Journal: bioRxiv

Article Title: Orally Delivered Milk-Derived Nanovesicles Loaded with Connexin 43 Peptides for Targeted Cardiac Ischemia-Reperfusion Therapy

doi: 10.1101/2025.01.01.630994

Figure Lengend Snippet: A) Schematic of mEV isolation protocol showing the steps of the procedure. Fractions referred to are those collected during Sepharose Column (SEC) fractionation. Fractions 7 through 9 (F7-F9) show highest levels of mEV enrichment and samples used in this study were pooled from F7-F9. Fractions >14 show enrichment for contaminating milk proteins such as casein, with low or no evidence of mEVs, and are typically discarded. B) Single Molecule Localization Microscopy (SMLM) image of an isolated mEV isolated by the protocol in (A) tagged for CD81 (red) and CD63 (teal). C) Negative stain transmission electron microscopy (TEM) indicating dense accumulations of mEVs generated by the isolation protocol. D) Calcein AM loading assay illustrating membrane intactness of isolated mEVs E) Western blotting of mEV isolates for CD81, CD9, TSG-101, and negativity and markedly reduced expression of Calnexin and Casein, respectively. Lysates from HeLa cells are included as a control. Sepharose fraction 16 (F16) shows elevated casein, but no evidence of EV markers. F) Nanoparticle Tracking Analysis (NTA) of an mEV isolate indicating >5×10 12 mEVs per mL, with average nanoparticle size of 125.0 nm, consistent with small EVs. G) SMLM-generated size distribution of CD9/CD81/CD63-positive mEVs, showing a similar histogram morphology to NTA results in panel F.

Article Snippet: Overnight primary antibody incubation was performed and primary antibodies were diluted in the blocking buffer as follows: CD81 (Cell Signaling Technology, 56039S, 1:1000) CD9 (Novus Biologicals, NB500-494, 1:1000), Calnexin (MilliporeSigma, AB2301, 1:5000), TSG101 (Bethyl Laboratories Inc. A303-506A, 1:5000), Casein (Abcam, Ab166596, 1:2000), Cx43 Cytoplasmic-Loop (Invitrogen, PA5-11632, 1:1000), and antibodies against the Cx43 CT (MilliporeSigma, C6219, 1:4000 and Abcam, Ab87645, 1:1000).

Techniques: Isolation, Fractionation, Microscopy, Staining, Transmission Assay, Electron Microscopy, Generated, Membrane, Western Blot, Expressing, Control

Journal: Frontiers in cardiovascular medicine

Article Title: Direct Cardiac Actions of Sodium-Glucose Cotransporter 2 Inhibition Improve Mitochondrial Function and Attenuate Oxidative Stress in Pressure Overload-Induced Heart Failure.

doi: 10.3389/fcvm.2022.859253

Figure Lengend Snippet: FIGURE 5 | EMPA treatment enhanced autophagy in failing hearts. (A) Transmission electron microscopy (TEM) showed autophagosomes in the hearts. Red arrows point to autophagosomes. (B) EMPA activated autophagy pathway by inhibiting mTOR pathway. (C) RT-PCR results showed that the mRNA expression of Beclin1, Atg7 and LC3 were increased by EMPA treatment after TAC. Results are expressed as mean ± SEM, n = 5–7, *p < 0.05 vs. corresponding sham group, †p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; Ulk, Unc-51 like autophagy activating kinase; Atg7, autophagy related 7; LC3, light chain 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Antibodies against phospho-AMP-activated protein kinase (p-AMPK) (Cell Signaling, 2535), AMPK (Cell Signaling, 5831), phospho-mammalian target of rapamycin (p-mTOR) (Cell Signaling, 2971), mTOR (Cell Signaling, 2983), phospho-Unc-51 like autophagy activating kinase (p-Ulk1) (Cell Signaling, 14202), Ulk1 (Cell Signaling, 8054), autophagy related 7 (Atg7) (Cell Signaling, 8558), Beclin1 (Novus, NB500-249), light chain 3 (LC3) A/B (Cell Signaling, 4108), Bcl-2 (Cell Signaling, 3498), Bax (Cell Signaling, 14796), cleaved caspase-3 (Cell Signaling, 9664), phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling, 4370), ERK (Cell Signaling, 4695), phospho-Bad (Cell Signaling, 9291), Bad (Cell Signaling, 9292), nuclear respiratory factor 1 (NRF-1) (Novus, NBP275597), the nuclear factor erythroid 2–related factor 2 (NRF-2) (Novus, H00004780-M02), heme oxygenase-1 (HO-1) (Cell Signaling, 86806), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) (Novus, NBP1-04676), mitochondrial transcription factor A (TFAM) (Novus, NBP219437), cyclooxygenase1 (COX1) (Cell Signaling, 9896), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling, 2118), were used as primary antibodies.

Techniques: Transmission Assay, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Cellular and molecular neurobiology

Article Title: Heterozygous FOXJ1 Mutations Cause Incomplete Ependymal Cell Differentiation and Communicating Hydrocephalus.

doi: 10.1007/s10571-023-01398-6

Figure Lengend Snippet: Fig. 3 Heterozygous Foxj1 mutation causes incomplete differentia- tion of ependymal cells. Lower magnification (a, g) and higher mag- nification (b, h) scanning electron microscopy images of the brain lateral ventricular surface of WT (Foxj1+/+) and Foxj1WT/CreERT2::GFP (Foxj1+/-) mice. c, i Immunohistochemistry of the lateral ventricu- lar wall revealed areas that lacked acetylated-tubulin (Ac-tubulin) immunoreactivity (a marker for motile cilia) in Foxj1WT/CreERT2::GFP mice when compared to WT mice. Each of the areas lacking motile

Article Snippet: These slices were stained for fluorescence immunohistochemistry using antibodies directed against acetylated α-tubulin (sc-23950, Lot# L1019, 1:500, Santa Cruz Biotechnology, USA), γ-tubulin (NB500-574, Lot#542972020916G, 1:500, Novus, USA), VANGL2 (sc515187, Lot# C0520, 1:100, Santa Cruz Biotechnology, USA; MABN750 clone 2G4, Lot#3,794,319, MilliporeSigma, USA), S100β (PA5-78,161, Lot#137187E, 1:500, Invitrogen, USA) FGFR1OP (FOP) (H00011116-M01, Lot#N2-131-2B1, Abnova, USA), alpha smooth muscle actin (αSMA) (48,938, Clone 1A4, Lot#2, 1:100, Cell Signaling Technology, USA), FOXJ1 (14–9965-82, Lot#2,488,476, 1:200, Invitrogen, USA), or GFAP (13–0300, Clone 2.2B10, Lot#TJ275318, 1:500, Invitrogen, USA).

Techniques: Mutagenesis, Electron Microscopy, Immunohistochemistry, Marker

Journal: Cellular and molecular neurobiology

Article Title: Heterozygous FOXJ1 Mutations Cause Incomplete Ependymal Cell Differentiation and Communicating Hydrocephalus.

doi: 10.1007/s10571-023-01398-6

Figure Lengend Snippet: Fig. 5 Heterozygous Foxj1 mutation disrupts translational and pla- nar cell polarity. a Fluorescence immunohistochemistry for the tight junction protein, ZO-1 (red), and the planar cell polarity pro- tein, VANGL2 (green), in ependymal cells on the lateral ventricu- lar surface of WT (Foxj1+/+ and Foxj1WT/CreERT2::GFP (Foxj1+/-) mice. Note the directionally asymmetric membrane association of VANGL2 immunoreactivity in ependymal cells from WT but not Foxj1WT/CreERT2::GFP mice. b Immunohistochemistry for basal foot marker FOP (red) and the basal body marker, γ-tubulin (green) in ependymal cells on the lateral ventricular surface of WT, and Foxj1WT/CreERT2::GFP mice. The rotational polarities are indicated by vectors (black arrows) connecting FOP-positive areas to the nearest γ-tubulin-positive areas. c Immunohistochemistry for ZO-1 (green) and the basal body marker γ-tubulin (red) in ependymal cells on the lateral ventricular surface of WT and Foxj1WT/CreERT2::GFP mice. Basal body clusters are directionally oriented and asymmetrically displaced

Article Snippet: These slices were stained for fluorescence immunohistochemistry using antibodies directed against acetylated α-tubulin (sc-23950, Lot# L1019, 1:500, Santa Cruz Biotechnology, USA), γ-tubulin (NB500-574, Lot#542972020916G, 1:500, Novus, USA), VANGL2 (sc515187, Lot# C0520, 1:100, Santa Cruz Biotechnology, USA; MABN750 clone 2G4, Lot#3,794,319, MilliporeSigma, USA), S100β (PA5-78,161, Lot#137187E, 1:500, Invitrogen, USA) FGFR1OP (FOP) (H00011116-M01, Lot#N2-131-2B1, Abnova, USA), alpha smooth muscle actin (αSMA) (48,938, Clone 1A4, Lot#2, 1:100, Cell Signaling Technology, USA), FOXJ1 (14–9965-82, Lot#2,488,476, 1:200, Invitrogen, USA), or GFAP (13–0300, Clone 2.2B10, Lot#TJ275318, 1:500, Invitrogen, USA).

Techniques: Mutagenesis, Fluorescence, Immunohistochemistry, Membrane, Marker

Journal: International Journal of Nanomedicine

Article Title: Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

doi: 10.2147/IJN.S446525

Figure Lengend Snippet: HLA class I-expressing CD9+ EVs quantified in plasma, whole urine, and ultracentrifuge-purified uEVs using IFCM. ( A – C ) The scatterplots present three final readouts from IFCM: CD9-APC single-positives (red dots), HLA class I (HLA-ABC-Alexa488) single-positives (green dots), and double-positives (“++”; blue dots). Each gate’s name shows the number and the percentage of gated objects. ( D – F ) The measurement of ( A ) – ( C ) after detergent (TritonX-100) treatment. ( G – I ) Isotype staining of all samples. Purified uEVs are obtained using ultracentrifugation and are concentrated 2.67 times compared to unprocessed urine. ( J & K ) The dilution-corrected concentration of CD9 & HLA class I double-positives ( J ) and HLA class I single-positives ( K ) in healthy plasma (n = 10, red bars), urine (n = 10, blue bars), and purified uEVs (n = 10, purple bars). Samples are collected from the same individuals. Data are presented as median [Q1 – Q3]. The black dot line depicts the limit of reliable detection of uEV concentration, representing the background levels observed in detergent-treated and isotype-staining controls (3 × 10 5 objects/mL). Created with BioRender.com. Marks : ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, no significant difference.

Article Snippet: For cryo-electron microscopy (cryo-EM) experiments, uEV samples isolated using ultracentrifugation were labeled with anti-HLA class I antibody (clone TP25.99SF, Bio-Techne) conjugated with 6-nm gold nanoparticles (NPs).

Techniques: Expressing, Purification, Staining, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

doi: 10.2147/IJN.S446525

Figure Lengend Snippet: TR-FIA. ( A ) Schematic overview of TR-FIA. ( B ) Summary of europium intensity of unprocessed plasma and urine. Plasma and urine are collected from the same healthy individuals (n = 10). EVs from samples are captured by anti-HLA class I or its isotype and then labeled by Europium (Eu)- conjugated anti-CD9 or anti-CD63. Data are presented as median [Q1 – Q3]. “HLA” means capturing EVs using anti-HLA class I antibody (Ab); “ISO” denotes capturing using isotype, IgG1. Created with BioRender.com. Marks : ***, p < 0.001; **, p < 0.01.

Article Snippet: For cryo-electron microscopy (cryo-EM) experiments, uEV samples isolated using ultracentrifugation were labeled with anti-HLA class I antibody (clone TP25.99SF, Bio-Techne) conjugated with 6-nm gold nanoparticles (NPs).

Techniques: Labeling

Journal: International Journal of Nanomedicine

Article Title: Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

doi: 10.2147/IJN.S446525

Figure Lengend Snippet: Immunogold labeling for HLA class I on EVs and visualization using cryo-EM. ( A ) Representative image of an HLA class I+ EV in T cell supernatant. ( B ) Purified uEV sample. Created with BioRender.com.

Article Snippet: For cryo-electron microscopy (cryo-EM) experiments, uEV samples isolated using ultracentrifugation were labeled with anti-HLA class I antibody (clone TP25.99SF, Bio-Techne) conjugated with 6-nm gold nanoparticles (NPs).

Techniques: Labeling, Cryo-EM Sample Prep, Purification

Journal: International Journal of Nanomedicine

Article Title: Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

doi: 10.2147/IJN.S446525

Figure Lengend Snippet: Location of HLA class I on the kidney biopsies. ( A – E ) HLA class I immunohistochemistry on kidney tissue obtained from discarded donor kidneys ( A – C ) and peritumoral kidney tissue ( D – E ). Two HLA class I antibodies are used. Black arrows point out the apical membrane of renal tubules, and purple arrows indicate their basal membrane side. Red arrows present vessels in the renal interstitium. ( F ) A cartoon illustrating the HLA class I location in the renal tubule and interstitium. Created with BioRender.com.

Article Snippet: For cryo-electron microscopy (cryo-EM) experiments, uEV samples isolated using ultracentrifugation were labeled with anti-HLA class I antibody (clone TP25.99SF, Bio-Techne) conjugated with 6-nm gold nanoparticles (NPs).

Techniques: Immunohistochemistry, Membrane

Journal: British Journal of Cancer

Article Title: Circulating tumour cells and PD-L1-positive small extracellular vesicles: the liquid biopsy combination for prognostic information in patients with metastatic non-small cell lung cancer

doi: 10.1038/s41416-023-02491-9

Figure Lengend Snippet: a Mode and mean size of sEVs. b Mean concentration of sEVs in the 53 samples. c Representative western blots showing the expression of sEV markers (ALIX, TSG101, CD63, CD81, CD9), PD-L1 and HSP70 in lysates of sEVs isolated from four different plasma samples (upper panels). GRP94 was used as a negative control for cellular material contamination, and the total protein extract of the human lung cancer cell line A549 as a positive control for this contamination (lower panel). d Representative distribution of the size of particles isolated from plasma samples of patients with NSCLC obtained by nanoparticle tracking analysis. e Representative transmission electron microscopy image of the isolated sEVs. Scale bar, 100 nm; sEVs small Extracellular Vesicles.

Article Snippet: Isolated sEVs were then incubated with antibodies against universal sEV markers: CD9 (sc-13118, Santa Cruz Biotechnology), ALIX (NB100-65678, Novus Bio), TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (NBP2-4225, BioTechne), CD81 (sc166028, Santa Cruz Biotechnology), HSP70 (ADI-SPA-810, Enzo Life science), PD-L1 (sc-50298, Santa Cruz Biotechnology), and GRP94 as negative control (ADI-SPA-850, Enzo Life Sciences).

Techniques: Concentration Assay, Western Blot, Expressing, Isolation, Negative Control, Positive Control, Transmission Assay, Electron Microscopy

Journal: Frontiers in Cardiovascular Medicine

Article Title: Direct Cardiac Actions of Sodium-Glucose Cotransporter 2 Inhibition Improve Mitochondrial Function and Attenuate Oxidative Stress in Pressure Overload-Induced Heart Failure

doi: 10.3389/fcvm.2022.859253

Figure Lengend Snippet: RT-qPCR primers.

Article Snippet: Antibodies against phospho-AMP-activated protein kinase (p-AMPK) (Cell Signaling, 2535), AMPK (Cell Signaling, 5831), phospho-mammalian target of rapamycin (p-mTOR) (Cell Signaling, 2971), mTOR (Cell Signaling, 2983), phospho-Unc-51 like autophagy activating kinase (p-Ulk1) (Cell Signaling, 14202), Ulk1 (Cell Signaling, 8054), autophagy related 7 (Atg7) (Cell Signaling, 8558), Beclin1 (Novus, NB500-249), light chain 3 (LC3) A/B (Cell Signaling, 4108), Bcl-2 (Cell Signaling, 3498), Bax (Cell Signaling, 14796), cleaved caspase-3 (Cell Signaling, 9664), phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling, 4370), ERK (Cell Signaling, 4695), phospho-Bad (Cell Signaling, 9291), Bad (Cell Signaling, 9292), nuclear respiratory factor 1 (NRF-1) (Novus, NBP2-75597), the nuclear factor erythroid 2–related factor 2 (NRF-2) (Novus, H00004780-M02), heme oxygenase-1 (HO-1) (Cell Signaling, 86806), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) (Novus, NBP1-04676), mitochondrial transcription factor A (TFAM) (Novus, NBP2-19437), cyclooxygenase1 (COX1) (Cell Signaling, 9896), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling, 2118), were used as primary antibodies.

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Direct Cardiac Actions of Sodium-Glucose Cotransporter 2 Inhibition Improve Mitochondrial Function and Attenuate Oxidative Stress in Pressure Overload-Induced Heart Failure

doi: 10.3389/fcvm.2022.859253

Figure Lengend Snippet: EMPA treatment enhanced autophagy in failing hearts. (A) Transmission electron microscopy (TEM) showed autophagosomes in the hearts. Red arrows point to autophagosomes. (B) EMPA activated autophagy pathway by inhibiting mTOR pathway. (C) RT-PCR results showed that the mRNA expression of Beclin1, Atg7 and LC3 were increased by EMPA treatment after TAC. Results are expressed as mean ± SEM, n = 5–7, * p < 0.05 vs. corresponding sham group, † p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; Ulk, Unc-51 like autophagy activating kinase; Atg7, autophagy related 7; LC3, light chain 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Antibodies against phospho-AMP-activated protein kinase (p-AMPK) (Cell Signaling, 2535), AMPK (Cell Signaling, 5831), phospho-mammalian target of rapamycin (p-mTOR) (Cell Signaling, 2971), mTOR (Cell Signaling, 2983), phospho-Unc-51 like autophagy activating kinase (p-Ulk1) (Cell Signaling, 14202), Ulk1 (Cell Signaling, 8054), autophagy related 7 (Atg7) (Cell Signaling, 8558), Beclin1 (Novus, NB500-249), light chain 3 (LC3) A/B (Cell Signaling, 4108), Bcl-2 (Cell Signaling, 3498), Bax (Cell Signaling, 14796), cleaved caspase-3 (Cell Signaling, 9664), phospho-extracellular signal-regulated kinase (p-ERK) (Cell Signaling, 4370), ERK (Cell Signaling, 4695), phospho-Bad (Cell Signaling, 9291), Bad (Cell Signaling, 9292), nuclear respiratory factor 1 (NRF-1) (Novus, NBP2-75597), the nuclear factor erythroid 2–related factor 2 (NRF-2) (Novus, H00004780-M02), heme oxygenase-1 (HO-1) (Cell Signaling, 86806), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) (Novus, NBP1-04676), mitochondrial transcription factor A (TFAM) (Novus, NBP2-19437), cyclooxygenase1 (COX1) (Cell Signaling, 9896), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling, 2118), were used as primary antibodies.

Techniques: Transmission Assay, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Affinity chromatography of VESmix on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). ( – )—absorbance on A 280 nm, mAU; P1—subfraction appeared in flow, P2—subfraction eluted with 0.5 NaCl, P3—subfraction eluted with 1.0 M NaCl, P4—subfraction eluted with 0.1 M Gly-HCl pH 2.6.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Transmission electron microscopy of horse milk exosomes on different stages of isolation and purification. P2 subfraction was obtained after affinity chromatography on anti-CD9-Sehparose ( A – D , I ) and anti-CD63-Sepharose ( E – H , J ). General view ( A , E ); exosomes of ≤ 100 nm ( B , F ); vesicles > 100 nm ( C ); exosomes (white arrow) and particles of medium electron density without outer membranes (ovals) ( D ); particles of medium electron density without outer membranes ( G ); vesicles, “non-vesicles”, particles of medium electron density without outer membranes ( H ); gold immunolabeling using anti-CD9-IgG ( I ) and anti-CD63-IgG ( J ). The length of the scale line corresponds to 100 nm ( B , D , F – J ) and 200 nm ( A , C , E ).

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Transmission Assay, Electron Microscopy, Isolation, Purification, Affinity Chromatography, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Flow cytometry analysis of P2 + P3 subfractions eluted from anti-CD9- and anti-CD63-Sepharose. Relative amounts of exosomes containing CD9 (left panel), CD63 (central panel), and CD81 (right panel) are shown.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Direct MALDI mass spectrometry analysis of untreated exosomes (P2 + P3) after affinity chromatography on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). The errors in determining the m / z value did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Mass Spectrometry, Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: MALDI mass spectrometry analysis of P2 + P3 exosome extracts obtained after affinity chromatography on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). The extracts were obtained after the destruction with TFA and acetonitrile. The error in determining the m / z values did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Mass Spectrometry, Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: MALDI mass spectra of P2 + P3 subfraction of horse milk exosome extracts obtained after affinity chromatography on anti-CD9- ( A ) and anti-CD63-Sepharose ( B ). The extracts were obtained by destruction of P2 subfraction with TFA and acetonitrile, and the compounds passed through the Amicon filter with a 3 kDa cut-off were analyzed. The error in determining the m / z values did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: MALDI mass spectra of horse milk exosome extract corresponding to peak P2 after affinity chromatography on anti-CD9-Sepharose before ( A ) and after proteolysis with trypsin ( B ), chymotrypsin ( C ), and proteinase K ( D ). The error in determining the m / z value did not exceed 0.5–1.0 Da.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Proteins and Peptides of Highly Purified CD9 + and CD63 + Horse Milk Exosomes Isolated by Affinity Chromatography

doi: 10.3390/ijms232416106

Figure Lengend Snippet: Molecular masses of major peptides corresponding to P2 + P3 peaks of VESmix isolated by affinity chromatography on anti-CD9/CD63-Sepharoses and extracts obtained using TFA and acetonitrile.

Article Snippet: Exosome preparations were applied to anti-CD9- and anti-CD63-Sepharoses equilibrated with 20 mM Tris-HCl pH 7.5; the sorbents were washed with the same buffer to zero A 280 density (peak P1).

Techniques: Isolation, Affinity Chromatography

Journal: Biology

Article Title: TLR9 Binding to Beclin 1 and Mitochondrial SIRT3 by a Sodium-Glucose Co-Transporter 2 Inhibitor Protects the Heart from Doxorubicin Toxicity

doi: 10.3390/biology9110369

Figure Lengend Snippet: Sodium-glucose co-transporter 2 inhibition increases the autophagic flux in mice hearts. ( A ) Temporal changes in LC3-II/I ratio and Beclin 1 protein abundances after empagliflozin feedings. Hearts were analyzed at different time points after intraperitoneal injection of 1.5 mg/kg BafA1 or normal saline for 2 h ( n = 3 per group. *** p < 0.001, ** p < 0.01, data were analyzed by one-way analysis of variance (ANOVA) with Dunnett post hoc analysis). ( B ) Representative fluorescence images of heart sections from CAG–RFP-EGFP–LC3 transgenic mice fed with 7-day empagliflozin and injected with BafA1 or normal saline for 2 h. Quantification of autophagosomes (yellow puncta) and autolysosomes (red puncta) numbers ( n = 6 mice per group and 10 slices per one mice, ** p < 0.01, * p < 0.05, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( C ) Changes in LC3-II/I ratio and Beclin 1 protein abundances after 7-day empagliflozin feedings and 1-dose intravenous doxorubicin injection (5 mg/kg) for 24 h. Hearts were analyzed after intraperitoneal injection of 1.5 mg/kg BafA1 or normal saline for 2 h ( n = 4 hearts per group with six microscopic fields (14,000 μm 2 ) per heart section analyzed. ** p < 0.01, * p < 0.05, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( D ) Representative fluorescence images of heart sections from CAG–RFP-EGFP–LC3 transgenic mice injected with doxorubicin (5 mg/kg) and fed with 7-day empagliflozin. Quantification of autophagosomes (yellow puncta) and autolysosomes (red puncta) numbers ( n = 6 per group. ** p < 0.01, * p < 0.05, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( E ) Representative transmission electron microscopy images of cardiomyocytes treated with doxorubicin and empagliflozin. Autophagosomes have two parallel membrane layers separated by a relatively narrower electron-translucent cleft. Autolysosomes have only one membrane and frequently contain electron dense cytoplasmic materials. Quantification of autophagosomes (yellow arrow) and autolysosomes (red arrow) numbers ( n = 6. * p < 0.05, ** p < 0.01, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( F ) Changes in LC3-II/I ratio and Beclin 1 protein abundances in cardiomyocytes treated with doxorubicin and empagliflozin ( n = 4 per group. * p < 0.05, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( G ) Changes in autophagy flux measured by RFP-GFP-LC3 tandem fluorescent-tagged LC3 in cardiomyocytes treated with doxorubicin and empagliflozin ( n = 4 per group. * p < 0.05, ** p < 0.01, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( H ) Long-lived protein degradation assay in cardiomyocytes ( n = 4 per group. * p < 0.05, data were analyzed by one-way ANOVA with Tukey post hoc analysis). Data are represented by mean ± s.e.m. (BafA1, bafilomycin A1; CAG-RFP-EGFP-LC3, CAG promoter-red fluorescent protein (RFP)–green fluorescent protein-LC3).

Article Snippet: The anti-LC3 antibody (ab48394, Abcam), anti-Beclin 1 (#3495, Cell Signaling, Danvers, MA, USA; NB500-249, Novus, Littleton, NH, USA), anti-GAPDH (#2118, Cell Signaling), anti-SIRT3 (#5490S, Cell Signaling), anti-TLR9 (ab17236, ab211012, Abcam; SC-47723, Santa Cruz; NBP2-24729, Novus), anti-ATP5A (ab110273, Abcam), anti-γ-H2AX (ab11174, Abcam), anti-Flag (M2-A8592, Sigma-Aldrich), and anti-HA(12013819001, Roche, Basel, Switzerland) were used.

Techniques: Inhibition, Injection, Fluorescence, Transgenic Assay, Transmission Assay, Electron Microscopy, Degradation Assay

Journal: Biology

Article Title: TLR9 Binding to Beclin 1 and Mitochondrial SIRT3 by a Sodium-Glucose Co-Transporter 2 Inhibitor Protects the Heart from Doxorubicin Toxicity

doi: 10.3390/biology9110369

Figure Lengend Snippet: Empagliflozin further protects doxorubicin cardiotoxicity in Beclin 1 deficient mice. ( A ) Left ventricular function determined by m-mode echocardiography at the indicated time points ( n = 7 per group. * p < 0.05, ** p < 0.01, data were analyzed by two-way analysis of variance (ANOVA) with Tukey post hoc analysis). ( B–F ) Mice were sacrificed immediately following the fourth injection at week 4. ( B ) Phosphorylation of H2A histone family member X (γ-H2AX) staining of left ventricular sections, *** p < 0.001. ( C ) Picrosirius red staining, *** p < 0.001. ( D ) Serum cardiac troponin-T, *** p < 0.001, ** p < 0.01. ( E ) BNP mRNA, ** p < 0.01, p = 0.06 for Beclin 1+/− + DOXO vs. Beclin 1+/− + DOXO/EMPA. ( F ) Col1a1 mRNA, * p < 0.05, NS , not significant ( n = 7 per group, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( G ) TUNEL staining of the neonatal cardiomyocytes. ( n = 4, ** p < 0.01, *** p < 0.001, data were analyzed by the Kruskal-Wallis one-way ANOVA). ( H ) Cellular ROS detected by H 2 DCFDA fluorescence ( n = 4, ** p < 0.01, *** p < 0.001, NS , not significant, data were analyzed by the two-way ANOVA with Tukey post hoc analysis). Data are represented by mean ± s.e.m. (WT, wild-type; EMPA, empagliflozin; DOXO, doxorubicin; ROS, reactive oxygen species).

Article Snippet: The anti-LC3 antibody (ab48394, Abcam), anti-Beclin 1 (#3495, Cell Signaling, Danvers, MA, USA; NB500-249, Novus, Littleton, NH, USA), anti-GAPDH (#2118, Cell Signaling), anti-SIRT3 (#5490S, Cell Signaling), anti-TLR9 (ab17236, ab211012, Abcam; SC-47723, Santa Cruz; NBP2-24729, Novus), anti-ATP5A (ab110273, Abcam), anti-γ-H2AX (ab11174, Abcam), anti-Flag (M2-A8592, Sigma-Aldrich), and anti-HA(12013819001, Roche, Basel, Switzerland) were used.

Techniques: Injection, Staining, TUNEL Assay, Fluorescence

Journal: Biology

Article Title: TLR9 Binding to Beclin 1 and Mitochondrial SIRT3 by a Sodium-Glucose Co-Transporter 2 Inhibitor Protects the Heart from Doxorubicin Toxicity

doi: 10.3390/biology9110369

Figure Lengend Snippet: Identification of TLR9 and SIRT3 as major empagliflozin-regulated components of the Beclin 1 complexes. ( A ) SYPRO Ruby staining of affinity-purified Beclin 1 complexes from Flag-Beclin 1-HA overexpressed 293 cell lines treated with vehicles (lane 2) or empagliflozin (lane 3). Specific Beclin 1-interacting protein bands were analyzed by mass spectrometry. ( B ) Coimmunoprecipitation of Beclin 1 with TLR9 or TLR9 with Beclin 1 from 293 cells with or without empagliflozin (200 nM) treatment. Western blot analysis of 293 whole-cell extracts transfected with Flag-TLR9 and Beclin 1-HA and immunoprecipitated with Beclin 1-HA (lane 1) or Flag-TLR9 (lane 2). ( C ) Beclin 1 interaction with TLR9 in mice hearts and regulated by empagliflozin. WT mice were fed with or without empagliflozin for 7 days. Heart lysates were immunoprecipitated with Beclin 1 or TLR9 antibody recognizing full-length 130 kDa or cleaved form 53 kDa and analyzed by western blot. ( D ) Protein domains involved in the Beclin-TLR9 interaction. Flag-tagged TLR9 and its indicated fragments (FL: full length TLR9; Cter: C-terminal TLR9; TIR: TIR domain of the TLR9; Nter: N-terminal TLR9; d-Hinge: full length TLR9 without Hinge domain) were coexpressed in 293 cells with Beclin 1-HA, and anti-HA immunoprecipitates were analyzed by anti-Flag immunoblotting. ( E ) Protein domains involved in the TLR9-SIRT3 interaction. Flag-tagged TLR9 and its indicated fragments (FL: full length TLR9; Cter: C-terminal TLR9; TIR: TIR domain of the TLR9; Nter: N-terminal TLR9; d-Hinge: full length TLR9 without Hinge domain) were coexpressed in 293 cells with Myc-Sirt3, and anti-Myc immunoprecipitates were analyzed by anti-Flag immunoblotting. ( F ) Left: Temporal changes in TLR9 and SIRT3 protein abundances after empagliflozin feedings. Hearts were analyzed at different time points after empagliflozin feedings. Right: Western blot analysis of changes in TLR9 and SIRT3 protein abundances after intravenous doxorubicin injections (5 mg/kg) and 7-day empagliflozin feedings. ( G ) Images of AC16 human cardiomyocytes stained for mitochondria (red), nucleus (blue), and overexpressed GFP-TLR9 (green). Cells were treated with indicated doses of empagliflozin before imaging. Increases of the orange signals (green + red) indicate the increases of mitochondrial localization of TLR9. ( H ) Western blot analysis of the mitochondria and whole-cell lysates from AC16 human cardiomyocytes treated with vehicle or empagliflozin (200 nM). ( I ) Images of AC16 human cardiomyocytes stained for mitochondria (red), nucleus (blue), and overexpressed GFP-TLR9 (green). Cells were treated with indicated doses of empagliflozin before imaging.

Article Snippet: The anti-LC3 antibody (ab48394, Abcam), anti-Beclin 1 (#3495, Cell Signaling, Danvers, MA, USA; NB500-249, Novus, Littleton, NH, USA), anti-GAPDH (#2118, Cell Signaling), anti-SIRT3 (#5490S, Cell Signaling), anti-TLR9 (ab17236, ab211012, Abcam; SC-47723, Santa Cruz; NBP2-24729, Novus), anti-ATP5A (ab110273, Abcam), anti-γ-H2AX (ab11174, Abcam), anti-Flag (M2-A8592, Sigma-Aldrich), and anti-HA(12013819001, Roche, Basel, Switzerland) were used.

Techniques: Staining, Affinity Purification, Mass Spectrometry, Western Blot, Transfection, Immunoprecipitation, Imaging

Journal: Biology

Article Title: TLR9 Binding to Beclin 1 and Mitochondrial SIRT3 by a Sodium-Glucose Co-Transporter 2 Inhibitor Protects the Heart from Doxorubicin Toxicity

doi: 10.3390/biology9110369

Figure Lengend Snippet: Reduction of empagliflozin effects in SIRT3 or TLR9 knockout mice and in humans with SIRT3 point mutation with reduced enzymatic activity. ( A ) Left ventricular function determined by m-mode echocardiography at the indicated time points ( n = 7 per group. * p < 0.05 for WT + DOXO + EMPA vs. SIRT3 KO + DOXO + EMPA or TLR9 KO + DOXO + EMPA, data were analyzed by two-way analysis of variance (ANOVA) with Tukey post hoc analysis). ( B – F ) Mice were sacrificed at 4 weeks after doxorubicin injection. ( B ) Phosphorylation of H2A histone family member X (γ-H2AX) staining of left ventricular sections, *** p < 0.001. ( C ) Picrosirius red staining, *** p < 0.001. ( D ) Serum cardiac troponin-T, * p < 0.05. ( E ) BNP mRNA, * p < 0.05. ( F ) Col1a1 mRNA, * p < 0.05 ( n = 7 per group, data were analyzed by one-way ANOVA with Tukey post hoc analysis). ( G ) Graphic representation of the SNP rs11246020 frequency in the prospective cohort of patients with DCM or control patients. CC corresponds to the normal alleles, CT to SNP heterozygosity, and TT to SNP homozygosity. ( H ) Western blots for TLR9 and SIRT3 performed on the cardiac biopsies before and after 28 days of empagliflozin treatment from patients with DCM (2 with SIRT3 rs11246020 CC and 1 with TT genotypes). ( I ) Basal mitochondrial respiration performed on the cardiac biopsies before and after 28 days of empagliflozin treatment from patients with DCM (2 with SIRT3 rs11246020 CC and 1 with TT genotypes). ( J ) Schematic of the empagliflozin promotes binding of the Beclin 1-TLR9 and trafficking to mitochondria toward SIRT3. In SIRT3 knockout mice, empagliflozin cannot enhance the trafficking of TLR9 toward mitochondria. Data are represented by mean ± s.e.m. (WT, wild-type, EMPA, empagliflozin; DOXO, doxorubicin, DCM, dilated cardiomyopathy).

Article Snippet: The anti-LC3 antibody (ab48394, Abcam), anti-Beclin 1 (#3495, Cell Signaling, Danvers, MA, USA; NB500-249, Novus, Littleton, NH, USA), anti-GAPDH (#2118, Cell Signaling), anti-SIRT3 (#5490S, Cell Signaling), anti-TLR9 (ab17236, ab211012, Abcam; SC-47723, Santa Cruz; NBP2-24729, Novus), anti-ATP5A (ab110273, Abcam), anti-γ-H2AX (ab11174, Abcam), anti-Flag (M2-A8592, Sigma-Aldrich), and anti-HA(12013819001, Roche, Basel, Switzerland) were used.

Techniques: Knock-Out, Mutagenesis, Activity Assay, Injection, Staining, Western Blot, Binding Assay