Journal: ACS Applied Bio Materials

Article Title: Hydrogel-Based Extracellular Vesicle Isolation Method from Various Biological Solutions

doi: 10.1021/acsabm.5c01838

Figure Lengend Snippet: Cellular uptake of isolated MSC-derived EVs isolated using the HAS method in different cell types. (A) Internalization of EVs in dermal papilla cells. EV membranes were labeled with ExoSparkler EX01 (green) and cell nuclei were stained with DAPI (blue). (B) Internalization of EVs in skin cells. EV proteins were labeled with ExoSparkler EX05 (red), and cell nuclei and cytoskeleton were stained with DAPI (blue) and phalloidin (green).

Article Snippet: Membranes of the isolated MSC-EVs were labeled with ExoSparkler EX01 (Dojindo Laboratories).

Techniques: Isolation, Derivative Assay, Labeling, Staining

Journal: Molecular Cancer

Article Title: Exosome-mediated lncRNA AFAP1-AS1 promotes trastuzumab resistance through binding with AUF1 and activating ERBB2 translation

doi: 10.1186/s12943-020-1145-5

Figure Lengend Snippet: Extracellular AFAP1-AS1 is transferred through incorporating into exosomes. a RNA fluorescence in situ hybridization (RNA-FISH) verified that AFAP1-AS1 was mainly distributed in nuclear of SKBR-3-TR and BT474-TR cells. b Extracellular AFAP1-AS1 was degraded by treatment with RNAse A and Triton simultaneously, ** P < 0.01. c TEM scanning showed the exosomes images released by SKBR-3-TR and SKBR-3 cells. d Size distribution of exosomes were analyzed by Zetasizer. e Exosomal protein markers (TSG101 and CD81) detection by Western blot from purified exosomes and exosome-depleted cell extracts. f qRT-PCR analysis of lncRNA AFAP1-AS1 expression in exosomes, culture medium with or without exosomes, *** P < 0.001. g RT-qPCR analysis showed that exosomal AFAP1-AS1 was upregulated in trastuzumab-resistant cells in contrast to sensitive cells, ** P < 0.01. h RNA immunoprecipitation assay using anti-HNRNPA2B1 antibody showed that AFAP1-AS1 was associated with HNRNPA2B1 protein. i HNRNPA2B1 was silenced by specific oligonucleotides in both transcript and protein levels, ** P < 0.01. j Knockdown of HNRNPA2B1 decreased AFAP1-AS1 expression in SKBR-3-TR and BT474-TR cells, * P < 0.05. k qRT-PCR and western blot verified the upregulation of HNRNPA2B1 by transfection of specific plasmids, ** P < 0.01. l AFAP1-AS1 was upregulated by overexpression of HNRNPA2B1 in SKBR-3-TR and BT474-TR cells, * P < 0.05

Article Snippet: Fig. 4 Extracellular AFAP1-AS1 is transferred through incorporating into exosomes. a RNA fluorescence in situ hybridization (RNA-FISH) verified that AFAP1-AS1 was mainly distributed in nuclear of SKBR-3-TR and BT474-TR cells. b Extracellular AFAP1-AS1 was degraded by treatment with RNAse A and Triton simultaneously, ** P < 0.01. c TEM scanning showed the exosomes images released by SKBR-3-TR and SKBR-3 cells. d Size distribution of exosomes were analyzed by Zetasizer. e Exosomal protein markers (TSG101 and CD81) detection by Western blot from purified exosomes and exosome-depleted cell extracts. f qRT-PCR analysis of lncRNA AFAP1-AS1 expression in exosomes, culture medium with or without exosomes, *** P < 0.001. g RT-qPCR analysis showed that exosomal AFAP1-AS1 was upregulated in trastuzumab-resistant cells in contrast to sensitive cells, ** P < 0.01. h RNA immunoprecipitation assay using anti-HNRNPA2B1 antibody showed that AFAP1-AS1 was associated with HNRNPA2B1 protein. i HNRNPA2B1 was silenced by specific oligonucleotides in both transcript and protein levels, ** P < 0.01. j Knockdown of HNRNPA2B1 decreased AFAP1-AS1 expression in SKBR-3-TR and BT474-TR cells, * P < 0.05. k qRT-PCR and western blot verified the upregulation of HNRNPA2B1 by transfection of specific plasmids, ** P < 0.01. l AFAP1-AS1 was upregulated by overexpression of HNRNPA2B1 in SKBR-3-TR and BT474-TR cells, * P < 0.05 Additional file 5: Dynamic video of exosome.

Techniques: Fluorescence, In Situ Hybridization, Western Blot, Purification, Quantitative RT-PCR, Expressing, RNA Immunoprecipitation, Knockdown, Transfection, Over Expression

Journal: Molecular Cancer

Article Title: Exosome-mediated lncRNA AFAP1-AS1 promotes trastuzumab resistance through binding with AUF1 and activating ERBB2 translation

doi: 10.1186/s12943-020-1145-5

Figure Lengend Snippet: Exosome mediated transfer of lncRNAAFAP1-AS1 induces trastuzumab resistance. a Intercellular trafficking of exosomes among different cell lines by isolated exosomes labeled with PKH26 dye. b Treatment with exosomes derived from SKBR-3-TR increased AFAP1-AS1 level, however this effect was abrogated when SKBR-3-TR cells were silenced with AFAP1-AS1, * P < 0.05. c-d CCK8 assay showed that SKBR-3-TR-derived exosomes induced trastuzumab resistance in SKBR-3 and BT474 cells, * P < 0.05. e Nano-sight particle tracking analysis of the size distributions and number of exosomes from SKBR-3-TR cells treated with nSMase, GW4869. f-g CCK8 assay showed that incubation with exosomes from SKBR-3-TR cells treated with GW4869 failed to confer trastuzumab resistance to recipient cells. h-i CCK8 assay verified that knockdown of AFAP1-AS1(h) or HNRNPA2B1(i) inhibited the ability of co-cultured parental cells to acquire trastuzumab resistance

Article Snippet: Fig. 4 Extracellular AFAP1-AS1 is transferred through incorporating into exosomes. a RNA fluorescence in situ hybridization (RNA-FISH) verified that AFAP1-AS1 was mainly distributed in nuclear of SKBR-3-TR and BT474-TR cells. b Extracellular AFAP1-AS1 was degraded by treatment with RNAse A and Triton simultaneously, ** P < 0.01. c TEM scanning showed the exosomes images released by SKBR-3-TR and SKBR-3 cells. d Size distribution of exosomes were analyzed by Zetasizer. e Exosomal protein markers (TSG101 and CD81) detection by Western blot from purified exosomes and exosome-depleted cell extracts. f qRT-PCR analysis of lncRNA AFAP1-AS1 expression in exosomes, culture medium with or without exosomes, *** P < 0.001. g RT-qPCR analysis showed that exosomal AFAP1-AS1 was upregulated in trastuzumab-resistant cells in contrast to sensitive cells, ** P < 0.01. h RNA immunoprecipitation assay using anti-HNRNPA2B1 antibody showed that AFAP1-AS1 was associated with HNRNPA2B1 protein. i HNRNPA2B1 was silenced by specific oligonucleotides in both transcript and protein levels, ** P < 0.01. j Knockdown of HNRNPA2B1 decreased AFAP1-AS1 expression in SKBR-3-TR and BT474-TR cells, * P < 0.05. k qRT-PCR and western blot verified the upregulation of HNRNPA2B1 by transfection of specific plasmids, ** P < 0.01. l AFAP1-AS1 was upregulated by overexpression of HNRNPA2B1 in SKBR-3-TR and BT474-TR cells, * P < 0.05 Additional file 5: Dynamic video of exosome.

Techniques: Isolation, Labeling, Derivative Assay, CCK-8 Assay, Incubation, Knockdown, Cell Culture

Journal: Molecular Cancer

Article Title: Exosome-mediated lncRNA AFAP1-AS1 promotes trastuzumab resistance through binding with AUF1 and activating ERBB2 translation

doi: 10.1186/s12943-020-1145-5

Figure Lengend Snippet: A scheme of the proposed mechanisms. Trastuzumab treatment increases AFAP1-AS1 expression; which upregulates HER-2 expression by guiding AUF1 to activate the translation of ERBB2 mRNA, inducing trastuzumab resistance. In addition, extracellular AFAP1-AS1 from trastuzumab-resistant cells was packaged into exosomes and disseminates trastuzumab resistance in trastuzumab sensitive cells

Article Snippet: Fig. 4 Extracellular AFAP1-AS1 is transferred through incorporating into exosomes. a RNA fluorescence in situ hybridization (RNA-FISH) verified that AFAP1-AS1 was mainly distributed in nuclear of SKBR-3-TR and BT474-TR cells. b Extracellular AFAP1-AS1 was degraded by treatment with RNAse A and Triton simultaneously, ** P < 0.01. c TEM scanning showed the exosomes images released by SKBR-3-TR and SKBR-3 cells. d Size distribution of exosomes were analyzed by Zetasizer. e Exosomal protein markers (TSG101 and CD81) detection by Western blot from purified exosomes and exosome-depleted cell extracts. f qRT-PCR analysis of lncRNA AFAP1-AS1 expression in exosomes, culture medium with or without exosomes, *** P < 0.001. g RT-qPCR analysis showed that exosomal AFAP1-AS1 was upregulated in trastuzumab-resistant cells in contrast to sensitive cells, ** P < 0.01. h RNA immunoprecipitation assay using anti-HNRNPA2B1 antibody showed that AFAP1-AS1 was associated with HNRNPA2B1 protein. i HNRNPA2B1 was silenced by specific oligonucleotides in both transcript and protein levels, ** P < 0.01. j Knockdown of HNRNPA2B1 decreased AFAP1-AS1 expression in SKBR-3-TR and BT474-TR cells, * P < 0.05. k qRT-PCR and western blot verified the upregulation of HNRNPA2B1 by transfection of specific plasmids, ** P < 0.01. l AFAP1-AS1 was upregulated by overexpression of HNRNPA2B1 in SKBR-3-TR and BT474-TR cells, * P < 0.05 Additional file 5: Dynamic video of exosome.

Techniques: Expressing

Journal: Journal of Agricultural and Food Chemistry

Article Title: Impact of Holder Pasteurization on Extracellular Vesicles and Immunoregulatory MicroRNAs in Human Breast Milk

doi: 10.1021/acs.jafc.5c11814

Figure Lengend Snippet: Schematic overview of the experimental procedure used to isolate purified exosomes and free-circulating RNA.

Article Snippet: Exosomes were isolated from 10 mL of human milk serum, obtained from both raw and pasteurized milk from each of the 6 samples, using the Plasma/Serum Exosome and Free-Circulating RNA Isolation Maxi Kit (Norgen Biotek Corp., Thorold, ON, Canada) following the manufacturer’s instructions ( ) and stored at −80 °C until further processing.

Techniques: Purification