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 – d Global gene expression analysis by RNA-seq using total RNA extracts from isolated primary islets of Ern1 fl/fl and Ern1 EC-KO mice after 16 weeks of HFD feeding ( n = 3 per group). a Gene ontology (GO) biological process (BP) analysis of 573 genes (average FPKM > 1; |log 2 fold-change (FC) | > 0.5) whose expression was significantly different as a result of IRE1α deficiency in ECs. b Volcano plot depicting the log 2 (FC) values ( Ern1 EC-KO versus Ern1 fl/fl ) versus -log 10 ( P -values) for genes (FPKM > 1) from the RNA-seq analysis of islets. Highlighted are up-regulated (red) or down-regulated (blue) genes ( | log 2 (FC) | > 0.5; P Value < 0.01) resulting from endothelial IRE1α deficiency. c List of top-ranked genes whose expression was significantly upregulated or downregulated in islets with EC IRE1α deficiency (FPKM > 1; |log 2 (FC) | > 0.5; P Value < 0.01). d Heat maps of 7 genes (average FPKM > 1; |log 2 (FC) | > 0.5; P Value < 0.01) involved in regulation of endothelial proliferation, which were differentially expressed in islets from HFD-fed Ern1 EC-KO mice versus their Ern1 fl/fl counterparts ( n = 3 mice per group). e , f Primary islets isolated from Ern1 fl/fl and Ern1 EC-KO mice after 16 weeks of HFD feeding. e Quantitative RT-PCR analysis of the mRNA abundance of islet Thbs1 (n = 7 per group) and Egfl7 ( Ern1 fl/fl , n = 6; Ern1 EC-KO , n = 7). f Immunoblot analysis of islet TSP1 protein levels ( Ern1 fl/fl , n = 5; Ern1 EC-KO , n = 6). α-Tubulin was used as a loading control. Data are shown as mean ± SEM by unpaired two-tailed Student’s t -test ( e , f ).

Article Snippet: Cells were treated with recombinant human TSP1 protein (ABclonal, RP01197) in the absence or presence of anti-CD47 neutralizing antibody (Bio X Cell- InVivo MAb, BE0283; Clone MIAP410) or IgG control (Bio X Cell- InVivo MAb, BE0083; Clone MOPC-21).

Techniques: Gene Expression, RNA Sequencing, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Control, 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 – 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: Cells were treated with recombinant human TSP1 protein (ABclonal, RP01197) in the absence or presence of anti-CD47 neutralizing antibody (Bio X Cell- InVivo MAb, BE0283; Clone MIAP410) or IgG control (Bio X Cell- InVivo MAb, BE0083; Clone MOPC-21).

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: Cells were treated with recombinant human TSP1 protein (ABclonal, RP01197) in the absence or presence of anti-CD47 neutralizing antibody (Bio X Cell- InVivo MAb, BE0283; Clone MIAP410) or IgG control (Bio X Cell- InVivo MAb, BE0083; Clone MOPC-21).

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

Journal: Cancer Research

Article Title: CD36 Drives Metastasis and Relapse in Acute Myeloid Leukemia

doi: 10.1158/0008-5472.CAN-22-3682

Figure Lengend Snippet: CD36 triggers blast migration. A, GO analysis of migration-related biological processes significantly enriched in the CD36 gene signature. B, Number of migrating shCtrl or shCD36 U937 cells in the lower chamber of the transwell assay (normalized to control; n = 5). C, As in B, but with CD36-blocking antibodies (FA6-152 or JC53.1; n = 4). D, Number of migrating cells in primary AML samples treated or not with CD36 blocking antibody (FA6-152; n = 7). E, Number of migrating shCtrl or shTSP1 U937 cells (normalized to control; n = 3). F, Number of migrating shCtrl or shCD36 U937 cells treated or not with TSP1-blocking antibody (A6.1; normalized to shCtrl; n = 5). G, Number of migrating U937 cells treated or not with recombinant TSP1 in the presence or not of CD36-blocking antibody (FA6-152; normalized to control; n = 3). H, As in D, but with TSP1-blocking antibody (A6.1; n = 5). I, Bioluminescence imaging of mice injected with the indicated AML cell line stably expressing a luciferase reporter from day 1 to day 64 after injection. J, Representative histologic section showing anti-human Ku-80 labeling of U937 AML blasts in the subcutaneous adipose tissue 17 days after xenograft. Positive brown nuclei highlight the infiltration of human blasts in murine adipose tissue either as dense infiltrate (top) or scattered isolated cells (bottom). Top scale bar, 50 μm; bottom, 10 μm. K, Percentage of viable OCIAML3 cells in the indicated tissues 14 days after orthotopic injection in the femur. L, Expression of CD36 [mean fluorescence intensity (MFI)] in viable OCIAML3 cells measured in the injected and contralateral femur and different organs of mice 14 days after orthotopic injection. Values are represented as mean ± SEM. B, C, and E, One sample t test. D and H, Paired t test. F and G, Ordinary one-way ANOVA with Tukey multiple comparisons test. L, Matched one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: As indicated, anti-CD36 blocking antibodies (1 μg/mL; FA6-152, Stem Cell Technologies 60084 or Cayman chemical JC63.1 10009893) and/or recombinant TSP1 (2 nmol/L; R&D Systems 3074-TH) were added in the upper chamber.

Techniques: Migration, Transwell Assay, Control, Blocking Assay, Recombinant, Imaging, Injection, Stable Transfection, Expressing, Luciferase, Labeling, Isolation, Fluorescence