EMBO/SourceData huggingface dataset:
Figure 3(A) Autophagosome-lysosome fusion is analysed in wild‐type and HDAC6 KO MEFs with or without starvation (6 h) using mCherry‐GFP‐LC3 as described in Figure 2A.(B) Wild‐type and HDAC6 KO MEFs were cultured in Hank's solution for 3 h followed by immunoblotting with an antibody for LC3, HDAC6, and GAPDH.(C) Long‐lived protein degradation in wild‐type and HDAC6 KO MEF cells. The degradation of [14C]‐valine labelled long‐lived protein was measured in the presence or absence of 3‐methyl adenine (3MA, inhibits the formation of autophagic vacuoles). The average of percentage degradation from three independent experiments is presented. The error bar represents the standard deviation.
Figure 4(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1.(B, C) Wild‐type and HDAC6 KO MEFs were transfected with mCherry‐GFP‐LC3, followed by treatment with LatA (100 nM) or nocodazole (250 nM) for 6 h and analysed as described in Figure 3A.(D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01.(E) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at *P0.05. The number of total fusion events/total number of vesicles for each condition was as follows: 169/1250; 87/972; 190/1120; and 175/1165.(F) Autophagosomes (APGs) and lysosomes (Lys) isolated from HDAC6 KO MEFs were treated or not with latrunculin (LatA) as indicated and subjected to in vitro fusion assay.
Figure 5(A) Biochemical characterization of autophagic compartments isolated from HDAC6 KO cells. Different subcellular fractions (75 μg protein) isolated from the wild type (WT) were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Hom, homogenate; APG, autophagosomes; APL, autophagolysosomes; Lys, lysosomes.(B) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed cells were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at **P0.01.(C) WT and ATG5 KO MEFs were treated with MG132 and immunostained with antibodies to ubiquitin (green) as indicated. F‐actin was detected by phalloidin (red). The arrows indicated ubiquitin‐positive protein aggregates.
Figure 6(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin (red), ubiquitin (green), and phalloidin for F‐actin (blue) as indicated. The arrows indicated ubiquitin‐positive aggregates that were colocalized with F‐actin and cortactin.(B) Wild‐type MEFs were transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for Lap‐1 (red, to label lysosome), or ubiquitin (green) and phalloidin for actin (blue). Note that F‐actin staining at protein aggregates was lost, but lysosomes remained concentrated in cortactin knockdown cells (arrow).(C) Wild‐type MEFs were transfected with control or cortactin siRNA, treated MG132 2.5 μM for 18 h, and subjected to filter‐trap assay using a ubiquitin antibody. The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel.(D) U2OS cells were transfected with control siRNA and cortactin siRNA. Autophagosome-lysosome fusion was analysed with or without starvation (6 h) using the mCherry‐GFP‐LC3 reporter as described in Figure 3A.(E) The mCherry‐GFP‐LC3 plasmid was cotransfected with wild type, 9KQ (acetylation‐mimic), or 9KR (deacetylation‐mimic) cortactin‐expressing plasmids into wild‐type MEFs. Autophagosome-lysosome fusion was analysed as described in Figure 3A.
Figure 7(A) HDAC6 KO mice accumulate ubiquitin‐positive protein aggregates in the brain. The hippocampus and cerebral cortex regions from 6‐month‐old wild‐type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin. The red arrows indicate ubiquitin‐positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin‐positive structures were rarely observed in control littermates. Scale bar, 50 μm(B) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining. Apoptotic cells were only observed in HDAC6 KO mice. Scale bar, 100 μm.(C) HDAC6 depletion in the Drosophila eye leads to ubiquitin‐positive pathology. Immunostaining for ubiquitin (green) in frontal eye sections of 1‐day‐old (d1) and 30‐day‐old (d30) fly eyes. The eyes of HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) developed ubiquitin‐ positive cytoplasmic inclusions that become more prominent at day 30 (d30). Blue, DAPI.(D) Depletion of HDAC6 in the Drosophila eye leads to age‐dependent degeneration. Light micrographs (left) and corresponding Richardson‐stained frontal eye sections (right) of 1‐day‐old and 30‐day‐old fly eyes. The eyes of control flies (GMR:GAL4/+) and HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) show normal highly organized ommatidial array at day 1; 30‐day‐old control animals also show no defects, but 30‐day‐old HDAC6‐depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80).
Figure 2(A, B) The mRNA levels of aub (A) and vasa (B) were quantified upon the depletion (RNAi) of L(3)mbt, Lint-1, CoRest, E2F2, Myb, Mip120, and Mip130, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.(C) Proteins immunoprecipitated with the anti-L(3)mbt antibody from the OSC lysates before (control) and after L(3)mbt knockdown (KD) were silver-stained. The bands corresponding to L(3)mbt-L and L(3)mbt-S are indicated.(F, G) The mRNA levels of vasa (F) and aub (G) were quantified upon the depletion (RNAi) of L(3)mbt, Atac3, Prod, CG2662/Lint-O, and CG2199, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.
Figure 3(B) Immunoprecipitation (IP)/western blotting shows that WT Lint-O, but not its ∆SAM mutant co-immunoprecipitated with L(3)mbt from the OSC lysates. n.i.: non-immune IgG.(C) Subcellular localization of WT Lint-O and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.(D) IP/western blotting shows that WT L(3)mbt-L, but not its ∆SAM mutant co-immunoprecipitated with Lint-O from the OSC lysates. n.i.: non-immune IgG.(E) Subcellular localization of WT L(3)mbt-L and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.(F, G) The mRNA levels of vasa (F) and aub (G) were quantified upon ectopic expression of EGFP, as well as WT Lint-O and its ∆SAM and 8CA mutants (Fig EV3A), in Lint-O-lacking OSCs (Lint-O RNAi) and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05. All t-tests were performed against samples with⎾ symbol.(H) IP/western blotting shows that L(3)mbt co-immunoprecipitated with WT Lint-O and its 8CA mutant, but not with ∆PHD mutant An asterisk shows background.(I) Subcellular localization of WT Lint-O and its 8CA and ∆PHD mutants (green). All proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.(J) ChIP-qPCR shows that L(3)mbt binding to the vasa promoter was weakened after the loss of Lint-O. Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.(K) Western blotting showing the amounts of L(3)mbt, Lint-O, and histone H3 (H3) in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs.(L) The lint-O mRNA levels in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs are shown by fragments per kilobase million (FPKM). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.
Figure 5(B) RT-qPCR analysis shows the mRNA levels of lint-O in y w and Lint-OKO ovaries. Data are expressed as mean and error bars represent SD. n = 2 biological replicates.(D) Confocal images of y w and Lint-OKO ovaries immunostained for Vasa. Scale bar: 50 μm.(E) Confocal images of y w and Lint-OKO ovarioles immunostained for Vasa. Vasa was ectopically expressed in follicle cells (white arrowheads). Scale bar: 50 μm.
Figure 6(A) Western blotting shows that Vasa, Piwi, and Aub were ectopically expressed in Lint-OKO and L(3)mbtts1 larval brains at 29ºC. Anti-Lint-O, anti-L(3)mbt, anti-Vasa, andi-Piwi, anti-Aub, and anti-Tubulin antibodies were used.(B) Confocal images of y w, Lint-OKO, and L(3)mbtts1 immunostained for Vasa (magenta), MIRA (green), and ELAV (green). Scale bar: 100 μm.(C) Quantification of brain lobe volume for the following genotypes: Lint-OKO -/+ (n = 42 biological replicates), Lint-OKO -/- (n = 36 biological replicates), and L(3)mbtts1 -/- (n = 34 biological replicates). Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively. P-values were calculated by the Student's t-test. (***P-value < 0.001).(D, E) The genomic regions harboring the vasa (D) and aub (E) genes. The RNA-seq reads in y w, L(3)mbtts1, and Lint-OKO brains are shown. The shading in gray corresponds to exons. The y-axis shows the number of RPM. RNA-seq samples were biological triplicates.
Figure 1RT‐PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam‐treated mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/flmice (right lane); the Syk‐ and Hprt‐specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control.Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl (right lane) mice both treated with Tam as described; blots were probed with anti‐Syk and anti‐GAPDH Ab, GAPDH being used as a loading control.Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk‐deficient and control mice.Genomic DNA analysis of Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/flmice (as indicated). Upper row: amplification of floxed (fl) and wt (+) alleles. Middle row: amplification of the deleted (d) allele. Lower row: loading control (TC21, a gene unaffected by the deletion of Syk). Established control DNA was used to visualize the individual bands (as indicated).
Figure 2A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/flmice (right) treated with Tam as described in the Materials and Methods section. The BM cells were stained with anti‐IgM and anti‐B220, and the SP cells with anti‐CD19 and anti‐CD93 or anti‐CD23 and anti‐CD21. The gated regions in the dot blots correspond to individual B‐cell populations: (A) Bone marrow: gate P (B220+IgM−) pro‐/pre‐B cells, gate I (B220loIgM+) immature B cells, gate M (B220hiIgM+) mature B cells; (B) spleen: gate T (CD19+ CD93+) transitional B cells (T), gate M (CD19+ CD93−) mature B cells; in CD93−splenic B cells, gate M (CD23hiCD21int CD93−) mature follicular B cells and gate MZ (CD23lo CD21hi CD93−) marginal zone B cells. The numbers in the dot plots indicate the mean relative frequency of cells in the gate.C-E Statistical analysis of absolute cell numbers per Tam‐treated mouse: filled circles indicating cells obtained from mb1‐CreERT2 control mice and open circles from mb1‐CreERT2;Sykfl/flmice. (C) Statistical analysis of the absolute cell numbers of pro‐/pre‐, immature and mature recirculating B cells in the BM. (D) Absolute cell numbers of transitional, M and MZ B cells found in the SP. (E) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1‐a and B1‐b. An asterisk (*) marks statistically significant differences (P 0.05), two asterisks (**) indicate P 0.01, P‐values were obtained using two‐tailed Student's t‐test. Cell numbers of four to five mice per group are shown, with each dot representing an individual animal.
Figure 3A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/flmice (solid line).B Flow cytometric analysis of tyrosine phosphorylation (pY) in splenic B cells from Tam‐treated mb1‐CreERT2 and mb1‐CreERT2;Sykfl/flmice stimulated with medium (dotted line) or 50 μM pervanadate (dashed and solid lines).C, D Flow cytometric analysis of the intracellular Ca2+ influx in purified splenic B cells derived from Tam‐treated mice. mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl were treated with (C) 10 μg/ml anti‐Kappa F(ab′)2 fragments or (D) 1 μM ionomycin.
Figure 4Ex vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). Purified splenic B cells were either left unstimulated (medium) or were stimulated with the indicated stimuli. The cells were analyzed by flow cytometry after 24 h. Shown are CD19 versus CD86 dot plots. 7‐AAD was included to distinguish dead from viable cells. The numbers in the quadrants indicate the relative frequencies of cells in the gate.Ex vivo proliferation assay with mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). The cells were incubated with the indicated stimuli for 90 h.
Figure 5A Flow cytometric analysis of splenic B cells from Rag2−/−;γc−/− mice. CD19 versus CD3ε dot plots are shown. The mice were injected i.v. with 5 × 106 splenic (CD19+ CD93−) B cells and (CD3ε+) T cells from Tam‐untreated mb1‐CreERT2 control or mb1‐CreERT2;Sykfl/fl mice. The recipient mice were treated with Tam beginning 1 day after transfer as described in the Materials and Methods section.B Quantitative analysis of B and T cells from Rag2−/−;γc−/− mice repopulated with mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl splenocytes. Each symbol represents an individual mouse. Filled circles and squares represent Syk+/+ B and T cells, respectively; open circles and squares represent Sykfl/fl B and T cells.
Figure 1Demonstration of translational readthrough of AGO1 using luciferase-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co-transfected renilla luciferase (RLuc). FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0001.Significance of the identity of the canonical stop codon in AGO1 translational readthrough. AGO1-ISR-FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above. FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0002; **, P = 0.0004.First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017.Demonstration of translational readthrough of AGO1 using fluorescence-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence. Scale bar, 50 um. The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs. Fluorescence intensity was measured by flowcytometry. ***, P = 0.007.
Figure 2Detection of endogenous Ago1x by Western blot in HEK293 cells using anti-Ago1x antibody and pre-immune serum.Western blot of lysates from HEK293 and HeLa cells using anti-Ago1x antibody pre-treated with Ago1x-specific peptide.Expression of Ago1x in HEK293 cells transfected with three AGO1-specific siRNAs (1,2,3). qRT-PCR analysis of AGO1 transcript expression in these cells is shown below.Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.).Expression of Ago1x in mouse organs.Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2-OS cells. Ribosome profiling data was taken from NCBI's sequence read archive (SRA Accession no. SRR1257257) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section. Zoomed area near the inter-stop codon region is shown in the inset. Position of canonical stop codon and the downstream in-frame stop codon are shown. Detection of Ago1x by Western blot in U2-OS cells is shown (right).
Figure 3Effect of let-7a inhibitor on translational readthrough of AGO1. let-7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.01.Effect of let-7a overexpression on translational readthrough of AGO1. HeLa cells were stably transfected with pri-let-7a overexpression construct. Ago1x expression was determined by Western blot. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.007.let-7a inhibitor reduces the AGO1 readthrough efficiency. HeLa cells were transfected with let-7a inhibitor or control inhibitor along with AGO1-TGA (or GCA)-ISR-FLuc construct. Readthrough was quantified by measuring FLuc activity, which was normalized to co-transfected renilla luciferase activity. FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown (Right). *, P = 0.027.AGO1 ISR with mutated let-7a binding site shows reduced translational readthrough activity. Assay was done as described above in HeLa cells. Mutated sequence is shown (top). ***, P = 0.008.AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.
Figure 4AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001.AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase (RLuc). AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells (left) and in vitro using rabbit reticulocyte lysate system (right). **, P = 0.006; ***, P = 0.0001.Tethering of N-peptide-tagged Ago1 or Ago1x can induce readthrough of AGO1. Two BoxB elements were cloned downstream of the AGO1 and upstream of the in-frame FLuc as shown in the schematic. This construct was transfected in HeLa cells expressing N-peptide-tagged or FLAG-tagged Ago proteins as indicated. Readthrough was measured as described above. *, P = 0.035 (with Welch correction); **, P = 0.016.
Figure 5Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells. Reads per million (RPM) values of 395 miRNAs, which were common to Ago1 and Ago1x are shown.qRT-PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show mean of three experiments ± S.E.RT-PCR results showing PTEN and MYC mRNAs co-immunoprecipitated with Ago1 or Ago1x in HeLa cells.Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells.
Figure 6Result of co-immunoprecipitation experiment in HeLa cells shows that GW182 is co-immunoprecipitated with Ago1, but not with Ago1x.Confocal immunofluoresence images of HeLa cells show co-localization of GW182 with Ago1, but not with Ago1x. White circles indicate zoomed areas.Confocal microscopy images of proximity ligation assay (PLA) in HeLa cells showing interaction of GW182 with Ago1, but not with Ago1x. Rabbit anti-GW182 and Mouse anti-FLAG antibodies were used to achieve proximity ligation. Interaction is indicated by red dots that results from proximity ligation. Quantification of dots per cell is shown (right). Each black circle represents a microscopic field. Mean ± S.E. are shown as horizontal lines (n=10 fields for vector control; n=12 fields for Ago1; n=11 fields for Ago1x). At least 100 cells were counted for dots in each category. ***, P < 0.0001 (Mann-Whitney test).
Figure 7Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Bar graph (mean ± S.E.) is a representative of four independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). *, P = 0.018; #, P = 0.041; $, P = 0.013. Numbers on the bars represent % change in relative luciferase activity.Expression of RLuc protein in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). RT-PCR analysis for RLuc mRNA level is shown (bottom). **, P = 0.001.Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged Ago2 (N-HA-Ago2) or N-HA-tagged chimeric Ago2 appended with the ISR region of Ago1x (N-HA-Ago2-ISRAgo1) as indicated. Bar graph (mean ± S.E.) is a representative of three independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). ***, P < 0.0001; **, P = 0.0004.
Figure 8Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test).[35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).