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