ago2 expression  (Sino Biological)

Journal: RNA Biology

Article Title: C-terminal tagging impairs AGO2 function

doi: 10.1080/15476286.2025.2534028

Figure Lengend Snippet: Creation and genomic characterisation of AGO2-HaloTag cell Lines(A) schematic of WT AGO2 and the C terminal AGO2-HaloTag fusion (including T2A site, puromycin resistant (PuroR) and Poly(A) sections) genotype to be generated by CRISPaint editing of A549 cells. Arrows indicate locations of forward and reverse primers designed to confirm editing. Blue = AGO2 WT last intron forward and reverse; Light Teal = HaloTag1 forward and reverse; Dark Teal = HaloTag2 forward and reverse.(B) agarose gel loaded with PCR products of A549 WT and two AGO2-HaloTag (AGO2-HaloTag C5 and AGO2-HaloTag C10) lines amplified with indicated combinations of AGO2 WT and HaloTag primers, as indicated in (A). Red arrows indicate gDNA containing HaloTag sequence which was purified and submitted for sequencing. Circled numbers 1–3 indicate gDNA containing C terminal non-HaloTagged AGO2 product which was purified and submitted for sequencing. (C) sequence (generated from TOPO-seq) alignments of WT and two AGO2-HaloTag clones at the AGO2-HaloTag junction. From several submitted TOPO clones, two variants of AGO2-HaloTag (one long and one short) were identified in AGO2-HaloTag cells. Asterisk (*) indicates STOP codon. (D) schematic to show known functionally important domains of AGO2, with a focus on C-terminal PIWI domain. CRISPaint mediated AGO2-HaloTag fusion generated a long and a short variant. (E) chromatograph of C terminal AGO2 sequence identified in WT, AGO2-HaloTag C5 and AGO2-HaloTag C10 cells (circled numbers 1–3 in (B)) showing the additional and premature STOP codon in both AGO2-HaloTag lines. (F) abundance of untagged AGO2 mRNA transcript in A549 WT, two UnTagged (UT C1 and UT C2) and two AGO2-HaloTag (AGO2-HaloTag C5 and AGO2-HaloTag C10) cells. AGO2 (non-HaloTagged) mRNA abundance normalized to B Actin mRNA abundance and made relative to levels in WT cells. Data represent mean ± SEM of experiments; n = 3 (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). (G & H) Western blot of whole-cell lysates from A549 WT, two UnTagged (UT C1 and UT C2) and two AGO2-HaloTag (AGO2-HaloTag C5 and AGO2-HaloTag C10) cell lines probed with antibodies against AGO2, HaloTag, and beta Actin.

Article Snippet: Single-cell clones were screened for loss of AGO2 expression by immunoblotting using rabbit anti-AGO2 (Sino Biological 101,620-T36).

Techniques: Generated, Agarose Gel Electrophoresis, Amplification, Sequencing, Purification, Clone Assay, Variant Assay, Western Blot

Journal: RNA Biology

Article Title: C-terminal tagging impairs AGO2 function

doi: 10.1080/15476286.2025.2534028

Figure Lengend Snippet: Initial characterisation of AGO2-HaloTag cells Lines(A) doubling time of A549 WT, two UnTagged (UT C1 and UT C2) and two AGO2-HaloTag (AGO2-HaloTag C5 and AGO2-HaloTag C10) measured using IncucyteZoom over 108 hours. Data represent mean ± SEM; n = 3 (ns p > 0.05). (B) Representative Western blot of whole-cell lysates from indicated cell lines (harvested during log-phase) probed with antibodies against AGO1, AGO2, AGO3, AGO4, Vinculin. (C) Representative Western blot of whole-cell lysates from indicated cell lines (harvested during log-phase) probed with antibodies against DDX6, TNRC6A, LIMD1, and beta Actin. (D) densitometry of AGO1/2/3/4 (normalized to loading control (Vinculin)) in indicated cell lines. Data represents mean ± SEM; n = 3 (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 according to one-way ANOVA test with Dunnett’s multiple comparisons). (E) densitometry analysis of drosha, exportin 5, Dicer, DGCR8, DDX6 andTNRC6A, (normalized to loading control (beta Actin or vinculin)) in indicated cell lines. Data represent mean ± SEM; n = 3 (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: Single-cell clones were screened for loss of AGO2 expression by immunoblotting using rabbit anti-AGO2 (Sino Biological 101,620-T36).

Techniques: Western Blot, Control

Journal: RNA Biology

Article Title: C-terminal tagging impairs AGO2 function

doi: 10.1080/15476286.2025.2534028

Figure Lengend Snippet: Characterising AGO2 interactions, AGO2 localization and miR-451a levels in AGO2-HaloTag clones. (A) endogenous co-immunoprecipitation of AGO2 with Dicer and TNRC6A in WT, UnTagged C1 , UnTagged C2, AGO2-HaloTag C5 and AGO2-HaloTag C10 cells. Note the increased AGO2-Dicer and decreased AGO2-TNRC6A co-immunoprecipitation in AGO2 HALO cells compared to in WT and UnTagged cells. Histogram shows mean densitometry for TNRC6A IP normalized to AGO2 IP densitometry relative to WT A549 cells. Data represents mean of four independent repeats with * indicating p ≤ 0.05 according to one-way ANOVA test with Dunnett’s multiple comparisons. (B) immunoblots of nuclear and cytoplasmic fractions from WT. UnTagged C1 , UnTagged C2, AGO2-HaloTag C5 and AGO2-HaloTag C10 cells. Whole cell lysate (WCL) from WT A549 was also blotted alongside. Beta tubulin and histone H3 were probed as loading controls for cytoplasmic and nuclear fractions, respectively. (C) MiR-451a abundance (normalized to U6 RNA and made relative to relevant WT) in indicated cell lines measured by RT-qPCR using the 2 –∆∆Ct method. Data represent mean ± SEM; n = 2 (* p ≤ 0.05 according to one-way ANOVA test with Dunnett’s multiple comparisons). (D) pri-miR-451 abundance (normalised to beta actin mRNA levels) in indicated cell lines measured by RT-qPCR using the standard curve method. Data represent mean ± SEM; n = 2 (ns = not significant according to one-way ANOVA test with Dunnett’s multiple comparisons).

Article Snippet: Single-cell clones were screened for loss of AGO2 expression by immunoblotting using rabbit anti-AGO2 (Sino Biological 101,620-T36).

Techniques: Clone Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR

Journal: RNA Biology

Article Title: C-terminal tagging impairs AGO2 function

doi: 10.1080/15476286.2025.2534028

Figure Lengend Snippet: Creation of AGO2 knockout A549, firefly luciferase assays and comparison of N - and C-terminally tagged AGO2. (A) Generation of AGO2 knockout A549 cells. Immunoblot of two AGO2 −/− clones alongside two control clones for AGO2 and beta actin as a loading control. (B) Relative firefly/Renilla luciferase activity in indicated cell lines transfected with reporter plasmids expressing firefly and Renilla luciferase and a siRNA against firefly luciferase (esi FFLuc ) or ‘non-targeting’ control (esi GFP ). The ratio between firefly and Renilla luciferase activity was measured 24 h after transfection. Data represent mean ± SEM; n = 3 (ns p > 0.05, **** p ≤ 0.0001 according to two-way ANOVA test with Sidak’s multiple comparisons). (C) Plasmid constructs for expression of untagged AGO2, EGFP-AGO2 and AGO2-EGFP. EGFP-VO is a control. (D) Relative firefly/Renilla luciferase activity in AGO2 −/− A549 transfected with reporter plasmids, AGO2 plasmids and an siRNA against firefly luciferase (esi FFLuc ) or ‘non-targeting’ control (scr). The ratio between firefly and Renilla luciferase activity was measured 24 h after transfection. Data represent mean ± SEM; n = 3 (ns p > 0.05; *** p ≤ 0.001, **** p ≤ 0.0001 according to one-way ANOVA test with Dunnett’s multiple comparisons). (E) Relative Renilla/firefly luciferase activity of a miR-100 targeted (T) reporter relative to a non-targeted (NT) reporter in AGO2 −/− A549 transfected with reporter plasmid, 15 nM miR-100 mimic and the indicated AGO2 constructs or EGFP-VO. Data represent mean ± SEM; n = 3 (ns p > 0.05; ** p ≤ 0.01 according to one-way ANOVA test with Dunnett’s multiple comparisons). (F) Immunoblots of nuclear and cytoplasmic fractions from AGO2 −/− A549 transfected with EGFP-VO, AGO2 UT, EGFP-AGO2 or AGO2-EGFP. Beta tubulin and histone H3 were probed as loading controls for cytoplasmic and nuclear fractions, respectively. (G) Fluorescence microscopy images for AGO2 −/− A549 transfected with EGFP-VO, EGFP-AGO2 or AGO2-EGFP. Cell nuclei are stained with DAPI and merged images show DAPI and EGFP signal combined. Scale bars indicate 10 µm. (H) UnTagged C1 and AGO2-HaloTag C10 cells treated with 100 nM HaloTag-TMR ligand visualized using confocal microscopy.

Article Snippet: Single-cell clones were screened for loss of AGO2 expression by immunoblotting using rabbit anti-AGO2 (Sino Biological 101,620-T36).

Techniques: Knock-Out, Luciferase, Comparison, Western Blot, Clone Assay, Control, Activity Assay, Transfection, Expressing, Plasmid Preparation, Construct, Fluorescence, Microscopy, Staining, Confocal Microscopy

Journal: RNA Biology

Article Title: C-terminal tagging impairs AGO2 function

doi: 10.1080/15476286.2025.2534028

Figure Lengend Snippet: Structural insights of the C-terminal of AGO2 for an explanation of impaired function upon HaloTag insertion. (A) Schematic composition of AGO2 showing 7 main domains and motifs. (B) C-terminal residue A859 contributes to miRNA binding (PDB code: 4OLB). Residues shown in stick format and residue type and sequence number annotated. Dashed lines show inter-atom distances 5.0 Å. (C) Surface representation of AGO2 (4OLB) with domains coloured as in (A). Bound miRNA shown in spheres with 5ʹ-3ʹ direction indicated. The approximate location of the buried C-terminal residue A859 is indicated. (D) Surface representation of AGO2 (4OLB) showing sites of tryptophan binding and the N-terminal most residue (A22) seen in the electron density. Residues 1–21 were not observed in the data. (E) pLDDT values for predictions of native AGO2 (solid black line) and C-terminal halo-tagged AGO2 and relative solvent accessible surface area (QASA, Å 2 ; red line) calculated using PDB entry 4OLB .

Article Snippet: Single-cell clones were screened for loss of AGO2 expression by immunoblotting using rabbit anti-AGO2 (Sino Biological 101,620-T36).

Techniques: Residue, Binding Assay, Sequencing, Solvent

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Ago2/CAV1 interaction analyzed using co-immunoprecipitation. In normal epithelial cells (blue) and cancer cells (red) of different tissues, Ago2 was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. Normal IgG was used as a negative control. ( B ) Direct interaction of Ago2 with CAV1 in vitro. In the mixture of Ago2-His and CAV1-His, Ago2-His was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. ( C ) In HEK293 cells, HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag and a series of truncated CAV1-Flag proteins was analyzed using anti-Flag antibodies. ( D ) In HEK293 cells, HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1 fragment-fused His-mRuby and His-mRuby was analyzed using anti-His antibodies. ( E ) Ago2/CAV1 interaction mediated by the CSD of CAV1. In HEK293 cells, HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CSD-mutated (F92A/V94A) or wild-type (Wt) CAV1-Flag was analyzed. ( F , G ) A series of C-terminal-deleted, N-terminal-deleted, and HA-tagged human Ago2 (HA-Ago2) coexpressed with Flag-labeled human CAV1 (CAV1-Flag) in HEK293 cells. HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag was analyzed using anti-Flag antibodies. ( H ) In HEK293 cells, HA-Ago2 or amino acid 175–226-deleted HA-Ago2 (∆175–226) was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag was analyzed using anti-Flag antibodies. ( I ) In HEK293 cells, Ago2 amino acid 175–226-fused eGFP-His [eGFP-Ago2(175–226)-His] or eGFP-His was immunoprecipitated with anti-His antibodies, and coprecipitation of CAV1 was analyzed. ( J ) Ago2 sequence alignment illustrating CBM conservation. Aromatic amino acids are marked in red, lysine is marked in blue, and conserved sequences are marked in gray. ( K ) Ago2/CAV1 interaction mediated by a conserved CBM in Ago2. In HEK293 cells, wild-type (Wt) or CBM-mutated (WF199/200/202/211 A) HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag was analyzed. ( L ) Accessible surface area (black and gray bars) and surface exposed ratio SER (red bar) of each residue of Ago2 were estimated using GetArea. The gray bar indicates the accessible surface area of the carbon, halogen, and nonpolar hydrogen atoms of the residue. The quantitation for Western blots in this figure is present in Fig. . .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Immunoprecipitation, Negative Control, In Vitro, Labeling, Sequencing, Residue, Quantitation Assay, Western Blot

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2) in the sample, relative to that of the cancer cell sample in each replicated experiment. Bars are means ± standard error of the mean (SEM, n = 3–6, biological replicates). Student’s t -test, ** P ≤ 0.01; **** P ≤ 0.0001. ( B ) Direct interaction of Ago2 with CAV1 in vitro. In the mixture of Ago2-His and CAV1-His, Ago2-His was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. The mixtures contain CAV1-His only, or IgG serves as negative controls. ( C ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to input CAV1 (inCAV1) in the sample, relative to that of the mixture of Ago2-His and CAV1-His (+Ago2-His) in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, **** P ≤ 0.0001. ( D ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with full-length HA-Ago2 (Full) in each replicated experiment. Bars are means ± SEM ( n = 4, biological replicates). Student’s t -test, ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. ( E ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with His-CAV1(1-102)-mRuby. n = 1. ( F ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with wild-type CAV1-Flag (Wt) in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, **** P ≤ 0.0001. ( G ) Schematic of human Ago2 deletion constructs. The full-length (Full) construct contains four characteristic domains, namely N-terminal (N), PAZ, MID, and C-terminal PIWI domains, where PAZ is an RNA-binding module and MID and PIWI are catalytic activity domains. ( H ) A series of HA-tagged human Ago2 (HA-Ago2), including C-terminal-deleted, N-terminal-deleted, and serine 387-substituted Ago2, coexpressed with Flag-labeled human CAV1 (CAV1-Flag) in HEK293 cells. Panel i: HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag was analyzed using anti-Flag antibodies. Panel ii: the quantitation of Western blots of experiments in panel i. Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with full-length HA-Ago2 (Full) in each replicated experiment. Bars are means ± SEM ( n = 2). ( I ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with full-length HA-Ago2 (Full) in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, * P ≤ 0.05. ( J ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with HA-Ago2(1–226) in each replicated experiment. Bars are means ± SEM ( n = 2). ( K ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with wild-type HA-Ago2 (Wt) in each replicated experiment. Bars are means ± SEM ( n = 6, biological replicates). Student’s t -test, **** P ≤ 0.0001. ( L ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated His-tagged eGFP (peGFP), relative to that of the mixture with eGFP-Ago2(175–226)-His in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, *** P ≤ 0.001. ( M ) Aromatic amino acids in Ago2 are displayed using Pepinfo. The black bars indicate the positions of aromatic residues among a total of 859 residues in Ago2. The red box indicates the CBM of Ago2 amino acids 199–212. ( N ) Changes in the protein thermodynamic stability (∆∆ G ) of the indicated amino acid mutations, estimated using the PoPMuSiC algorithm. Aromatic amino acids are marked in red, and lysine is marked in blue. SER: surface exposed ratio. SERs exceeding 45% are indicated by a yellow background, suggesting an exposed amino acid. SERs below 20% are indicated by a gray background, suggesting a buried amino acid. Mutation-inducing protein thermodynamic instability is indicated by a red background. ( O ) Effects of Ago2 CBM mutation on Ago2/CAV1 interaction. In HEK293 cells, wild-type (Wt) or mutated (WF199/200AA, F202A, W211A) HA-Ago2 was coexpressed with CAV1-Flag. Panel i: in HEK293 cells, HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag was analyzed using anti-Flag antibodies. The mixture contains HA-Ago2 serves as a negative control. Panel ii: the quantitation of Western blots of experiments in panel i. Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the mixture with wild-type HA-Ago2 (Wt) in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, * P ≤ 0.05; ** P ≤ 0.01. ( P ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to input CAV1 (inCAV1) in the sample, relative to that of the mixture with wild-type HA-Ago2 (Wt) in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, **** P ≤ 0.0001.

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Quantitation Assay, Western Blot, In Vitro, Immunoprecipitation, Construct, RNA Binding Assay, Activity Assay, Labeling, Mutagenesis, Negative Control

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Amino acid sequences of blocking peptides P1, P2, and P3, which are designed in accordance with the amino acid sequence 175–226 of Ago2. Cell-penetrating sequence GRKKRRQRRRPQ was added to the blocking peptides. The red box indicates an aromatic amino acid, and the blue box indicates a charged amino acid. ( B ) Disruption of Ago2/CAV1 interaction with blocking peptides P1 and P2 was analyzed using co-immunoprecipitation. In A549 (panel i) and HCC1806 (panel ii) cancer cells treated with P1 and P2 peptides, respectively, Ago2 was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. Normal IgG was used as a negative control for precipitation, and HMLE and MCF10A cells were used as negative controls for the interaction. ( C ) Disruption of Ago2/CAV1 interaction by peptides. In A549 (panel i) and HCC1806 (panel ii) cancer cells treated with P2 and P2S peptides, respectively, Ago2 was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. Panel iii: The quantitation of Western blots of experiments in panel i and ii. Each spot describes the ratio of coprecipitated CAV1 (pCAV1) to precipitated Ago2 (pAgo2), relative to that of the sample treated with P2S in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, * P ≤ 0.05; **** P ≤ 0.0001. ( D ) Expression of Ago2 in CRISPER/Cas9 gene-edited A549 cancer cells. A549 Ago2-KO : A549 cells with Ago2 knocked out by CRISPER/Cas9 gene editing. A549 Ago2-KO/HA-Ago2Wt : Ago2-knockout A549 cells expressing HA-tagged wild-type Ago2. A549 Ago2-KO/HA-Ago2∆ : Ago2-knockout A549 cells expressing HA-tagged CBM-deleted (amino acids 175–226) Ago2. The expression of Ago2 was analyzed using anti-Ago2 and anti-HA antibodies, with GAPDH used as a loading control. ( E ) Distribution of Ago2 (green), endosome marker EEA1 (purple, left panel), and ER marker CANX (purple, right panel) analyzed using immunofluorescence in normal epithelial cells BEAS-2B and cancer cells A549, A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2∆ . Cell nuclei were stained with Hoechst (blue). Scale bar = 20 µm. ( F ) Distribution of Ago2 (green) and CAV1 (red) was analyzed using immunofluorescence in normal epithelial cells and cancer cells. Lipid rafts were stained with CTB (purple), and cell nuclei were stained with Hoechst (blue). The arrowheads indicate the colocalization of Ago2 and CAV1, and the arrows indicate CAV1 without colocalization with Ago2. Scale bar = 25 µm. ( G ) Western blot analysis of HA-Ago2(Wt), HA-Ago2(∆175–226), CAV1, and GAPDH in cell membrane fractions enriched in lipid rafts (LRF, fractions 2–5) and non-lipid rafts (non-LRF, fractions 7–9). HA-Ago2(Wt) and HA-Ago2(∆175–226) were expressed in HEK293 cells. Panel i: proteins were detected in these membrane fractions through Western blotting, with ganglioside-GM1 (CTB dot blotting) and CAV1 being markers of lipid rafts and GAPDH being a marker of non-lipid rafts. Panel ii: The quantitation of Western blots of experiments in panel i. Each spot describes the level of Ago2 in the fraction, relative to that of the 9th fraction in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01. ( H ) Distribution of eGFP-His, eGFP-Ago2(175–226)-His (green), and CAV1 (purple) in HEK293 cells analyzed using immunofluorescence in panel i. Lipid rafts were stained with CTB (red). The arrows indicate protein colocalization. Scale bar = 25 µm. Panel ii: the quantification of eGFP-His/eGFP-Ago2(175–226)-His association with CAV1 (left) and CTB (right) on the plasma membrane of normal epithelial cells and cancer cells in panel i. Each spot indicates the level of Ago2 overlapping with cell surface CAV1/CTB, normalized with total mCAV1/CTB, respectively. Each condition was quantified from 2 and 4 fields per replicate, 2 replicates per experiment. Bars are means ± SEM ( n = 4 and 8). Student’s t -test, * P ≤ 0.05, *** P ≤ 0.001.

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Blocking Assay, Sequencing, Disruption, Immunoprecipitation, Negative Control, Quantitation Assay, Western Blot, Expressing, Knock-Out, Marker, Immunofluorescence, Staining, Membrane

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Membrane association of Ago2 in cancer cells treated with P2. The levels of Ago2, GW182, Dicer, CAV1, and α-tubulin were analyzed in the membrane fractions (MFs) and cytosolic fractions (CFs) of A549 cells treated with P2 or P2S. α-Tubulin: CF marker; CAV1: MF marker. The quantitation is present in panel ii. Each spot describes the ratio of the protein in the membrane fraction to that of the cytosolic fraction. Bars are means ± standard error of the mean (SEM; n = 3, biological replicates). Student’s t -test, * P ≤ 0.05. ( B ) Levels of HA-Ago2Wt, HA-Ago2∆, CAV1, and α-tubulin analyzed in the MFs and CFs of A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2∆ cells. The quantitation is present in panel ii. Each spot describes the ratio of the protein in membrane fraction over cytosolic fraction. Bars are means ± SEM ( n = 4, biological replicates). Student’s t -test, * P ≤ 0.05. ( C ) Distribution of Ago2 (green) and CAV1 (purple) analyzed using immunofluorescence in normal epithelial cells BEAS-2B and cancer cells A549, A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2∆ . Cell nuclei were stained with Hoechst (blue). The arrowheads indicate the colocalization of Ago2 and CAV1. Scale bar = 10 µm. ( D ) Quantification of Ago2 localization with CAV1, endosome marker EEA1, and ER marker Calnexin (CANX). Ago2 distribution is evaluated with the percentage of Ago2 overlapping with CAV1, EEA1, or CANX in BEAS-2B, A549, A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2∆ cells. Images of representative samples are shown in panel C and Fig. . Each condition was quantified from four fields per replicate, three biological replicates per experiment. Each spot indicates the percentage of an individual field. Bars are means ± SEM ( n = 12). Student’s t -test, * P ≤ 0.05, **** P ≤ 0.0001. ( E ) Levels of Ago2 were analyzed in the plasma membrane (PM) and cellular organelle (Org) fractions of BEAS-2B and A549 cells. CALR: calreticulin as an ER/organelle fraction marker. CAV1: PM marker. The quantitation is present in panel ii. Each spot describes the relative Ago2 protein level of the sample to that of the BEAS-2B sample. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01. ( F ) Distribution of Ago2 (green) and CAV1 (red) was analyzed using immunofluorescence in normal epithelial cells and cancer cells. Lipid rafts were stained with CTB (purple), and cell nuclei were stained with Hoechst (blue). The arrowheads indicate the colocalization of Ago2 and CAV1, and the arrows indicate CAV1 without colocalization with Ago2. Scale bar = 25 µm. ( G ) Quantification of Ago2 association with CAV1 on the plasma membrane of normal epithelial cells and cancer cells. The association is evaluated with the level of Ago2 overlapping with cell surface CAV1 (mCAV1), normalized with total mCAV1. Images of representative samples are shown in panel F and Fig. . Each condition was quantified from three fields per replicate, three biological replicates per experiment. Each spot indicates the level of an individual field. Bars are means ± SEM ( n = 9). Student’s t -test, ** P ≤ 0.01, **** P ≤ 0.0001. ( H ) Distribution of Ago2 in cancer cells treated with P2. In cancer cells treated with P2 or P2S peptides, the distribution of Ago2 (green) and CAV1 (red) was analyzed using immunofluorescence. Cell nuclei were stained with Hoechst (blue). The closed arrowheads indicate the colocalization of Ago2 and CAV1, and the open arrowheads indicate CAV1 without colocalization with Ago2. Scale bar = 10 µm. ( I ) Quantification of Ago2 association with CAV1 on the plasma membrane of cancer cells treated with P2 and P2S peptides. The association is evaluated with the level of Ago2 overlapping with cell surface CAV1 (mCAV1), normalized with total mCAV1. Images of representative samples are shown in panel ( H ). Each condition was quantified from three fields per replicate, three biological replicates per experiment. Each spot indicates the level of an individual field. Bars are means ± SEM ( n = 9). Student’s t -test, * P ≤ 0.05, **** P ≤ 0.0001. .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Membrane, Marker, Quantitation Assay, Immunofluorescence, Staining

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Blockage of Ago2/CAV1 interaction increased the resistance of cancer cells to trypsinization. A549 cells were treated with P2 or P2S peptides (10 μM daily) for 2 days. Treated cells were evaluated in terms of resistance to 0.05% trypsin. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, *** P ≤ 0.001. ( B ) Blockage of Ago2/CAV1 interaction decreased the resistance of cancer cells to anoikis. The numbers of P2/P2S-treated A549 cells in suspension were evaluated in terms of resistance to anoikis. The red line indicates the initial cell number on day 0. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01. Scale bar = 50 µm. ( C ) Blockage of Ago2/CAV1 interaction increased the sensitivity of cancer cells to chemotherapeutic agents. P2- and P2S-treated cells were exposed to increasing doses of Paclitaxel (Pac) or Gefitinib (Gef), and their viability was measured using an MTT assay. Each condition was quantified from two technical replicates per biological replicates, and three biological replicates per experiment Bars are means ± SEM ( n = 6). Student’s t -test, **** P ≤ 0.0001. ( D ) Blockage of Ago2/CAV1 interaction decreased tumorsphere formation. A549 (panel i) and HCC1806 (panel ii) cells were treated with P2 or P2S peptides. The cells were subjected to tumorsphere assays, and treatment was suspended during the assays. Bars are means ± SEM ( n = 3 and 4, biological replicates). Student’s t -test, ** P ≤ 0.01; *** P ≤ 0.001. ( E ) Blockage of Ago2/CAV1 interaction decreased the invasion and migration of cancer cells. A549 (panel i) and HCC1806 (panel ii) cells were treated with P2 or P2S peptides. The cells were subjected to invasion and migration assays, and treatment was suspended during the assays. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01, *** P ≤ 0.001. Scale bar = 100 µm. .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Suspension, MTT Assay, Migration

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Blockage of Ago2/CAV1 interaction with AID (see “Materials and Methods”). In A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ and HCC1806 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells, OsTIR1 E3 ligase increased the ubiquitylation of HA-AID-Ago2Wt in the presence of IAA, which in turn resulted in protein degradation. IAA-treated cells expressed only HA-Ago2∆, which does not bind to CAV1. Ago2/CAV1 interaction was blocked in IAA-treated cancer cells with an auxin-inducible degron (AID) system. ( B ) Expression of HA-Ago2 and HA-Ago2∆ of A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ under auxin-inducible degradation. A549 Ago2-KO : A549 cells with Ago2 knocked out by CRISPER/Cas9 gene editing. A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ : Ago2-knockout A549 cells expressing HA-AID-tagged wild-type Ago2 and HA-tagged CBM-deleted (amino acids 175–226) Ago2. Panel i: The expression of Ago2 was analyzed using anti-HA antibodies, with GAPDH used as a loading control. Panel ii: The quantitation of Western blots of experiments in panel i. Each spot describes the level of Ago2 in the sample, relative to that of vehicle (Ctrl)-treated A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells in each replicated experiment. Bars are means ± SEM ( n = 4, biological replicates). Student’s t -test, *** P ≤ 0.001, **** P ≤ 0.0001 ( C ) Panel: In vehicle (Ctrl)- and IAA-treated A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells, HA-AID-Ago2Wt and HA-Ago2∆ proteins were immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1 was analyzed. A549 Ago2-KO was used as a negative control. Panel ii: The quantitation of Western blots of experiments in panel i. Each spot describes the level of the precipitated protein, relative to that of vehicle (Ctrl)-treated A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells in each replicated experiment. Bars are means ± SEM ( n = 4, biological replicates). Student’s t -test, **** P ≤ 0.0001. ( D ) Expression of miR-3613-3p, miR-6126 (panel i), and SCAI mRNAs (panel ii) in PBS- and IAA-treated A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells. Each spot describes the relative miRNA or mRNA expression level of the sample to that of the PBS-treated sample. miRNA data normalized to U6 snRNA and mRNA data normalized to GAPDH mRNA. Bars are means ± SEM ( n = 3 and 4, biological replicates). Student’s t -test, * P ≤ 0.05; ** P ≤ 0.01. ( E ) Expression of SCAI (left panel) and GRP78 (right panel) mRNAs in PBS- and IAA-treated A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells with miR-3613-3p or miR-6126 mimics. Each spot describes the relative mRNA expression level of the sample to that of the A549 cells without miRNA mimics (Ctrl). Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, * P ≤ 0.05. ( F ) Blockage of Ago2/CAV1 interaction increased the resistance of cancer cells to trypsinization. A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells were treated with vehicle or IAA. Treated cells were evaluated in terms of resistance to 0.05% trypsin. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, * P ≤ 0.05. ( G ) Blockage of Ago2/CAV1 interaction decreased the resistance of cancer cells to anoikis. The numbers of IAA/PBS-treated A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ cells in suspension were evaluated in terms of resistance to anoikis. The red line indicates the initial cell number on day 0. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, *** P ≤ 0.001. Scale bar = 50 µm. ( H ) Blockage of Ago2/CAV1 interaction decreased tumorsphere formation. A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ (panel i) and HCC1806 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ (panel ii) cells were treated with vehicle or IAA. The cells were subjected to tumorsphere assays, and treatment was suspended during the assays. Bars are means ± SEM ( n = 4, biological replicates). Student’s t -test, * P ≤ 0.05; *** P ≤ 0.001. ( I ) Blockage of Ago2/CAV1 interaction decreased the invasion and migration of cancer cells. A549 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ (panel i) and HCC1806 Ago2-KO/HA-AID-Ago2Wt/HA-Ago2∆ (panel ii) cells were treated with vehicle or IAA. The cells were subjected to invasion assays, and treatment was suspended during the assays. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01, *** P ≤ 0.001. Scale bar = 100 µm.

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Expressing, Knock-Out, Quantitation Assay, Western Blot, Immunoprecipitation, Negative Control, Suspension, Migration

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Scheme of animal experiments, including lung metastasis of subcutaneous tumor cells (panel i), lung targeting of circulating cancer cells (panel ii), and lung colonization of circulating cancer cells (panel iii), indicating the time points and routes of PBS/IAA injection, time points and routes of A549 Ago2KO/HA-AID-Ago2Wt/HA-Ago2∆ injection, and time points of IVIS imaging and tissue collection. ( B ) Growth of PBS- and IAA-treated subcutaneous A549 Ago2KO/HA-AID-Ago2Wt/HA-Ago2∆ tumors expressing luciferase (fLuc), monitored using noninvasive imaging. Lung metastasis was detected using PCR, which specifically amplifies human GAPDH gDNA (hGAPDH). Mouse GAPDH gDNA was used as a loading control (mGAPDH). ( C ) Quantification of lung metastasis of PBS- and IAA-treated A549 Ago2KO/AID-Ago2Wt/Ago2∆ tumors in panel B by qPCR. Each spot describes the number of tumor cells per 25 mg of lung tissue in an individual mouse. Bars are means ± SEM ( n = 6). Mann–Whitney U -test, * P ≤ 0.05. ( D ) Role of Ago2/CAV1 interaction in the lung targeting of cancer cells. Quantification of lung targeting by PBS- and IAA-treated A549 Ago2KO/HA-AID-Ago2Wt/HA-Ago2∆ tumors through qPCR at 96 h after intravenous (IV) injection. Each spot describes the number of tumor cells per 25 mg of lung tissue in an individual mouse. Bars are means ± SEM ( n = 5). Student’s t -test, * P ≤ 0.05. Mann–Whitney U -test, P = 0.06. ( E ) Role of Ago2/CAV1 interaction in the colonization of cancer cells. Panel i: Tumor colonization in the lungs was monitored using noninvasive imaging. Panel ii: The luminescent counts of lung area in the images of panel i were calculated. Each spot describes the counts of an individual mouse. Bars are means ± SEM ( n = 6). Mann–Whitney U -test, ** P ≤ 0.01. Panels iii and iv: The number of tumor nodules, indicated with arrows, in the lung sections of mice injected with PBS- and IAA-treated A549 Ago2KO/AID-Ago2Wt/Ago2∆ cells was quantified. Each spot describes the number of tumor nodules in the lung sections of an individual mouse. Bars are means ± SEM ( n = 6). Mann–Whitney U -test, ** P ≤ 0.01. Scale bar = 0.2 cm. Panel v- Quantification of lung colonization of PBS- and IAA-treated A549 Ago2KO/HA-AID-Ago2Wt/HA-Ago2∆ cells through qPCR. Each spot describes the number of tumor cells per 25 mg of lung tissue in an individual mouse. Bars are means ± SEM ( n = 6). Mann–Whitney U -test, ** P ≤ 0.01. ( F ) Role of Ago2/CAV1 interaction in the growth and metastasis of breast tumors. Panels (i) and (ii): Breast tumors formed by PBS- or IAA-treated HCC1806 Ago2KO/HA-AID-Ago2Wt/HA-Ago2∆ cells in fat pads, as indicated in Appendix Fig. S , monitored using noninvasive imaging (panel i, top). Breast tumors isolated from mice were weighed (panel i [bottom] and panel ii). Each spot describes tumor weight at each injection site. Bars are means ± SEM ( n = 14). Mann–Whitney U -test, * P ≤ 0.05. Panels (iii) and (iv): qPCR quantification of lymph node metastasis (panel iii) and lung metastasis (panel iv) of tumors formed by PBS- and IAA-treated HCC1806 Ago2KO/HA-AID-Ago2Wt/HA-Ago2 cells. Each spot describes the number of tumor cells in an individual mouse. Bars are means ± SEM [ n = 7 for panel (iii) and n = 14 for panel (iv); see “Materials and Methods”]. Mann–Whitney U- test, * P ≤ 0.05. .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Injection, Imaging, Expressing, Luciferase, MANN-WHITNEY, IV Injection, Isolation

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) In HEK293 cells, wild-type (Wt) or mutated (E186F, E197F, K212A, and D217F) HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag was analyzed. ( B ) Interaction of Ago2, Ago2K212R, and Ago2K212A with CAV1. In the indicated cells, HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1-Flag or endogenous CAV1 was analyzed. 293 in panel (i), A549 Ago2-KO in panel (ii), and HCC1806 Ago2-KO in panel (iii) were used as negative controls. ( C ) Ago2/CAV1 interaction and Ago2 acetylation attenuated by mutations in Ago2 lysine 212 (K212). In A549 Ago2-KO/HA-Ago2Wt , A549 Ago2-KO/HA-Ago2K212A (panel i), HCC1806 Ago2-KO/HA-Ago2Wt , and HCC1806 Ago2-KO/HA-Ago2K212A cells (panel ii), HA-Ago2 was immunoprecipitated with anti-HA antibodies. Acetylation of HA-Ago2 and coprecipitation of CAV1-Flag were analyzed. ( D ) Expression of deacetylase in cancer cells (A549 and HCC1806) and normal epithelial cells (BEAS-2B and HMLE), analyzed using Western blotting. GAPDH was used as a loading control. ( E ) In cancer cells treated with a vehicle or a deacetylase inhibitor (Nexturastat A, Sirtinol, EX-527, or TM), Ago2 was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. ( F ) In A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2K212R cells treated with vehicle or TM (10 µM), HA-Ago2 was immunoprecipitated with anti-HA antibodies, and coprecipitation of CAV1 was analyzed (panel i). A549 Ago2-KO and A549 Ago2-KO/HA-Ago2K212A cells were used as negative controls. The ratio of precipitated Cavl to precipitated HA-Ago2 was measured in three rounds of immunoprecipitation experiments (Panel ii). Bars are means ± SEM ( n = 3, biological replicates). Student’s t - test, * P ≤ 0.05; ** P ≤ 0.01. ( G ) Levels of Ago2 in the plasma membrane (PM) and endosome (Endo) fractions of A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2K212A cells and in the whole cells were analyzed with Western blots. EEA1: Endo marker. CAV1: PM marker. ( H ) The miRNA fluctuation resulting from disruption of Ago2/CAV1 interaction by P2 peptides, deletion of Ago2 CBM, and Ago2K212A substitution analyzed with miRNA arrays. Panel i: The Venn diagrams of upregulated (left) and downregulated (right) miRNAs in P2-treated A549, A549 Ago2-KO/HA-Ago2∆ (Ago2Dm), and A549 Ago2-KO/HA-Ago2K212A (Ago2K212A) cancer cells. Panel ii: There are ten shared upregulated miRNAs (red) and nine shared downregulated miRNAs (green) in P2-treated A549, A549 Ago2-KO/HA-Ago2∆ (Ago2Dm), and A549 Ago2-KO/HA-Ago2K212A (Ago2K212A) cancer cells, with a fold-change >1.5. ( I ) Expression of miR-3613-3p miRNA (left panel) and SCAI mRNA (right panel) in A549 Ago2-KO/HA-Ago2Wt (Wt), and A549 Ago2-KO/HA-Ago2K212A (K212A) cells. Each spot describes the relative miRNA level of the sample to that of the A549 Ago2-KO/HA-Ago2Wt sample. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, * P ≤ 0.05; **** P ≤ 0.0001. ( J ) Expression of SCAI proteins in A549 Ago2-KO/HA-Ago2Wt , A549 Ago2-KO/HA-Ago2∆ , and A549 Ago2-KO/HA-Ago2K212A cancer cells. The levels of proteins were analyzed in the MFs (M), CFs (C), and NFs (N) of the cells. α-Tubulin: CF marker; CAV1: MF marker; HDAC1: NF marker. ( K ) Expression of N-cadherin and fibronectin in A549 Ago2-KO/HA-Ago2Wt , A549 Ago2-KO/HA-Ago2∆ , and A549 Ago2-KO/HA-Ago2K212A cancer cells. The quantitation for Western blots in this figure is presented in Appendix Fig. S . .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Immunoprecipitation, Expressing, Histone Deacetylase Assay, Western Blot, Membrane, Marker, Disruption, Quantitation Assay

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) A549 Ago2-KO/HA-Ago2Wt (Wt), A549 Ago2-KO/HA-Ago2K212R (K212R), and A549 Ago2-KO/HA-Ago2K212A (K212A) cells were subjected to migration (panels i and ii) and tumorsphere formation (panel iii) assays. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01; *** P ≤ 0.001. Scale bar = 100 µm. ( B ) A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2K212R cells were subjected to migration assay under vehicle or TM treatment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bar = 100 µm. ( C ) A549 Ago2-KO/HA-Ago2Wt , A549 Ago2-KO/HA-Ago2K212R , and A549 Ago2-KO/HA-Ago2K212A cells were subjected to a tumorsphere formation assay after vehicle or TM treatment. Bars are means ± SEM ( n = 4, biological replicates). Student’s t -test, *** P ≤ 0.001; **** P ≤ 0.0001. A549 Ago2-KO/HA-Ago2K212A was used as a negative control. ( D ) Panel (i): Scheme of an animal experiment, indicating the time points of IV injection of A549 Ago2-KO/HA-Ago2Wt , A549 Ago2-KO/HA-Ago2K212R , and A549 Ago2-KO/HA-Ago2K212A cells and tissue collection. Panel (ii): Quantification of lung targeting by A549 cells at 96 h after administration. qPCR was used to detect A549 cell genomic DNA in tissues. Each spot describes the number of tumor cells per 25 mg of lung tissue in an individual mouse. Bars are means ± SEM ( n = 10). Mann–Whitney U -test, ** P ≤ 0.01, *** P ≤ 0.001. ( E ) Panel i: Scheme of an animal experiment, indicating the time points of IV injection of A549 Ago2-KO/HA-Ago2Wt , A549 Ago2-KO/HA-Ago2K212R , and A549 Ago2-KO/HA-Ago2K212A cells and IVIS imaging and tissue collection. Panel (ii): Tumor colonization was monitored using noninvasive imaging at 4 weeks after injection. Panel (iii): Luminescent counts of lung area in the images of panel (ii). Panels (iv) and (v): Number of tumor nodules, indicated with arrows, in the lung sections of mice. Scale bar = 0.1 cm. Panels (vi) and (vii): Numbers of tumor cells in the lungs (panel vi) and lymph nodes (panel vii) of mice were quantified using qPCR for human gDNA. Each spot describes the counts of an individual mouse. Bars are means ± SEM ( n = 6). Mann–Whitney U -test, ** P ≤ 0.01. .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Migration, Tube Formation Assay, Negative Control, IV Injection, MANN-WHITNEY, Imaging, Injection

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) In human primary breast tumors (P) and paired lymphatic metastases (M), Ago2 was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. Each number indicates an individual patient. ( B ) The ratio of coprecipitated CAV1 to precipitated Ago2 was calculated for each sample in panel A ( n = 10, patient number). Student’s t -test, ** P ≤ 0.01. ( C ) Distribution of Ago2 in A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2∆ tumors. Ago2 was stained with anti-Ago2 antibodies (Ago2, red), and cytokeratin was stained with anti-cytokeratin 8/18 antibodies (Cytokeratin, green). Cell nuclei were stained with Hoechst 33258 (blue). Scale bar = 25 µm. ( D ) In human primary breast tumors and paired metastases, Ago2 was stained with anti-Ago2 antibodies (Ago2, green), and cytokeratin was stained with anti-cytokeratin 8/18 antibodies (Cytokeratin, red). Cell nuclei were stained with Hoechst 33258 (blue). Images of representative samples are shown. Each number indicates an individual patient. Scale bar = 50 µm. ( E ) Ago2 distribution is evaluated with the percentage of Ago2 overlapping with cytokeratin 8/18 in primary breast tumors (P) and paired metastases (Meta). BM bone metastasis, LM lung metastasis, Meta without labeling: lymphatic metastasis. ( n = 18, patient number). Student’s t -test, *** P ≤ 0.001. ( F ) Ago2 distribution in images of normal breast tissue, inflamed breast tissue (mastitis), and breast tumors in a breast disease spectrum array. Representative immunofluorescence images are depicted in Fig. . Each spot describes the percentage of Ago2 overlapping with cytokeratin 8/18 in a tissue sample. Bars are means ± SEM. The sample size ( n ) of each category is depicted on the x -axis. Student’s t -test, P values are indicated in each plot. .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Immunoprecipitation, Staining, Labeling, Immunofluorescence

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Ago2 distribution (red) in tissues in the breast disease spectrum array, analyzed using immunofluorescence. Cytokeratin was recognized using anti-cytokeratin 8/18 (green). Images of representative samples are shown. Scale bar = 50 µm. Representative immunofluorescence images of tissues in the breast disease spectrum array are depicted. Scale bar = 50 µm. ( B ) Percentage of Ago2 overlaps with cytokeratin 8/18 in images of tissues in the breast disease spectrum array. Data were identical to those shown in Fig. and are re-sorted by the cancer stage along the x -axis. Each spot describes the percentage of Ago2 overlaps with cytokeratin 8/18 in a tissue sample. Bars are means ± SEM. The sample size ( n , patient number) of each category is depicted on the x -axis. Student’s t -test, * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. ( C ) Ago2 distribution in lung tissues and lung carcinomas in the lung disease spectrum array. Each spot describes the percentage of Ago2 overlaps with cytokeratin 8/18 in a tissue sample. Bars are means ± SEM ( n = 13 and 88, patient number). Student’s t -test, P values are indicated on each plot.

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Immunofluorescence

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) Ago2 in the fraction containing small extracellular vesicles (EVs). Ago2, CD63, TSG101, CD81, and GAPDH proteins were measured using Western blotting in the P4 and P3 fractions of A549 cell–conditioned media. ( B ) Ago2/CAV1 interaction in A549 cell-derived EVs. In EVs, Ago2 was immunoprecipitated with anti-Ago2 antibodies, and coprecipitation of CAV1 was analyzed. ( C ) Ago2 amino acids 1–226, containing a CAV1-interacting domain, sorted into EVs, unlike Ago2 amino acids 226–859. The EVs of HEK293 cells expressing HA-Ago2(1–226) or HA-Ago2(226–859) were collected and subjected to Western blotting to measure HA-Ago2 fragments. The EVs of HEK293 cells were used as negative controls. ( D ) CBM of Ago2 is necessary for its sorting into EVs. In the cell and EV fractions of A549 Ago2-KO/HA-Ago2Wt and A549 Ago2-KO/HA-Ago2∆ cells, HA-Ago2Wt, HA-Ago2∆, TSG101, and GAPDH were measured using Western blotting. ( E ) Levels of EV miR-3613-3p in A549 Ago2-KO/HA-Ago2Wt (Wt) and A549 Ago2-KO/HA-Ago2∆ (Dm) cells measured using qPCR, normalized to 5S RNA, in EVs. Each spot describes the relative EV miR-3613-3p level of the sample to that of the A549 Ago2-KO/HA-Ago2Wt cells. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01. ( F , G ) Levels of miRNAs in the plasma EVs of mice bearing A549 Ago2-KO/HA-Ago2Wt (Wt) and A549 Ago2-KO/HA-Ago2∆ (Dm) tumors. The same amount of plasma was collected from the mice and subjected to EV isolation and miRNA array assays. Panel ( F ) illustrates EV miRNAs that differed significantly between Wt and Dm mice by more than 1.5 folds, measured by miRNA array assays. The green indicates downregulated miRNAs and the red indicates upregulated miRNAs within the plasma EVs of mice bearing A549 Ago2-KO/HA-Ago2∆ (Dm) tumors. The miRNAs altered in both A549 Ago2-KO/HA-Ago2Wt cells vs A549 Ago2-KO/HA-Ago2∆ cells and in plasma EVs of mice bearing A549 Ago2-KO/HA-Ago2Wt (Wt) and A549 Ago2-KO/HA-Ago2∆ (Dm) tumors are labeled in blue. In panel ( G ), differences are validated using qPCR. Each spot describes relative EV miRNAs in the plasma of mice to the average amount of mice not bearing tumor (Crtl). Bars are means ± SEM ( n = 5–7, biological replicates). Student’s t -test, * P ≤ 0.05; ** P ≤ 0.01. ( H ) Levels of exosomal miRNAs in the plasma of patients with breast tumors at different stages. Plasma EV RNA was collected and analyzed using qPCR. Each spot describes the relative EV miRNA in the plasma of patients to the average amount of patients at stage DCIS. Bars are means ± SEM ( n = 8 and 10, patient number). Student’s t -test, * P ≤ 0.05; ** P ≤ 0.01. .

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Western Blot, Derivative Assay, Immunoprecipitation, Expressing, Isolation, Labeling

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: ( A ) The quantitation of Western blots of experiments in Fig. . Each spot describes the protein level of the sample normalized by GAPDH, relative to that of the fourth fraction sample in each replicated experiment. Bars are means ± SEM ( n = 2). ( B ) P3 and P4 fractions of A549 cell–conditioned media were collected and subjected to NanoSight nanoparticle tracking analysis, revealing their particle size and concentration. ( C ) Ago2 and small EVs in the same fraction of A549 cell–conditioned media separated with a density gradient. Ago2, CAV1, CD63, TSG101, CD9, and CD81 were measured using Western blotting in each fraction of the density gradient. ( D ) Proteinase K protection assay revealing Ago2 in EVs. A549 cell-derived EVs were treated using RIPA lysis buffer or protease K as indicated. Ago2, CAV1, and CD81 were measured using Western blotting in the treated EVs. Right panel: the quantitation of Western blots. Each spot describes the protein level, relative to that of PBS(-)-treated sample in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, *** P ≤ 0.001. ( E ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of precipitated CAV1 (pCAV1) to input CAV1 (inCAV1) in the sample, relative to that of the sample precipitated by anti-Ago2 antibodies in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, **** P ≤ 0.0001. ( F ) The quantitation of Western blots of experiments in Fig. . Each spot describes the exosomal Ago2 protein level of the sample normalized by exosome marker CD81, relative to that of the exosome sample of HEK293 cells expressing HA-Ago2(1–226) in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, ** P ≤ 0.01. ( G ) The quantitation of Western blots of experiments in Fig. . Each spot describes the ratio of exosomal Ago2 to cellular Ago2 in the sample, relative to that of A549 Ago2KO/HA-Ago2Wt (Ago2 Wt ) sample in each replicated experiment. Bars are means ± SEM ( n = 3, biological replicates). Student’s t -test, **** P ≤ 0.0001.

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Quantitation Assay, Western Blot, Concentration Assay, Derivative Assay, Lysis, Marker, Expressing

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet: Left panel: In normal epithelial cells, K212-acetylated Ago2 does not interact with CAV1 on the plasma membrane. SCAI is expressed to repress gene expression required for tumor promotion and metastasis (blue arrows). Right panel: In cancer cells, Ago2 is deacetylated by SIR2 at K212 and is recruited to the plasma membrane through Ago2/CAV1 interaction (red arrows). MiR-3613-3p is generated through the maturation process catalyzed by CAV1-associated Ago2. During miRNA maturation, miR-3613-5p is cleaved by CAV1-associated Ago2 and degraded (red arrows). Subsequently, miR-3613-3p-mediated SCAI translational suppression (blue arrows) and release of miR-3613-3p-containing EVs (black arrows) happen in an Ago2/CAV1-dependent manner.

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: Membrane, Expressing, Generated

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet:

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques:

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet:

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques:

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet:

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques:

Journal: EMBO Reports

Article Title: Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

doi: 10.1038/s44319-024-00132-7

Figure Lengend Snippet:

Article Snippet: Portions of Ago2 (HG11079-NY, Sino Biological) and Caveolin-1 (HG11440-CF, Sino Biological) were generated as HindIII-XbaI restriction fragments by PCR, using appropriate synthetic primers, and then cloned into the pCMV3-N-HA and pCMV3-C-FLAG (Sino Biological), respectively.

Techniques: