Journal: Cell reports

Article Title: Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning

doi: 10.1016/j.celrep.2021.109293

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To assay human RUNX2 isoform overexpression we subcloned human RUNX2-I (MRIPV isoform, GeneCopoeia #EX-I2457-Lv105) into pCIB (Addgene #119863), and the human-equivalent ERK-target sites were made using Quikchange and subcloning: wild-type pCIB-hsRUNX2, pCIB-hsRUNX2-S280A-S298A (RUNX2-SA) and hsRUNX2-S280E-S298E (RUNX2-SE) which were used for additional in vitro validations (Figures S9B and S9C; note that human RUNX2-SA does not run faster by SDS-PAGE than human RUNX2-WT or human RUNX2-SE, as is observed with mouse isoforms, yet the effects on target gene expression is consistent for both species).

Techniques: Recombinant, Membrane, Enzyme-linked Immunosorbent Assay, Software

Journal: JCI Insight

Article Title: Palmitate impairs autophagic degradation via oxidative stress/perilysosomal Ca 2+ overload/mTORC1 activation pathway in pancreatic β cells

doi: 10.1172/jci.insight.192827

Figure Lengend Snippet: ( A and B ) Expression profile of TRPML channels in mouse pancreatic islets ( A ) and MIN6 cells ( B ). ( C ) Lower expression of TRPML1 in pancreatic islets from patients with type 2 diabetes compared with controls in separate public datasets (black square) and their meta-analyzed fold-change between control and diabetes (red diamond) calculated by Inverse-Variance Weighted method. Heatmaps of TRPMLs show the z values (top) calculated by meta-analysis and t values from each dataset by DESeq2 analysis. ( D ) GCaMP3-TRPML1 fluorescent probe detecting perilysosomal Ca 2+ level. ( E and F ) MLSA1 (10 μM), a TRPML agonist, and GPN (100 μM), a lysosomotrope, elevated perilysosomal (GCaMP3-TRPML1) and cytosolic (Fura-2) Ca 2+ levels in MIN6 cells. ( G ) MLSA1 decreased ER Ca 2+ content measured by G-CEPIA1er, suggesting ER Ca 2+ release. ( H ) Cytosolic Ca 2+ rose upon extracellular Ca 2+ supplementation in MLSA1-treated group, indicating store-operated Ca 2+ entry. ( I ) Fluorescence imaging of MIN6 cells expressing GCaMP3-TRPML1 treated with BSA or PA. Scale bar: 1 μm ( I ). ( J and K ) PA elevated the basal level but abolished MLSA1 response of perilysosomal ( J ) or cytosolic ( K ) Ca 2+ in MIN6 cells. ( L and M ) The oxidants, menadione (100 μM) ( L ) or H 2 O 2 ( M ), like PA, elevated the baseline Ca 2+ and abolished the MLSA1 response. [Ca 2+ ] max implies cells in normal Ca 2+ Krebs–Ringer bicarbonate buffer (KRBB) with ionomycin. Data are presented as means ± SD, and ( n ) is the number of analyzed cells from more than 3 independent experiments, except those in L and M (from 2–3 independent experiments). Statistical significance was determined using unpaired 2-tailed Student’s t test ( E , F , H , J , and L ) or 1-way ANOVA with post hoc Tukey multiple-comparison test ( M ). ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To measure ER Ca 2+ and lysosomal Ca 2+ , G-CEPIA1er (Addgene plasmid #105012) and GCaMP3-TRPML1 provided by Haoxing Xu (University of Michigan, Ann Arbor, Michigan, USA), respectively, were introduced by transfection 48 hours prior to the experiment.

Techniques: Expressing, Control, Fluorescence, Imaging, Comparison