Journal: Nature Communications

Article Title: Endothelial IRE1α promotes thrombospondin-1 mRNA decay and supports metabolic stress adaptation of pancreatic islets

doi: 10.1038/s41467-025-68276-1

Figure Lengend Snippet: a – f MS1 endothelial cells ( a , c , e ) or primary HUVECs ( b , d , f ) were infected with lentiviruses encoding a shRNA directed against IRE1α ( shErn1 or shERN1 ) or a scramble control ( shCtrl ) for 48 hours. a , b Quantitative RT-PCR analysis of Xbp1 mRNA splicing and Thbs1/THBS1 mRNA abundance in MS1 cells ( a ) or HUVECs ( b ) pre-cultured for 4 hours at 5 mM glucose or following stimulation for 24 hours with high glucose at 16 mM ( n = 3 independent experiments). c , d Immunoblot analysis of IRE1α and TSP1 protein levels in MS1 cell lysates ( c ) or HUVEC lysates ( d ) when cultured at 5 mM versus 16 mM glucose for 24 hours. α-Tubulin was used as a loading control (n = 3 independent experiments). e , f Immunoblot analysis of TSP1 protein in culture medium of MS1 cells ( e ) or HUVECs ( f ). Cell lysate α-Tubulin was used as a loading control ( n = 3 independent experiments). g Cell proliferation and viability analysis by cell-count assay or using the CCK-8 Kit for lentivirus-infected MS1 cells cultured at 16 mM glucose (n = 2 independent experiments). h , i MS1 cells were infected with shCtrl , shErn1 , or both shErn1 and shThbs1 lentiviruses for 48 hours. h Immunoblot analysis of IRE1α and TSP1 proteins in MS1 cell lysates. i MS1 cell proliferation analysis by cell-count assay or using the CCK-8 Kit when cultured at 16 mM glucose ( n = 2 independent experiments). j Proliferation analysis by the CCK-8 Kit of MS1 cells that were cultured at 16 mM glucose and treated with PBS (Vehicle, Veh.) or 1 μg/mL recombinant human TSP1 protein in the absence or presence of 1 μg/mL anti-CD47 neutralizing antibody for 48 hours ( n = 6 independent treatment experiments). Data are shown as mean ± SEM by two-way ANOVA ( a – d , g , i , * indicates shCtrl versus shErn1 ; # indicates shErn1 versus shErn1/shThbs1 in i ) or unpaired two-tailed Student’s t -test ( e , f , j ).

Article Snippet: Murine islet-derived MS1 endothelial cells were from ATCC (CRL-2279) and cultured in DMEM with 5% FBS.

Techniques: Infection, shRNA, Control, Quantitative RT-PCR, Cell Culture, Western Blot, Cell Characterization, CCK-8 Assay, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Endothelial IRE1α promotes thrombospondin-1 mRNA decay and supports metabolic stress adaptation of pancreatic islets

doi: 10.1038/s41467-025-68276-1

Figure Lengend Snippet: a MS1 cells were pre-cultured at 5 mM glucose for 4 hours before stimulation with 16 mM glucose for 12 hours in the absence or presence of 10 μM 4μ8C. Quantitative RT-PCR analysis of Xbp1 mRNA splicing and Thbs1 mRNA abundance (n = 3 independent experiments). b HEK293T cells with IRE1α depletion (HEK293T-KO) were transiently transfected for 36 hours with vector control (-) or THBS1-Myc plasmid, or co-transfected with THBS1-Myc plus IRE1α or XBP1s-Flag plasmids. Cells transfected as indicated were also treated with 10 μM 4μ8C for 12 hours. Immunoblot analysis of IRE1α, XBP1s and TSP1-Myc protein. Shown also is quantification of TSP1-Myc protein level after normalization to β-Actin control ( n = 3 independent experiments). c , d HEK293T-KO cells were transiently transfected for 36 hours with vector control, IRE1α-WT or its RNase-deficient K907A mutant plasmids. c Quantitative RT-PCR analysis of human XBP1 s, BLOC1S1 and THBS1 mRNA levels ( n = 3 independent experiments). d Immunoblot analysis of IRE1α, TSP1-Myc and XBP1 protein with α-Tubulin as a loading control. Shown also is quantification of TSP1-Myc protein levels (n = 3 independent experiments). e Consensus sequence (underlined) of the putative RIDD region in human and mouse TSP1 mRNAs within the stem-loop structure predicted by RNAstructure Version 6.2. f Agarose gel analysis of IRE1α-mediated cleavage of human THBS1 mRNA. In vitro transcription-derived mRNA fragments of THBS1 (nt 1-1770 and nt 1771-3510) were incubated for 1 hour with recombinant human IRE1α protein (1 μg) in the presence of DMSO (-) or 10 μM 4μ8C, followed by 3% agarose gel analysis. Human XBP1 mRNA was used as a positive control. Red arrows indicate the probable major RNA cleavage products. g Synthetic RNA substrates for wild-type THBS1 WT (nt 2771-2808) or THBS1 Mut with the indicated G-to-C mutation were incubated for 1 hour with human IRE1α protein (1 μg) pre-mixed for 1 hour with DMSO (-) or 10 μM 4μ8C, followed by 20% TBE-Urea PAGE gel analysis. The red arrow indicates the RNA cleavage product. Data are representative of 2 independent experiments in ( f , g ). Results are shown as mean ± SEM by unpaired two-tailed Student’s t -test ( a – d ).

Article Snippet: Murine islet-derived MS1 endothelial cells were from ATCC (CRL-2279) and cultured in DMEM with 5% FBS.

Techniques: Cell Culture, Quantitative RT-PCR, Transfection, Plasmid Preparation, Control, Western Blot, Mutagenesis, Sequencing, Agarose Gel Electrophoresis, In Vitro, Derivative Assay, Incubation, Recombinant, Positive Control, Two Tailed Test