 h, and 24 h post heatstroke. The data are expressed as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, vs. Con.

Article Snippet: The membrane was further incubated overnight at 4°C with primary antibodies against MMP9 (TA326652; ORIGENE, USA), MMP2 (TA326260; ORIGENE, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 2118L; CST, USA).

Techniques: Functional Assay, Concentration Assay, Injection, Expressing