Journal: Experimental & Molecular Medicine

Article Title: Advanced glycation end products promote meniscal calcification by activating the mTOR-ATF4 positive feedback loop

doi: 10.1038/s12276-024-01190-6

Figure Lengend Snippet: In the left panel, a normal meniscus without abnormal neovascularization was observed, exhibiting well-aligned fibers and optimal biomechanical properties. Most of the ATF4 protein in meniscal cells is degraded via the ubiquitination pathway, and there is no significant mTOR activation. The right panel shows that meniscal vessels introduce a significant amount of AGEs with increasing age or glucose levels, which promotes neoangiogenesis and osteogenic differentiation of meniscal cells. AGEs and RAGE activate the ATF4-mTOR positive feedback loop, where mTOR inhibits the degradation of ATF4 by decreasing its ubiquitination. Simultaneously, ATF4 activated mTOR by elevating arginine uptake, eventually leading to calcification of the meniscus and impairment of its biomechanical properties. The schematic was constructed using Figdraw.

Article Snippet: Antigen repair was performed using citric acid repair solution (P0083, Beyotime) at 65 °C for 16 h, after which the sections were blocked in 10% goat serum for 30 min. Antigen recognition was achieved by incubating primary antibodies against AGEs (bs-1158R, Bioss, RRID: AB_10857744) and RAGE (16346-1-AP, Proteintech, RRID: AB_2878246) overnight at 4 °C and then with goat anti-rabbit Alexa Fluor 488 (Abmart) as a secondary antibody for 30 min at 37 °C.

Techniques: Activation Assay, Introduce, Construct

Journal: Annals of Hematology

Article Title: Outcome of COVID-19 infection in 50 multiple myeloma patients treated with novel drugs: single-center experience

doi: 10.1007/s00277-021-04594-w

Figure Lengend Snippet: Patient characteristics: cohort of 50 MM pts with COVID-19 infection

Article Snippet: None of the other studied risk factors was associated with COVID-19 death (age, gender, ISS stage, immunoparesis, type of anti-myeloma treatment, COVID-19 treatment with remdesivir and convalescent plasma).

Techniques: Infection

Journal: Disease Models & Mechanisms

Article Title: Comparison of the pathogenesis of SARS-CoV-2 infection in K18-hACE2 mouse and Syrian golden hamster models

doi: 10.1242/dmm.049632

Figure Lengend Snippet: Identification of SARS-CoV-2-infected cells in the lungs of K18-hACE2 mice and Syrian golden hamsters. (A) Representative immunofluorescence images for N protein, AGER (type 1 alveolar cell marker), LAMP3 (type 2 alveolar cell marker) and 4′,6-diamidino-2-phenylindole (DAPI; nuclear marker) in the lungs of K18-hACE2 mice 2 days after SARS-CoV-2 infection. Scale bars: 40 µm. (B) Percentage of infected cells (N protein + ) per total positive cells in the alveolar lineage (AGER + or LAMP3 + ) in SARS-CoV-2-infected K18-hACE2 mice at a 40× HPF ( n =6 per group). All data are presented as the mean±s.e.m. P -values were obtained by two-tailed unpaired Student's t -test (*** P <0.001). (C) Representative immunofluorescence images for N protein, SCGB1A1 (club cell marker), acetylated TUBA1A (Ac-TUBA1A; ciliated cell marker) and DAPI in the lungs of Syrian golden hamsters 2 days after SARS-CoV-2 infection. Scale bars: 40 µm. (D) Percentage of infected cells (N protein + ) per total number of positive cells in the bronchus lineage (SCGB1A1 + or Ac-TUBA1A + or MUC5AC + or CHGA + ) in SARS-CoV-2-infected Syrian golden hamsters in a 40× HPF ( n =6 per group). All data are presented as the mean±s.e.m. P -values were obtained by two-tailed unpaired Student's t -test (*** P <0.001, versus goblet cells; ### P <0.001, versus neuroendocrine cells). (E) Representative in situ hybridization images of human ACE2 (h ACE2 ) in the lungs of non-infected K18-hACE2 mice and Syrian golden hamsters. Scale bars: 50 µm. (F) Co-staining images of in situ hybridization for S gene and immunohistochemical images of TUNEL in the lungs of K18-hACE2 mice and Syrian golden hamsters 2 days after SARS-CoV-2 infection. The black arrows indicate TUNEL + / S gene + cells. Scale bars: 50 µm. (G) Percentage of TUNEL + / S gene + cells relative to total S gene + cells in SARS-CoV-2-infected K18-hACE2 mice and Syrian golden hamsters in a 40× HPF ( n =6 per group). All data are presented as the mean±s.e.m. P -values were obtained by two-tailed unpaired Student's t -test (*** P <0.001).

Article Snippet: The sections were incubated with the following primary antibodies overnight at 4°C in a humidity chamber: anti-N protein (NB100-56576, Novus, 1:1000 and 40143-MM08, Sino Biological, 1:1000), anti-LAMP3 (DDX0190P, Novus, 1:500), anti-AGER (MAB1179, R&D Systems, 1:200), anti-SCGB1A1 (ABS1673, Millipore, 1:200), anti-acetylated TUBA1A (T7451, Sigma-Aldrich, 1:1000), anti-MUC5AC (MA5-12178, Thermo Fisher Scientific, 1:1000), anti-PTPRC (ab64100, Abcam, 1:1000), anti-CD3D (ab5690, Abcam, 1:200), anti-Ly-6G/Ly-6C (ab2557, Abcam, 1:250), anti-p-AKT (9271S, Cell Signaling Technology, 1:500), anti-CHGA (ab15160, Abcam, 1:1000), anti-CD4 (25229S, Cell Signaling Technology, 1:200) and anti-CD8 (14-0808-82, Thermo Fisher Scientific, 1:200).

Techniques: Infection, Immunofluorescence, Marker, Two Tailed Test, In Situ Hybridization, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Cell proliferation

Article Title: Deciphering the Immunomodulatory Function of GSN + Inflammatory Cancer-Associated Fibroblasts in Renal Cell Carcinoma Immunotherapy: Insights From Pan-Cancer Single-Cell Landscape and Spatial Transcriptomics Analysis.

doi: 10.1111/cpr.70062

Figure Lengend Snippet: FIGURE 6 | Silencing of GSN in CAFs inhibits intra-tumoral CD8+ T cells function and drives them towards a dysfunctional state. (A) tSNE plot of iCAF marker gene GSN, and expression of GSN in iCAFs from responders and non-responders. (B) Survival analysis of GSN (in terms of OS) in TCGA-KIRC cohort. (C) Immunohistochemistry (IHC) staining in tumour samples of three non-responders and three responders. (D) Workflow of the co-culture system with primary CAFs (NC, siGSN-1 and siGSN-2), primary ccRCC tumour cells and CD8+ T cells. (E) qRT-PCR analysis of GSN mRNA in three groups (NC, siGSN-1 and siGSN-2). (F) ELISA analysis of the IFN-γ and TNF-α levels in supernatant from the co-culture system. (G) Flow cytometry analysis of IFN-γ, TNF-α, GZMB and Perforin in CD8+ T cells isolated from the co-culture system. (H) GSEA analysis in TCGA-KIRC cohort (low GSN vs. high GSN), as well as in pan-cancer scRNA-seq landscape of ICI therapy (non-responders vs. responders). (I) Representative western blot of GSN, p65, p-p65, IKB, p-IKB and IKKα + β protein expression levels in the co-culture system. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: These primary antibodies were used in our western blot analysis: p65 (8242, Cell Signalling Technology, CST), p- p65 (3033, CST), IKB (4812, CST), p- IKB (2859, CST), IKKα + β (2697, CST), anti- human GSN (11644- 2- AP, Protein tech), β- tubulin (10094- 1- AP, Protein tech).

Techniques: Marker, Expressing, Immunohistochemistry, Co-Culture Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Isolation, Western Blot