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 Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Schematic illustration of the fabrication and evaluation of Treg-Exo-IKVAV for traumatic SCI.( a ) The isolation of Treg-Exo and the preparation process of Treg-Exo-IKVAV. ( b ) Mechanism underlying Treg-Exo-IKVAV’s immunoregulatory and neuro-regenerative capabilities. ( c ) Treg-Exo-IKVAV spatiotemporally sequentially reshaped the neuro-immune microenvironment, and then promoted functional recovery of SCI mice

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Isolation, Functional Assay

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV. Treg-Exo was stained by DiO and IKVAV was labeled by Cy5.5. ( i-j ) The physicochemical stability of Treg-Exo and Treg-Exo-IKVAV after a 7-day incubation period in phosphate-buffered saline (PBS) using time-dependent measurements of particle size distribution and zeta potential values ( n = 3). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( f , g ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Western Blot, Flow Cytometry, Fluorescence, Staining, Labeling, Incubation, Saline, Zeta Potential Analyzer, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: In vitro immunomodulatory effects of Treg-Exo-IKVAV.( a ) Uptake of green, fluorescent FITC-labeled Treg-Exo and Treg-Exo-IKVAV into Raw 264.7 macrophages. ( b ) The typical images of iNOS (green) and CD206 (red) immunostaining. ( c ) Quantitative analysis of immunostaining ( n = 5). (d) Flow cytometry analysis of CD86 and CD206 positive cells after Treg-Exo and Treg-Exo-IKVAV treatments. ( e ) Quantification of the flow cytometry results ( n = 5). ( f ) RT-PCR detection of macrophage polarization and inflammation-related gene expression ( n = 5). ( g ) KEGG enrichment analysis. ( h ) GO analysis. ( i ) Schematic illustration of the immunoregulatory mechanism of Treg-Exo-IKVAV on macrophage. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( f ) or two-way ANOVA ( c , e ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: In Vitro, Labeling, Immunostaining, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV promoted neuronal differentiation of NSCs in vitro.( a ) Schematic diagram of the experiment evaluating the direct effect on NSCs differentiation. ( b ) Representative images of MAP2 (green) and GFAP (red) immunostaining. ( c ) Quantification of MAP2 and GFAP immunostaining of ( b ). ( d ) Schematic diagram of the experiment evaluating the immunoregulatory effect on NSCs differentiation. ( e ) Representative images of MAP2 (green) and GFAP (red) immunostaining. ( f ) Quantification of MAP2 and GFAP immunostaining of ( e ). ( g ) Neuronal differentiation related gene expression determined by RT-PCR. ( h ) KEGG analysis. ( i ) GO analysis. ( j ) Schematic illustration of the immunoregulatory mechanism of Treg-Exo-IKVAV on NSCs differentiation. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA with Tukey’s multiple comparison test ( c , f , g ). ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: In Vitro, Immunostaining, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV targeted inflammation and promoted motor functional behavior recovery of SCI mice. ( a ) Schematic illustration of the in vivo experiments. ( b ) Typical fluorescence images of ICG-labeled Treg-Exo and Treg-Exo-IKVAV. ( c ) Quantitative analysis of ( b ) ( n = 3). ( d ) Ex vivo ICG signals of spinal cords harvested from SCI mice at 7. days after injection with Treg-Exo and Treg-Exo-IKVAV. ( e ) Ex vivo ICG signals of major organs (heart, liver, spleen, lung and kidney) harvested at 7. days after injection with Treg-Exo and Treg-Exo-IKVAV. ( f ) Quantitative analysis of relative fluorescence intensity in spinal cords and major organs ( n = 3). ( g ) Representative images of footprint assay on day 28 after Treg-Exo and Treg-Exo-IKVAV treatments, and quantitative analysis of toe dragging and stride length ( n = 5). ( h ) Typical images of the swimming test on day 28. ( i ) Quantification of the swimming test using Louisville Swim Scale ( n = 3). ( j ) BMS scores in the different groups ( n = 5). ( k ) H&E staining of bladder tissues in different groups on day 28, and quantitative analysis of bladder volume and bladder weight ( n = 3). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( g , k ) or two-way ANOVA with Tukey’s multiple comparison test ( c , f , i , j ). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Functional Assay, In Vivo, Fluorescence, Labeling, Ex Vivo, Injection, Staining, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV attenuated macrophage infiltration and inhibited macrophage-induced inflammation response secondary to SCI.( a ) Sagittal T2-MRI images on day 7. ( b ) Immunostaining of CD68 in different groups on day 7. ( c ) Quantification of CD68 immunostaining ( n = 3). ( d ) Co-immunostaining of CD68 (green) and iNOS (red), and CD68 (green) and Arg1 (red) in the different groups. ( e-f ) Quantification of CD68, iNOS, and Arg1 immunofluorescence staining ( n = 3). ( g ) RT-PCR detection of the gene expression (iNOS, TNF-α, IL-1β, IL-10, and Arg1) ( n = 3). ( h ) Levels of TNF-α, IL-6, CXCL10, IL-4 and IL-10 detected by ELISA ( n = 5). Data are presented as mean ± SD. Statistical significance was tested by one-way ANOVA ( c , e , f , g , h ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Immunostaining, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV promoted neuronal differentiation of NSCs, and axonal regeneration and remyelination.( a ) Immunofluorescence staining of NeuN to label the mature neurons on day 28. ( b ) Quantification of NeuN + cells in Z1-Z4 regions adjacent to the lesion core ( n = 3). ( c ) Immunostaining of TUJ1 to label the newly born neurons on day 28. ( d ) Percent of TUJ1 + /DAPI + cells ( n = 5). ( e ) Quantification analysis of ( f ) co-immunostaining of NF-200 (green) and GFAP (red), and ( g ) co-immunostaining of MBP (green) and GFAP (red) in the different groups at 28 days after SCI. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( d ) or two-way ANOVA with Tukey’s multiple comparison test (c). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Immunofluorescence, Staining, Immunostaining, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: In vitro and in vivo biosafety of Treg-Exo-IKVAV nanovesicles.( a ) Typical images of Live/Dead staining. ( b ) Viability of NSCs after cultured with Treg-Exo or Treg-Exo-IKVAV for 24 h determined by CCK-8 ( n = 5). ( c ) H&E staining of heart, liver, spleen, lung, and kidney tissues in the different groups. ( d ) Biochemical markers relevant to hepatic (ALT, AST) and kidney (UA, BUN) function ( n = 5). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA (b, d) with Tukey’s multiple comparison test. *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: In Vitro, In Vivo, Staining, Cell Culture, CCK-8 Assay, Comparison

Journal: Journal of Extracellular Biology

Article Title: TDO2‐augmented fibroblasts secrete EVs enriched in immunomodulatory Y‐derived small RNA

doi: 10.1002/jex2.73

Figure Lengend Snippet: NT4 is a short non‐coding RNA fragment with immunomodulatory function derived from human Y RNA‐4 (hY4). (a) Schematic of the hY4 sequence and functional regions. (b) Sequence of NT4 and its position within the hY4 fragment EV‐YF1. (c) qPCR showing NT4 enrichment in CCM of fibroblasts transduced with TDO2‐activating lentivirus (TDO2‐CCM) compared to those from fibroblasts transduced with a lentivirus containing a control vector (VC‐CCM). (d) QPCR of NT4 from SEC‐isolated EVs compared to the EV‐depleted fraction. (e) Absolute quantification (NT4 copies/10 3 particles) of TDO2‐CCM, VC‐CCM, SEC‐isolated TDO2‐EVs and the EV‐depleted fraction. (f) RNAse protection assay demonstrating NT4 is encapsulated inside TDO2‐EVs. (g) Broad expression of the monocyte marker, CD68, in bone marrow‐derived macrophages. (h) Equivalent uptake of TDO2‐ and HDF‐EVs (EV ceramide dye; red, phalloidin: green and nuclei: blue) by bone marrow derived macrophages at 30, 60 and 180 min post exposure as measured by internalisation of red signals inside macrophages and, (i) area under the curve measurement of cumulative signal at all three time points. (j) Representative image of TDO2‐ and HDF‐EVs (EV ceramide dye; red, phalloidin: green and nuclei: blue) by bone marrow derived macrophages at 1‐h post‐exposure. (k–m) TDO2‐EV and NT4 pre‐treatment lowered expression of inflammatory genes il1b and il6 , as well as pro‐senescence p21 / cdkn1a . Suppression of NT4 in TDO2‐CCM abrogated the suppressive effect of TDO2‐CCM on il1b , il6 and p21/cdkn1a in LPS‐stimulated macrophages. Scale bars: 100 μm. Statistical analysis between two groups was done using Student's T test and comparisons between three or more groups was done using one‐way ANOVA with Sidak's multiple comparison test. Error bars represent standard deviation,* p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: RNA was isolated using the urine EV RNA isolation kit (Norgen Biotek, cat# 47200). qPCR was performed to assess NT4 expression levels.

Techniques: Derivative Assay, Sequencing, Functional Assay, Transduction, Control, Plasmid Preparation, Isolation, Quantitative Proteomics, Rnase Protection Assay, Expressing, Marker, Comparison, Standard Deviation