Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance.

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: Figure 3. The effects of CD44-knockdown on NMR OPCs (A) A representative immunoblot of CD44 in mouse and NMR OPCs. (B) A representative immunoblot of CD44 in NMR OPCs transfected with siLuciferase, siCD44-1, or siCD44-2. Cells were collected 3 days after transfection. (C) The heatmap shows the relative levels of genes that are differently expressed between control and CD44-knockdown NMR OPCs (q value <0.05). (D) The boxplot shows the effects of CD44 knockdown on the expression levels of the indicated gene sets in NMR OPCs. The effects are shown for whole genes, the genes that were differentially expressed in NMR OPCs compared with OPCs of all other species, and the genes that were differentially expressed in both NMR and human OPCs compared with mouse and rat OPCs. (E) Functional enrichment analyses of genes that were differentially expressed between control and CD44 knockdown NMR OPCs. (F) The results of transcription factor target enrichment analysis of genes that were downregulated by CD44 knockdown in NMR OPCs. Top five overrepresented transcription factors obtained by the search of ARCHS4 TFs. Co-expression database and TRRUST TF-target regulatory interaction database are shown. (G) The heatmap shows the relative expression levels of differentially expressed genes that are associated with the terms highlighted in (E). (H) A representative immunoblot of CD44 in mouse OPCs transfected with siLuciferase, siCD44-1, or siCD44-2. Cells were collected 3 days after transfection. (I) Cell survival rate of mouse and NMR OPCs transfected with siLuc, siCD44-1, and siCD44-2 after 6 days of 0.5 mg/mL tunicamycin treatment. Transfection was performed 2 days before starting tunicamycin treatment (n = 8).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 500 kDa-HA Echelon Cat#HYA-500KEF 1000 kDa-HA Echelon Cat#HYA-1000KEF FGF-b Peprotech Cat#100-18B PDGF-AA Sigma Cat#H8291 N2 supplement Thermo Fisher Scientific Cat#17502048 B27 supplement Thermo Fisher Scientific Cat#17504044 0.01% poly-L-ornithine solution Sigma Cat#A-0040-C Laminin Sigma Cat#L2020 Accutase Innovative Cell Technologies Cat#AT104 Insulin Sigma Cat#91077 T3 Sigma Cat#T2877 Liberase TM Sigma Cat#5401119001 PEI-MAX transfection reagent Polysciences Inc Cat#24765 RNAiMAX transfection reagent Thermo Fisher Scientific Cat#13778075 Protease inhibitor cocktail Nacalai Tesque Cat#04080-11 CellRox Deep Red Thermo Fisher Scientific Cat#C10422 Critical commercial assays CyQUANT Cell Proliferation Assay kit Thermo Fisher Scientific Cat#C7026 Neural Tissue Dissociation Kit Miltenyi Biotech Cat#130-092-628 Deposited data RNA-Seq, NMR OPCs https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-9160 RNA-Seq, NMR OPCs transfected with siLuc or siCD44 https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-9166 RNA-Seq, IMR90-hTert cells expressing shLuc or shCD44 https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-12936 RNA-Seq, human OPCs https://www.ncbi.nlm.nih.gov/geo/ GSE181404 RNA-Seq, mouse OPCs https://www.ncbi.nlm.nih.gov/geo/ GSE79244 RNA-Seq, rat OPCs https://www.ncbi.nlm.nih.gov/geo/ GSE72739 RNA-Seq, NMR whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE67542 RNA-Seq, human whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE120795 RNA-Seq, mouse whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE96644 RNA-Seq, rat whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE53960 RNA-Seq, IMR90 cells overexpressing CD44 https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-8943 Mass spectrometry, proteins co-immunoprecipitated with CD44 in IMR90 cells https://www.ebi.ac.uk/pride/ PXD013445 Experimental models: Cell lines IMR90 Japanese Collection of Research Bioresources Cat#JCRB9054 U2OS ATCC Cat#HTB-96 HEK293T ATCC Cat#CRL-3216 Experimental models: Organisms/strains Naked Mole Rat Our lab N/A C57BL/6J mouse The Jackson Laboratory N/A Oligonucleotides Please see Table S5 for sequences of PCR primers, siRNAs, shRNAs, and gRNAs.

Techniques: Knockdown, Western Blot, Transfection, Control, Expressing, Functional Assay

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance.

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: Figure 6. CD44 localizes to the ER and interacts with multiple ER proteins (A) Protein-protein interactions among the 41 CD44-associated proteins plus IRE1, PERK, and ATF6 were mapped using the STRING software. Only highest confidence interactions (interaction score R0.9) that are experimentally determined (purple line) or stored in curated databases (blue line) were included in the analysis. IRE1, PERK, and ATF6 are shown as red nodes and the 16 CD44-associated ER proteins are shown as blue nodes. CD44-associated proteins are defined here as proteins that are detected by cross-linked immunoprecipitation-mass spectrometry of IMR90 cells using anti-CD44 antibody with at least 10-fold greater abundance than in control experiment using normal rabbit IgG antibody. (B) Representative immunoblots of CALR, HSP47, and CD44 in whole cell lysate and in CD44-immunoprecipitates prepared from IMR90 cells. The experiment was repeated once with similar results. (C) Representative confocal images of IMR90 cells co-stained with antibodies against CD44 and organelle markers (mitochondrial marker mtTFA and ER markers ERp57, HSP47, and RPN2). (D) The dot plot shows Pearson correlation coefficients between immunofluorescence signals of CD44 and organelle markers. Each dot represents a single cell (n = 30). Error bars are presented as mean ± SD values. Scale bars, 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 500 kDa-HA Echelon Cat#HYA-500KEF 1000 kDa-HA Echelon Cat#HYA-1000KEF FGF-b Peprotech Cat#100-18B PDGF-AA Sigma Cat#H8291 N2 supplement Thermo Fisher Scientific Cat#17502048 B27 supplement Thermo Fisher Scientific Cat#17504044 0.01% poly-L-ornithine solution Sigma Cat#A-0040-C Laminin Sigma Cat#L2020 Accutase Innovative Cell Technologies Cat#AT104 Insulin Sigma Cat#91077 T3 Sigma Cat#T2877 Liberase TM Sigma Cat#5401119001 PEI-MAX transfection reagent Polysciences Inc Cat#24765 RNAiMAX transfection reagent Thermo Fisher Scientific Cat#13778075 Protease inhibitor cocktail Nacalai Tesque Cat#04080-11 CellRox Deep Red Thermo Fisher Scientific Cat#C10422 Critical commercial assays CyQUANT Cell Proliferation Assay kit Thermo Fisher Scientific Cat#C7026 Neural Tissue Dissociation Kit Miltenyi Biotech Cat#130-092-628 Deposited data RNA-Seq, NMR OPCs https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-9160 RNA-Seq, NMR OPCs transfected with siLuc or siCD44 https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-9166 RNA-Seq, IMR90-hTert cells expressing shLuc or shCD44 https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-12936 RNA-Seq, human OPCs https://www.ncbi.nlm.nih.gov/geo/ GSE181404 RNA-Seq, mouse OPCs https://www.ncbi.nlm.nih.gov/geo/ GSE79244 RNA-Seq, rat OPCs https://www.ncbi.nlm.nih.gov/geo/ GSE72739 RNA-Seq, NMR whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE67542 RNA-Seq, human whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE120795 RNA-Seq, mouse whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE96644 RNA-Seq, rat whole brain https://www.ncbi.nlm.nih.gov/geo/ GSE53960 RNA-Seq, IMR90 cells overexpressing CD44 https://www.ebi.ac.uk/biostudies/arrayexpress E-MTAB-8943 Mass spectrometry, proteins co-immunoprecipitated with CD44 in IMR90 cells https://www.ebi.ac.uk/pride/ PXD013445 Experimental models: Cell lines IMR90 Japanese Collection of Research Bioresources Cat#JCRB9054 U2OS ATCC Cat#HTB-96 HEK293T ATCC Cat#CRL-3216 Experimental models: Organisms/strains Naked Mole Rat Our lab N/A C57BL/6J mouse The Jackson Laboratory N/A Oligonucleotides Please see Table S5 for sequences of PCR primers, siRNAs, shRNAs, and gRNAs.

Techniques: Protein-Protein interactions, Software, Immunoprecipitation, Mass Spectrometry, Control, Western Blot, Staining, Marker