Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 4. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) RNA-seq analyses in human SMSCs after static and CTS treatment [(A1) volcano plot of differentially expressed genes (DEGs) in CTS group versus static group; (A2) KEGG pathway enrichment, red boxes represent Hippo and TGFβ signaling]. (B) Volcano plot of differentially expressed genes in regenerated tissue of PCL scaffold + synovium transplant group versus native porcine menisci [(B1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (B2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (B3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (C) Heatmap of differential gene cluster in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (C2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (C3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (D) KEGG pathway enrichment in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(D1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (D2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (D3) PCL scaffold + synovium transplant group at 2 versus 4 months, the red boxes represent Hippo and TGFβ signaling]. (E) Protein and mRNA levels in human SMSCs after static and CTS treatment [(E1) Western blot analysis; (E2) semiquantitative analysis of Western blot, n = 3, unpaired t test; (E3) mRNA levels, n = 8, unpaired t test]. (F) Cell immunofluorescence of human SMSCs after static and CTS treatment [(F1) cell immunofluorescence; (F2) six regions are randomly selected for semiquantitative analysis; the intensity of immunofluorescence is calculated by ImageJ software, n = 6, unpaired t test] (continued with Fig. 5). **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: RNA Sequencing, Western Blot, Immunofluorescence, Software

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 5. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) Protein and mRNA levels in human SMSCs after being treated with dihy- drexidine and SIS3 [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (A3) the alcian blue staining; (A4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (A5) cell immunofluorescence; (A6) the semiquantitative analysis of cell immunofluorescence, Dihy represents dihydrexidine, n = 6, one-way ANOVA; (A7) the mRNA levels, n = 8, one-way ANOVA]. (B) Immunofluorescent colocalization of YAP and Smad2/3 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) immunofluorescent colocalization of YAP and Smad2/3; (B2) immu- nofluorescence overlap coefficient, n = 9]. (C) Immunofluorescent colocalization of pSmad2/3 and SOX9 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) immunofluorescent colocalization of pSmad2/3 and SOX9; (C2) immunofluorescence overlap coefficient, n = 9]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 6. Piezo1 mediates mechanotransduction on YAP-pSmad2/3-SOX9 axis through concerted activation of calcineurin and NFATc1 during MSCs chondro- genesis. (A) Protein and mRNA levels of Piezo1 in human SMSCs after static and CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A3) the cell immunofluorescence; (A4) the semiquantitative analysis of cell immunofluorescence, n = 6, unpaired t test; (A5) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of Piezo1 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (C) Protein and mRNA levels in human SMSCs after being treated with YODA1 or GsMTx-4 [(C1) the Western blot analysis; (C2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (C3) the mRNA levels, n = 8, unpaired t test; (C4) the alcian blue staining; (C5) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (C6) the cell immunofluorescence; (C7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. (D) Assessment of intracellular calcium content in human SMSCs [(D1) intracellular calcium content after static and CTS treatment; (D2) the semiquantitative analysis of calcium content, n = 5, unpaired t test; (D3) intracellular calcium content after being treated with YODA1 or GsMTx-4; (D4) the semiquantitative analysis of calcium content, n = 5, one-way ANOVA]. (E) Protein and mRNA levels in human SMSCs after CsA treatment [(E1) Western blot analysis, the red arrow represents dephosphorylated NFATc1, the blue arrows represent phosphorylated NFATc1; (E2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (E3) the alcian blue staining; (E4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (E5) the mRNA levels, n = 8, unpaired t test; (E6) the cell immunofluorescence; (E7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing, Staining

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 7. The effect of matrix stiffness on YAP-pSmad2/3-SOX9 axis. (A) Protein and mRNA levels in human SMSCs after being treated with soft or stiff matrix [(A1) the mechanical properties of silicon rubber and polystyrene culture plate, n = 3; (A2) Western blot analysis, the red arrow indicates dephosphorylated NFATc1, the blue arrows indicate phosphorylated NFATc1; (A3) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A4) the alcian blue staining; (A5) the semiquantitative analysis of alcian blue staining, n = 3, unpaired t test; (A6) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of FAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) the immunofluorescence of FAK; (B2) the semiquantitative analysis of FAK, n = 6, one-way ANOVA]. (C) Immunofluorescent assessment of pFAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) the immunofluorescence of pFAK; (C2) the semiquantitative analysis of pFAK, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 8. The biomechanical stimulus and matrix stiffness regulate the expression of collagen cross-link enzymes (LOX and LH) through Piezo1. (A) Protein and mRNA levels of LOX and LH in human SMSCs after static or CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (A3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (A4) the cell immunofluorescence of LOX; (A5) the semiquantitative analysis of LOX immunofluorescence, n = 6, unpaired t test; (A6) the cell immunofluorescence of LH2; (A7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, unpaired t test; (A8) the mRNA levels, n = 8, unpaired t test]. (B) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with YODA1 or GsMTx-4 [(B1) the Western blot analysis; (B2) the semiquantitative analysis of LOX, n = 3, one-way ANOVA; (B3) the semiquantitative analysis of LH2, n = 3, one-way ANOVA; (B4) the cell immunofluorescence of LOX; (B5) the semiquantitative analysis of LOX immunofluorescence, n = 6, one-way ANOVA; (B6) the cell immunofluorescence of LH2; (B7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, one-way ANOVA; (B8) the mRNA levels, n = 8, one-way ANOVA]. (C) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with soft or stiff matrix [(C1) the Western blot analysis; (C2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (C3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (C4) the mRNA levels, n = 8, unpaired t test]. (D) Immunofluorescent assessment of LOX expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (E) Immunofluorescent assessment of LH2 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: iScience

Article Title: Complement is primarily activated in the lung in a mouse model of severe COVID-19

doi: 10.1016/j.isci.2025.111930

Figure Lengend Snippet: Schematic of complement cascade and components upregulated by SARS-CoV-2 infection Green text indicates proteins with increased mRNA transcripts in mice following SARS2-N501Y MA30 infection, red text indicates proteins with reduced mRNA transcript abundance in mice following SARS2-N501Y MA30 infection. As depicted, the alternative pathway is continuously and spontaneously activated at low levels (tick-over) to generate C3bBb (C3 convertase), though rapid self-enhancement occurs by feedback amplification loop once activation is initiated. The lectin and classical pathways have different triggering substrates (glycoproteins and immunoglobulins, respectively), but both converge on the generation of C4 and C2, which join to create the C3 convertase C4bC2a. Although the lectin and classical pathways are initiated via separate mechanisms, a majority of their activity ultimately converges on C3bBb (C3 convertase), which cleaves C3 into C3a (anaphylatoxin) and C3b (opsonin). All three pathways coalesce with C5 convertase cleavage of C5, and the generation of C5a (anaphylatoxin) and C5b (joins with C6-C9 to form C5b-9, membrane attack complex). In addition, there are various cofactor proteins that help regulate cascade activity, such as properdin (FP) and complement factor H (FH), which can augment or inhibit further activation, respectively. FB, factor B; MBL, mannose binding lectin; MASP, MBL-associated serine protease; FD, factor D.

Article Snippet: The enriched RNA pool was fragmented, converted to cDNA, and ligated to sequencing adaptors using the TruSeq stranded mRNA sample preparation kit (RS-122-2101, Illumina).

Techniques: Infection, Amplification, Activation Assay, Activity Assay, Membrane, Binding Assay

Journal: iScience

Article Title: Complement is primarily activated in the lung in a mouse model of severe COVID-19

doi: 10.1016/j.isci.2025.111930

Figure Lengend Snippet: Messenger RNA expression analysis suggests local production of complement proteins in lung tissue following SARS2-N501Y MA30 infection (A) RNAscope for S-protein RNA and C3 RNA in lung tissue sections on 2 dpi following 5,000 PFU of SARS2-N501Y MA30 . White arrows indicate C3 RNA and SARS2-N501Y MA30 S-protein RNA, red = C3, green = S-protein, and blue = DAPI. (B) Heatmap depicting the results of bulk RNA sequencing for genes associated with complement pathways from lung tissue collected 5 dpi following inoculation with 1,000 PFU SARS2-N501Y MA30 . (C) Mean log 2 fold change in transcript abundance post-infection. Complement pathway indicated by bar color: red = alternative pathway specific genes, purple = lectin pathway specific genes, green = classical pathway specific genes, gray = common pathway genes, blue = shared classical and lectin pathway genes. n = 4. dpi, days post-infection. Error bars represent mean ± SEM.

Article Snippet: The enriched RNA pool was fragmented, converted to cDNA, and ligated to sequencing adaptors using the TruSeq stranded mRNA sample preparation kit (RS-122-2101, Illumina).

Techniques: RNA Expression, Infection, RNAscope, RNA Sequencing

Journal: iScience

Article Title: Complement is primarily activated in the lung in a mouse model of severe COVID-19

doi: 10.1016/j.isci.2025.111930

Figure Lengend Snippet:

Article Snippet: The enriched RNA pool was fragmented, converted to cDNA, and ligated to sequencing adaptors using the TruSeq stranded mRNA sample preparation kit (RS-122-2101, Illumina).

Techniques: Virus, Cell Culture, Recombinant, Sample Prep, RNAscope, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell Reports Medicine

Article Title: Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas

doi: 10.1016/j.xcrm.2023.101082

Figure Lengend Snippet:

Article Snippet: For near-complete knockdown of NFKBIA , G418-resistant primary human astrocytes transduced to stably express wildtype IDH1 or mutant IDH1- ( R132H ) were transfected with Accell human NFKBIA small interfering (si)RNA or non-targeting control siRNA (Dharmacon), at 20nM concentration using lipofectamine 2000 reagent (Invitrogen) at 1:1 ratio for 48 h. For complete clustered regularly interspaced short palindromic repeats (CRISPR) knockout of NFKBIA , astrocytes were transfected with IκBα Double Nickase Plasmid (h) (sc-400034-NIC, Santa Cruz)—consisting of a pair of plasmids each encoding a D10A mutated Cas9 nuclease and a target-specific 20-nucleotide (nt) guide RNA (gRNA)—using plasmid transfection medium (sc-108062) and Ultra-Cruz transfection reagent (sc-395739) at 1:1.5 ratio and were incubated for 72 h. After incubation, cells were screened for GFP-positivity to select for successfully transfected cells.

Techniques: Plasmid Preparation, Recombinant, Transfection, Activation Assay, Methylation, Marker, DNA Methylation Assay, Sequencing, Empire Assay, Software