Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: Activated UPR in CTC clusters protects cells from cell death. (A) Schematic diagram of the experimental design for isolating CTCs from mice for RNA sequencing at week 4 following fat pad inoculation with MDA‐MB‐231/LM2 cells. (B) GO pathway enrichment analysis of upregulated gene sets in CTC clusters compared to single CTCs ( n = 3). (C) GSVA enrichment scores of single CTCs and CTC clusters from breast cancer patients and PDX‐Br16 mouse model. The single‐cell RNA sequencing data were obtained from the GEO database ( GSE111065 ). (D) Relative mRNA expression levels of UPR‐related genes in single CTCs and CTC clusters from the NCG‐MDA‐MB‐231/LM2 mouse model ( n = 3). (E) Representative flow cytometry plots showing the gating strategy for identifying ER tracker intensity in single CTCs and CTC clusters from the blood of NCG‐MDA‐MB‐231/LM2 mice. (F) MFI of TPE‐MI in single CTCs and CTC clusters, as determined by flow cytometry ( n = 5). G. Percentage of Annexin V + single and clustered tumor cells pretreated with thapsigargin ( n = 3). (H) Cell viability of single and clustered tumor cells under thapsigargin treatment ( n = 3). (I) Schematic diagram of the experimental design. Clustered tumor cells were pretreated with vehicle or azoramide for 6 h prior to tail vein injection into NCG mice, followed by assessment of disseminated tumor cells in the lung at 24 h. (J‐K) Bioluminescence images (left) and quantification of fluorescence signal intensity (right) of mice (J) and the lungs (K) to assess lung metastasis at 24 h after tail vein injection of clustered MDA‐MB‐231/LM2‐CTC cells ( n = 5). Results represent mean ± SD. Student's t‐test in C, D, F, J, and K, two‐way ANOVA test in G and H. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: CTCs, circulating tumor cells; RNA‐seq, RNA sequencing; GO, gene ontology; GSVA, gene set variation analysis; PDX‐Br16, patient‐derived xenograft‐Br16; GEO, ​gene expression omnibus​; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; TPE‐MI, tetraphenylethene‐maleimide; MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; UPR, unfolded protein response; HSPA5, heat shock protein family A member 5; XBP1, x‐box binding protein 1; DDIT3, DNA damage inducible transcript 3; DNAJB1, dnaJ heat shock protein family member B1; ATF3, activating transcription factor 3; ATF4, activating transcription factor 4; PPP1R15A, protein phosphatase 1 regulatory subunit 15A; ER Tracker, endoplasmic reticulum tracker; MFI, mean fluorescence intensity; Azo, azoramide; IV, intravenous; ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Injection, Fluorescence, Derivative Assay, Binding Assay

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: PERK is a key factor in promoting CTC cluster survival. (A) Relative mRNA expression levels of UPR‐related genes in single CTCs and CTC clusters isolated from NCG‐MDA‐MB‐231/LM2 (left) and C57BL/6‐B16F10 (right) mouse models ( n = 3). (B) Percentage of Annexin V + single and clustered MDA‐MB‐231/LM2‐CTC (left) and B16F10 (right) cells transfected with siRNAs against PERK and IRE1α and treated with or without thapsigargin ( n = 3). (C) Percentage of Annexin V + clustered MDA‐MB‐231/LM2‐CTC (left) and B16F10 (right) cells treated with AMG44 prior to thapsigargin ( n = 3). (D) Relative PERK expression in single CTCs and CTC clusters isolated from BC patients and PDX mouse models (PDX‐1, PDX‐2, and PDX‐3) ( n = 15 for patients; n = 10 for PDX‐1; n = 8 for PDX‐2 and PDX‐3). (E) Schematic design of the experimental design. MDA‐MB‐231/LM2 cells were orthotopically implanted into mice, and lung metastases were analyzed via IHC at week 4. Representative IHC images of pPERK in lung metastases. The arrow indicates single CTCs; the dashed circle indicates CTC cluster. (F‐I) Representative immunofluorescence images of single CTCs and CTC clusters from a patient and the NCG‐MDA‐MB‐231/LM2 mouse model. Cells were stained for pPERK (red), EGFR (green) (F) or TUNEL (purple) (H), GFP (green) (G) or TUNEL (purple) (I), and DAPI (blue). (J) Schematic design of the experiment (left). MDA‐MB‐231/LM2‐CTC cells were pretreated with Vehicle, MK‐28 or AMG44 and then cultured in suspension for 24 h. Percentage of Annexin V + tumor cell clusters was analyzed by flow cytometry (right) ( n = 5). Results represent mean ± SD. Student's t‐test in A and D, one‐way ANOVA test in B, C, and J. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: UPR, unfolded protein response; CTCs, circulating tumor cells; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; ATF6, activating transcription factor 6; PERK, protein kinase R (PKR)‐like endoplasmic reticulum kinase; IRE1α, inositol‐requiring enzyme 1 alpha; BC, breast cancer; PDX, patient‐derived xenograft; IHC, immunohistochemistry; pPERK, phosphorylated PERK; DAPI, 4′,6‐diamidino‐2‐phenylindole; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling; siRNA, small interfering RNA;ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: Expressing, Isolation, Transfection, Immunofluorescence, Staining, TUNEL Assay, Cell Culture, Suspension, Flow Cytometry, Derivative Assay, Immunohistochemistry, Small Interfering RNA

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: PERK signaling regulates MAT2A via ATF4 to support enhanced methionine metabolism. (A) KEGG enrichment analysis of upregulated genes in scramble control tumor cell clusters compared to PERK‐KO clusters ( n = 3). (B) Volcano plot showing PERK‐related metabolites identified through targeted metabolomics ( n = 6). (C) Heatmap showing metabolites involved in the methionine cycle in scramble and PERK‐KO tumor cell clusters ( n = 6). (D) Intracellular metabolites in NC‐oe and PERK‐oe tumor cell clusters ( n = 6). (E) The schematic diagram for the conversion of [ 13 C 5 ]‐methionine into various metabolites (left) and LC‐MS quantification of M+5 methionine, M+5 SAM, and M+4 SAH following a 16 h incubation with [ 13 C 5 ]‐methionine in scramble and PERK‐KO tumor cell clusters (right) ( n = 4). (F) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC cells cultured in CM or medium lacking Ser, Gly, Met, or Cys for 24 h ( n = 5). (G‐I) Western blot analysis of metabolic enzyme expression in MDA‐MB‐231/LM2‐CTC and B16F10 tumor cell clusters with or without PERK (G), ATF4 expression in MDA‐MB‐231/LM2‐CTC with or without PERK (H), and ATF4 and MAT2A expression in MDA‐MB‐231/LM2‐CTC with or without ATF4 (I). (J) Relative MAT2A expression in MDA‐MB‐231/LM2‐CTC transfected with shNC or shATF4 #2 ( n = 3). (K) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC transduced with shNC or shMAT2A #3 and treated with or without SAM for 24 h (left) or pretreated with PF9366 prior to SAM (right) ( n = 3). (L) Schematic of the in vivo experiment design. (M) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). (N) Representative H&E staining images (left) and quantification of pulmonary nodules (right) in NCG mice at 2 weeks after tail vein injection of MDA‐MB‐231/LM2‐CTC clusters (shNC and shMAT2A #3) ( n = 5). (O) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters were cultured in CM or MRM for 24 h before tail vein injection into NCG mice, with lung metastases assessed at 24 h. (P) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). Results represent mean ± SD. Student's t‐test in D, E, J, M, and P; one‐way ANOVA test in F and K. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; FC, fold change; Sc, scramble; PERK, Protein kinase RNA‐like endoplasmic reticulum kinase; PERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; LC‐MS, liquid chromatography‐mass spectrometry; Met, methionine; SAM; S‐adenosylmethionine; SAH, S‐adenosylhomocysteine; CM, control medium; Ser, serine; Gly, glycine; Cys, cysteine; PSAT1, phosphoserine aminotransferase 1; PHGDH, phosphoglycerate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase; MAT2A, methionine adenosyltransferase 2A; ATF4, activating transcription factor 4; shNC, negative control short hairpin RNA; NC‐oe, negative control‐overexpression; PERK‐oe, PERK‐overexpression; shATF4, short hairpin RNA of ATF4; shMAT2A, short hairpin RNA of MAT2A; MRM, methionine‐restricted medium; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; CTCs, circulating tumor cells; BLI, lung Bioluminescence imaging; H&E, hematoxylin and eosin; ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Incubation, Cell Culture, Western Blot, Expressing, Transfection, Transduction, In Vivo, Imaging, Fluorescence, Injection, Staining, Knock-Out, Liquid Chromatography, Mass Spectrometry, Negative Control, shRNA, Over Expression

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: Methionine metabolism promotes H3K4me3 methylation modification to upregulate PDGFB expression in CTC clusters. (A) Western blot analysis of PERK, MAT2A, and H3K4me3 levels in control and PERK‐KO MDA‐MB‐231/LM2‐CTC clusters treated with or without PF9366 or SAM. (B) The volcano plot depicting differentially expressed genes affected by methionine ( n = 3). (C) Metaplot comparing H3K4me3 enrichment profiles in MDA‐MB‐231/LM2‐CTC clusters cultured in CM or MRM. The plot is centered on TSS (±3.0 Kb) ( n = 3). (D) Bioinformatics analysis filtered out 10 survival‐related genes as downstream targets of H3K4me3. (E) Heatmap showing survival‐associated gene expression in MDA‐MB‐231/LM2‐CTC clusters under CM or MRM conditions ( n = 3). (F) Genome browser tracks of H3K4me3 at the PDGFB gene locus by ChIP‐seq ( n = 3). (G) TEM images of MDA‐MB‐231/LM2‐CTC cluster. (H‐J) Representative immunofluorescence images (left) and quantification (right) of PDGFB in CTC clusters from NCG‐MDA‐MB‐231/LM2 mouse model (H, n = 20); or in the complete or dispersed CTC clusters (I, n = 15); or in control or PDGFB‐knockdown CTC clusters (J, n = 15). Arrows indicate cell‐cell border; circle indicates intracellular regions. (K) Representative images of flow cytometry (left) and quantification of AnnexinV + MDA‐MB‐231/LM2‐CTC clusters (right) in control (shNC) and PDGFB‐knockdown (shP#1/2/3) groups ( n = 5). (L) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters (shNC and shPDGFB #3) were injected into NCG mice via the tail vein, and metastatic burden was assessed by bioluminescence imaging at 24 h. (M‐N) Bioluminescence images (left) and quantification of fluorescence intensity (right) showing lung metastasis after tail vein injection at 24 h ( n = 5). Results represent mean ± SD. One‐way ANOVA test in A and K; student's t‐test in H, I, J, M, and N. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; CTCs, circulating tumor cells; FC, fold change; PERK, PRKR‐like endoplasmic reticulum kinase; ERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; MAT2A, methionine adenosyltransferase 2A; H3K4me3, trimethylation of histone H3 at lysine 4; SAM, S‐adenosylmethionine; CM, control medium; MRM, methionine‐restricted medium; No., number; TSS, transcription start site; ChIP‐seq, chromatin immunoprecipitation sequencing; RNA‐seq, RNA sequencing; PDGFB, platelet‐derived growth factor subunit B; IGF2BP3, insulin‐like growth factor 2 mRNA‐binding protein 3; PHGDH, phosphoglycerate dehydrogenase; ANXA11, annexin A11; ITGA6, integrin subunit alpha 6; ITGB4, integrin subunit beta 4; LOX, lysyl oxidase; PIK3C2B, phosphatidylinositol‐4‐phosphate 3‐kinase catalytic subunit type 2 beta; IRF9, interferon regulatory factor 9; ATP2A3, ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 3; TEM, transmission electron microscopy; DAPI, 4′,6‐diamidino‐2‐phenylindole; shNC, negative control short hairpin RNA; shP#, short hairpin RNA targeting PDGFB; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; BLI, lung Bioluminescence imaging; ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: Methylation, Modification, Expressing, Western Blot, Control, Cell Culture, Gene Expression, ChIP-sequencing, Immunofluorescence, Knockdown, Flow Cytometry, Injection, Imaging, Fluorescence, Knock-Out, RNA Sequencing, Derivative Assay, Binding Assay, Transmission Assay, Electron Microscopy, Negative Control, shRNA

Journal: STAR Protocols

Article Title: Generation of mixed murine organoids to model cellular interactions

doi: 10.1016/j.xpro.2021.100997

Figure Lengend Snippet:

Article Snippet: 4′,6-Diamidino-2-phenylindole Dihydrochloride (DAPI) , Toronto Research Chemicals , D416050; CAS RN: 28718-90-3.

Techniques: Recombinant, Membrane, Software, Microscopy, Transferring, Polymer

Journal: Reviews in Cardiovascular Medicine

Article Title: Targeting Tumor Necrosis Factor-α Mitigates Glucose Fluctuation-Induced Aortic Valve Fibrosis: Insights From Diabetic Rat Models

doi: 10.31083/RCM42804

Figure Lengend Snippet: Colocalization of CD3, CD68, and JAK1 in the aortic valves of diabetic rat models . (A,B) The coimmunostaining analysis of the HG and GF groups demonstrated the presence of CD3, CD68, and JAK1 proteins, indicating that JAK1 expression is primarily localized in T lymphocytes and macrophages. Notably, this expression pattern could be altered through the inhibition of TNF- α . Scale bar = 50 µm. DAPI, 4 ′ ,6-diamidino-2-phenylindole.

Article Snippet: In addition, the concentrated SABC-POD (Mouse/Rabbit IgG) kit (SA2010, BOSTER Biological Technology Co., Ltd., Wuhan, China), DyLight 488 conjugated AffiniPure goat anti-mouse IgG (H + L) (BA1126, BOSTER Biological Technology Co., Ltd., Wuhan, China), DyLight 594 conjugated AffiniPure goat anti-rabbit IgG (H + L) (BA1142, BOSTER Biological Technology Co., Ltd., Wuhan, China), ethylenediaminetetraacetic acid (EDTA) antigen retrieval solution (AR0023, BOSTER Biological Technology Co., Ltd., Wuhan, China), 4 ′ ,6-diamidino-2-phenylindole (DAPI) staining solution (AR1176, BOSTER Biological Technology Co., Ltd., Wuhan, China), human TNF- α enzyme-linked immunosorbent assay (ELISA) kit (EK0525, BOSTER Biological Technology Co., Ltd., Wuhan, China), and human TGF- β 1 ELISA kit (EK0513, BOSTER Biological Technology Co., Ltd., Wuhan, China) were obtained from Boster, China.

Techniques: Expressing, Inhibition