antibody against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibody against ago2
    Antibody Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibodies against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against ago2
    Antibodies Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibody against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibody against ago2
    Antibody Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibody against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibody against ago2
    Antibody Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gated antibodies against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gated antibodies against ago2
    Gated Antibodies Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibodies against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against ago2
    Antibodies Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibodies against argonaute risc catalytic component 2 ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against argonaute risc catalytic component 2 ago2
    Antibodies Against Argonaute Risc Catalytic Component 2 Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibodies against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against ago2
    hsa_circ_0006692 played its roles via sponging hsa-miR-205-5p and regulation of CDK19. ( A ) After the detection of <t>ago2</t> or IgG RIP assay, hsa_circ_0006692 and miR-205-5p levels was determined by qRT-PCR; ( B ) After RNA pull-down analysis using Bio-miR-NC and Bio-miR-205-5p in A549 and H1299 cells, expression of hsa_circ_0006692 was measured by qRT-PCR; ( C – E ) CDK19 expression level was increased in A549/circ-0006692-OE cells and decreased in A549/circ-0006692-SH cells analyzed by qRT-PCR and WB; ( C , D ) miR-205-5p expression was decreased in A549/circ-0006692-OE cells and increased in A549/circ-0006692-SH cells analyzed by qRT-PCR; ( F ) miR-205-5p expression in A549 cells transfected with miR-205-5p mimics or inhibitors was analyzed using qRT-PCR; ( G , H ) qRT-PCR analysis of hsa_circ_0006692 and CDK19 expression in A549 cells transferred with miR-205-5p mimics or inhibitor. *** p < 0.001; **** p < 0.0001.
    Antibodies Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Hsa_circ_0006692 Promotes Lung Cancer Progression via miR-205-5p/CDK19 Axis"

    Article Title: Hsa_circ_0006692 Promotes Lung Cancer Progression via miR-205-5p/CDK19 Axis

    Journal: Genes

    doi: 10.3390/genes13050846

    hsa_circ_0006692 played its roles via sponging hsa-miR-205-5p and regulation of CDK19. ( A ) After the detection of ago2 or IgG RIP assay, hsa_circ_0006692 and miR-205-5p levels was determined by qRT-PCR; ( B ) After RNA pull-down analysis using Bio-miR-NC and Bio-miR-205-5p in A549 and H1299 cells, expression of hsa_circ_0006692 was measured by qRT-PCR; ( C – E ) CDK19 expression level was increased in A549/circ-0006692-OE cells and decreased in A549/circ-0006692-SH cells analyzed by qRT-PCR and WB; ( C , D ) miR-205-5p expression was decreased in A549/circ-0006692-OE cells and increased in A549/circ-0006692-SH cells analyzed by qRT-PCR; ( F ) miR-205-5p expression in A549 cells transfected with miR-205-5p mimics or inhibitors was analyzed using qRT-PCR; ( G , H ) qRT-PCR analysis of hsa_circ_0006692 and CDK19 expression in A549 cells transferred with miR-205-5p mimics or inhibitor. *** p < 0.001; **** p < 0.0001.
    Figure Legend Snippet: hsa_circ_0006692 played its roles via sponging hsa-miR-205-5p and regulation of CDK19. ( A ) After the detection of ago2 or IgG RIP assay, hsa_circ_0006692 and miR-205-5p levels was determined by qRT-PCR; ( B ) After RNA pull-down analysis using Bio-miR-NC and Bio-miR-205-5p in A549 and H1299 cells, expression of hsa_circ_0006692 was measured by qRT-PCR; ( C – E ) CDK19 expression level was increased in A549/circ-0006692-OE cells and decreased in A549/circ-0006692-SH cells analyzed by qRT-PCR and WB; ( C , D ) miR-205-5p expression was decreased in A549/circ-0006692-OE cells and increased in A549/circ-0006692-SH cells analyzed by qRT-PCR; ( F ) miR-205-5p expression in A549 cells transfected with miR-205-5p mimics or inhibitors was analyzed using qRT-PCR; ( G , H ) qRT-PCR analysis of hsa_circ_0006692 and CDK19 expression in A549 cells transferred with miR-205-5p mimics or inhibitor. *** p < 0.001; **** p < 0.0001.

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection

    antibodies against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against ago2
    CircRHBDD1 activates PI3K/AKT signaling by augmenting YTHDF1-mediated translation of PIK3R1 (A) Western blotting analysis showing the expression levels of p-PI3K, PI3K, p-AKT, and AKT in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (B) Expression levels of PIK3R1 in circRHBDD1-silenced HCCLM3 and MHCC97H cells were determined by western blotting. (C) Expression levels of PIK3R1 and p-AKT in the tumor tissues of PDX were analyzed using immunohistochemistry. (D) PIK3R1 mRNA levels were detected by qRT-PCR in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (E) The effects of circRHBDD1 knockdown on the degradation rate of PIK3R1 protein in HCCLM3 cells pretreated with cycloheximide (CHX) were analyzed by western blotting. (F) Quantitative analysis of (E). (G) The effects of MG132 and chloroquine (CQ) on circRHBDD1 knockdown-induced PIK3R1 downregulation were determined by western blotting. (H) The amount of PIK3R1 mRNA in various polysome fractions was analyzed by qRT-PCR. (I) An <t>AGO2-RIP</t> assay was performed to detect the levels of circRHBDD1 in the AGO2 IP pellet. (J) Results from the CPAT database indicating the absence of coding potential for circRHBDD1. (K) Mass spectrometry analysis was conducted to determine the proteins that could bind to circRHBDD1. YTHDF1 was revealed as a potential circRHBDD1-interacting protein. (L) RNA pull-down assays followed by western blotting for YTHDF1 in HCCLM3 cells. (M) The interaction between YTHDF1 and circRHBDD1 was verified by RIP assays. (N) FISH for circRHBDD1 and immunofluorescence for YTHDF1 in HCCLM3 cells. The profiles of colocalization are also provided. (O) TCGA data suggested that the expression level of YTHDF1 was upregulated in HCC tissues compared with peritumoral tissues (p < 0.05). (P) YTHDF1 was associated with unfavorable overall survival in HCC patients based on the TCGA data (hazard ratio = 1.8). ∗∗∗p < 0.001; ns, no significance.
    Antibodies Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CircRHBDD1 augments metabolic rewiring and restricts immunotherapy efficacy via m 6 A modification in hepatocellular carcinoma"

    Article Title: CircRHBDD1 augments metabolic rewiring and restricts immunotherapy efficacy via m 6 A modification in hepatocellular carcinoma

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2022.02.021

    CircRHBDD1 activates PI3K/AKT signaling by augmenting YTHDF1-mediated translation of PIK3R1 (A) Western blotting analysis showing the expression levels of p-PI3K, PI3K, p-AKT, and AKT in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (B) Expression levels of PIK3R1 in circRHBDD1-silenced HCCLM3 and MHCC97H cells were determined by western blotting. (C) Expression levels of PIK3R1 and p-AKT in the tumor tissues of PDX were analyzed using immunohistochemistry. (D) PIK3R1 mRNA levels were detected by qRT-PCR in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (E) The effects of circRHBDD1 knockdown on the degradation rate of PIK3R1 protein in HCCLM3 cells pretreated with cycloheximide (CHX) were analyzed by western blotting. (F) Quantitative analysis of (E). (G) The effects of MG132 and chloroquine (CQ) on circRHBDD1 knockdown-induced PIK3R1 downregulation were determined by western blotting. (H) The amount of PIK3R1 mRNA in various polysome fractions was analyzed by qRT-PCR. (I) An AGO2-RIP assay was performed to detect the levels of circRHBDD1 in the AGO2 IP pellet. (J) Results from the CPAT database indicating the absence of coding potential for circRHBDD1. (K) Mass spectrometry analysis was conducted to determine the proteins that could bind to circRHBDD1. YTHDF1 was revealed as a potential circRHBDD1-interacting protein. (L) RNA pull-down assays followed by western blotting for YTHDF1 in HCCLM3 cells. (M) The interaction between YTHDF1 and circRHBDD1 was verified by RIP assays. (N) FISH for circRHBDD1 and immunofluorescence for YTHDF1 in HCCLM3 cells. The profiles of colocalization are also provided. (O) TCGA data suggested that the expression level of YTHDF1 was upregulated in HCC tissues compared with peritumoral tissues (p < 0.05). (P) YTHDF1 was associated with unfavorable overall survival in HCC patients based on the TCGA data (hazard ratio = 1.8). ∗∗∗p < 0.001; ns, no significance.
    Figure Legend Snippet: CircRHBDD1 activates PI3K/AKT signaling by augmenting YTHDF1-mediated translation of PIK3R1 (A) Western blotting analysis showing the expression levels of p-PI3K, PI3K, p-AKT, and AKT in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (B) Expression levels of PIK3R1 in circRHBDD1-silenced HCCLM3 and MHCC97H cells were determined by western blotting. (C) Expression levels of PIK3R1 and p-AKT in the tumor tissues of PDX were analyzed using immunohistochemistry. (D) PIK3R1 mRNA levels were detected by qRT-PCR in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (E) The effects of circRHBDD1 knockdown on the degradation rate of PIK3R1 protein in HCCLM3 cells pretreated with cycloheximide (CHX) were analyzed by western blotting. (F) Quantitative analysis of (E). (G) The effects of MG132 and chloroquine (CQ) on circRHBDD1 knockdown-induced PIK3R1 downregulation were determined by western blotting. (H) The amount of PIK3R1 mRNA in various polysome fractions was analyzed by qRT-PCR. (I) An AGO2-RIP assay was performed to detect the levels of circRHBDD1 in the AGO2 IP pellet. (J) Results from the CPAT database indicating the absence of coding potential for circRHBDD1. (K) Mass spectrometry analysis was conducted to determine the proteins that could bind to circRHBDD1. YTHDF1 was revealed as a potential circRHBDD1-interacting protein. (L) RNA pull-down assays followed by western blotting for YTHDF1 in HCCLM3 cells. (M) The interaction between YTHDF1 and circRHBDD1 was verified by RIP assays. (N) FISH for circRHBDD1 and immunofluorescence for YTHDF1 in HCCLM3 cells. The profiles of colocalization are also provided. (O) TCGA data suggested that the expression level of YTHDF1 was upregulated in HCC tissues compared with peritumoral tissues (p < 0.05). (P) YTHDF1 was associated with unfavorable overall survival in HCC patients based on the TCGA data (hazard ratio = 1.8). ∗∗∗p < 0.001; ns, no significance.

    Techniques Used: Western Blot, Expressing, Immunohistochemistry, Quantitative RT-PCR, Mass Spectrometry, Immunofluorescence

    rabbit mono antibody against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit mono antibody against ago2
    Rabbit Mono Antibody Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibodies against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against ago2
    a Hypoxia inhibited mRNA loading to <t>AGO2.</t> Cumulative fraction analysis of RIP-Seq for mRNA transcripts bound to AGO2 ( n = 7638 mRNA transcripts) in stable HeLa cells expressing Flag-AGO2 under hypoxia (1% O 2 ) for 24 h. b , c Hypoxia suppressed the interaction of let-7a-targeted c-MYC and HMGA2 -3′-UTR-mut with AGO2. b HeLa cells treated with hypoxia were performed by RIP assay, let-7a and c-MYC associated with AGO2 were detected by northern blotting and qRT-PCR, respectively. Data were mean ± s.d., n = 4 biologically independent samples, and P -values were determined by unpaired two-sided t-test. c Cell lysates from 293 T cells treated with hypoxia were co-incubated with let-7a duplex mimics and biotin-tagged HMGA2 -3′-UTR-mut for in vitro targeted mRNA pull-down assay. AGO2 associated with HMGA2 -3′-UTR-mut was pulled down by streptavidin beads, and then detected by WB. d – f Hypoxia decreased the interaction of AGO2 with mRNAs targeted by only top 10 non-difference miRNAs. Cumulative fraction analysis of AGO2 bound mRNA transcripts ( n = 559 mRNA transcripts) targeted by only top 10 non-difference miRNAs but not by hypoxia-reduced miRNAs in stable HeLa cells expressing Flag-AGO2 under hypoxia ( d ). Coverage plots showing the abundance of MYC ( e ) and HMGA2 ( f ) mRNAs binding to AGO2 by RIP-Seq. g , h Hypoxia increased the accumulation of mRNA transcripts targeted by top 10 non-difference miRNAs ( g ) and their corresponding 6mer, 7mer and 8mer seed sequences with a single site ( h ). Cumulative fraction analyses were performed with RNA-Seq data in HeLa-Flag-AGO2 stable cell lines under normoxia and hypoxia. i Hypoxia inhibited the let-7a-miRISC activity. 293T cells were transfected psiCHECK2-4xlet-7a-BS and then treated with hypoxia or CoCl 2 (300 μM) for 12 and 24 h. The let-7a-miRISC activity was measured by the dual-luciferase assay. Data were mean ± s.e.m., n = 4 (left panel) or n = 3 (right panel) biologically independent experiments, and P -values were determined by unpaired two-sided t-test. In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P -values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis ( a , d , g , h ).
    Antibodies Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against ago2/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
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    antibodies against ago2 - by Bioz Stars, 2023-03
    96/100 stars

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    1) Product Images from "Hypoxia regulates overall mRNA homeostasis by inducing Met 1 -linked linear ubiquitination of AGO2 in cancer cells"

    Article Title: Hypoxia regulates overall mRNA homeostasis by inducing Met 1 -linked linear ubiquitination of AGO2 in cancer cells

    Journal: Nature Communications

    doi: 10.1038/s41467-021-25739-5

    a Hypoxia inhibited mRNA loading to AGO2. Cumulative fraction analysis of RIP-Seq for mRNA transcripts bound to AGO2 ( n = 7638 mRNA transcripts) in stable HeLa cells expressing Flag-AGO2 under hypoxia (1% O 2 ) for 24 h. b , c Hypoxia suppressed the interaction of let-7a-targeted c-MYC and HMGA2 -3′-UTR-mut with AGO2. b HeLa cells treated with hypoxia were performed by RIP assay, let-7a and c-MYC associated with AGO2 were detected by northern blotting and qRT-PCR, respectively. Data were mean ± s.d., n = 4 biologically independent samples, and P -values were determined by unpaired two-sided t-test. c Cell lysates from 293 T cells treated with hypoxia were co-incubated with let-7a duplex mimics and biotin-tagged HMGA2 -3′-UTR-mut for in vitro targeted mRNA pull-down assay. AGO2 associated with HMGA2 -3′-UTR-mut was pulled down by streptavidin beads, and then detected by WB. d – f Hypoxia decreased the interaction of AGO2 with mRNAs targeted by only top 10 non-difference miRNAs. Cumulative fraction analysis of AGO2 bound mRNA transcripts ( n = 559 mRNA transcripts) targeted by only top 10 non-difference miRNAs but not by hypoxia-reduced miRNAs in stable HeLa cells expressing Flag-AGO2 under hypoxia ( d ). Coverage plots showing the abundance of MYC ( e ) and HMGA2 ( f ) mRNAs binding to AGO2 by RIP-Seq. g , h Hypoxia increased the accumulation of mRNA transcripts targeted by top 10 non-difference miRNAs ( g ) and their corresponding 6mer, 7mer and 8mer seed sequences with a single site ( h ). Cumulative fraction analyses were performed with RNA-Seq data in HeLa-Flag-AGO2 stable cell lines under normoxia and hypoxia. i Hypoxia inhibited the let-7a-miRISC activity. 293T cells were transfected psiCHECK2-4xlet-7a-BS and then treated with hypoxia or CoCl 2 (300 μM) for 12 and 24 h. The let-7a-miRISC activity was measured by the dual-luciferase assay. Data were mean ± s.e.m., n = 4 (left panel) or n = 3 (right panel) biologically independent experiments, and P -values were determined by unpaired two-sided t-test. In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P -values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis ( a , d , g , h ).
    Figure Legend Snippet: a Hypoxia inhibited mRNA loading to AGO2. Cumulative fraction analysis of RIP-Seq for mRNA transcripts bound to AGO2 ( n = 7638 mRNA transcripts) in stable HeLa cells expressing Flag-AGO2 under hypoxia (1% O 2 ) for 24 h. b , c Hypoxia suppressed the interaction of let-7a-targeted c-MYC and HMGA2 -3′-UTR-mut with AGO2. b HeLa cells treated with hypoxia were performed by RIP assay, let-7a and c-MYC associated with AGO2 were detected by northern blotting and qRT-PCR, respectively. Data were mean ± s.d., n = 4 biologically independent samples, and P -values were determined by unpaired two-sided t-test. c Cell lysates from 293 T cells treated with hypoxia were co-incubated with let-7a duplex mimics and biotin-tagged HMGA2 -3′-UTR-mut for in vitro targeted mRNA pull-down assay. AGO2 associated with HMGA2 -3′-UTR-mut was pulled down by streptavidin beads, and then detected by WB. d – f Hypoxia decreased the interaction of AGO2 with mRNAs targeted by only top 10 non-difference miRNAs. Cumulative fraction analysis of AGO2 bound mRNA transcripts ( n = 559 mRNA transcripts) targeted by only top 10 non-difference miRNAs but not by hypoxia-reduced miRNAs in stable HeLa cells expressing Flag-AGO2 under hypoxia ( d ). Coverage plots showing the abundance of MYC ( e ) and HMGA2 ( f ) mRNAs binding to AGO2 by RIP-Seq. g , h Hypoxia increased the accumulation of mRNA transcripts targeted by top 10 non-difference miRNAs ( g ) and their corresponding 6mer, 7mer and 8mer seed sequences with a single site ( h ). Cumulative fraction analyses were performed with RNA-Seq data in HeLa-Flag-AGO2 stable cell lines under normoxia and hypoxia. i Hypoxia inhibited the let-7a-miRISC activity. 293T cells were transfected psiCHECK2-4xlet-7a-BS and then treated with hypoxia or CoCl 2 (300 μM) for 12 and 24 h. The let-7a-miRISC activity was measured by the dual-luciferase assay. Data were mean ± s.e.m., n = 4 (left panel) or n = 3 (right panel) biologically independent experiments, and P -values were determined by unpaired two-sided t-test. In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P -values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis ( a , d , g , h ).

    Techniques Used: Expressing, Northern Blot, Quantitative RT-PCR, Incubation, In Vitro, Pull Down Assay, Binding Assay, RNA Sequencing Assay, Stable Transfection, Activity Assay, Transfection, Luciferase, MANN-WHITNEY

    a Scatter distribution plot of proteins interacting with AGO2 under hypoxia and normoxia conditions were analyzed by mass spectrometry (MS). HeLa cells expressing Flag-AGO2 were treated with hypoxia or normoxia for 24 h, respectively. Cell lysates were used for co-IP with anti-Flag antibody, and followed by MS analysis. b , c Ectopically expressed Flag-HOIP or Flag-HOIL-1L interacted with AGO2. Myc-AGO2 was co-transfected with Flag-HOIP ( b ) or Flag-HOIL-1L ( c ) into 293T cells, the association of AGO2 with HOIP or HOIL-1L was determined by co-IP/WB. d HOIP, HOIL-1L and SHARPIN interacted with AGO2. Lysates from HeLa cells were used by co-IP with anti-AGO2 antibody, and followed by WB. e SHARPIN associated strongly with HOIP, HOIL-1L but weakly with AGO2. Lysates form HeLa cells were used by co-IP with anti-SHARPIN antibody, and followed by WB. f Hypoxia promoted the interaction of AGO2 with HOIL-1L and HOIP. HeLa cells treated with hypoxia were lysed for co-IP with anti-AGO2 antibody, and followed by WB with anti-HOIP and anti-HOIL-1L antibodies. g Increasing expression of HOIL-1L promoted AGO2 recruiting more LUBAC. Flag-HOIL-1L was transfected in a dose-increasing manner with HA-AGO2 into 293T cells. co-IP was performed with anti-HA antibody, and then endogenous HOIP and Flag-HOIL-1L bound to AGO2 were conducted by WB. h Knockdown of HOIL-1L decreased the interaction of AGO2 with HOIP. Stable HOIL-1L-knockdown HeLa cells were used for co-IP/WB with indicated antibodies. i AGO2 prolyl-4-hydroxylation did not influence its interaction with HOIL-1L and HOIP. 293T cells transfected with Flag-AGO2-WT or prolyl-4-hydroxylation mutant Flag-AGO2-P700A were treated with hypoxia for indicated times, and then lysed for co-IP/WB with indicated antibodies. j Hypoxia enhanced the association of HOIL-1L with HOIP and AGO2. HeLa cells treated with hypoxia for indicated times were lysed for co-IP/WB with indicated antibodies. k , l Analyses of HOIP and HOIL-1L binding domains with AGO2. Various truncated forms of Flag-HOIP ( k ) or Flag-HOIL-1L ( l ) were individually co-transfected with HA-AGO2 into 293T cells, and then lysates were used by co-IP with anti-HA antibody, followed by WB.
    Figure Legend Snippet: a Scatter distribution plot of proteins interacting with AGO2 under hypoxia and normoxia conditions were analyzed by mass spectrometry (MS). HeLa cells expressing Flag-AGO2 were treated with hypoxia or normoxia for 24 h, respectively. Cell lysates were used for co-IP with anti-Flag antibody, and followed by MS analysis. b , c Ectopically expressed Flag-HOIP or Flag-HOIL-1L interacted with AGO2. Myc-AGO2 was co-transfected with Flag-HOIP ( b ) or Flag-HOIL-1L ( c ) into 293T cells, the association of AGO2 with HOIP or HOIL-1L was determined by co-IP/WB. d HOIP, HOIL-1L and SHARPIN interacted with AGO2. Lysates from HeLa cells were used by co-IP with anti-AGO2 antibody, and followed by WB. e SHARPIN associated strongly with HOIP, HOIL-1L but weakly with AGO2. Lysates form HeLa cells were used by co-IP with anti-SHARPIN antibody, and followed by WB. f Hypoxia promoted the interaction of AGO2 with HOIL-1L and HOIP. HeLa cells treated with hypoxia were lysed for co-IP with anti-AGO2 antibody, and followed by WB with anti-HOIP and anti-HOIL-1L antibodies. g Increasing expression of HOIL-1L promoted AGO2 recruiting more LUBAC. Flag-HOIL-1L was transfected in a dose-increasing manner with HA-AGO2 into 293T cells. co-IP was performed with anti-HA antibody, and then endogenous HOIP and Flag-HOIL-1L bound to AGO2 were conducted by WB. h Knockdown of HOIL-1L decreased the interaction of AGO2 with HOIP. Stable HOIL-1L-knockdown HeLa cells were used for co-IP/WB with indicated antibodies. i AGO2 prolyl-4-hydroxylation did not influence its interaction with HOIL-1L and HOIP. 293T cells transfected with Flag-AGO2-WT or prolyl-4-hydroxylation mutant Flag-AGO2-P700A were treated with hypoxia for indicated times, and then lysed for co-IP/WB with indicated antibodies. j Hypoxia enhanced the association of HOIL-1L with HOIP and AGO2. HeLa cells treated with hypoxia for indicated times were lysed for co-IP/WB with indicated antibodies. k , l Analyses of HOIP and HOIL-1L binding domains with AGO2. Various truncated forms of Flag-HOIP ( k ) or Flag-HOIL-1L ( l ) were individually co-transfected with HA-AGO2 into 293T cells, and then lysates were used by co-IP with anti-HA antibody, followed by WB.

    Techniques Used: Mass Spectrometry, Expressing, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Binding Assay

    a LUBAC catalyzed M1-Ubi of AGO2. LUBAC related plasmids were transfected into 293T cells and subsequently performed by M1-SUB pull-down assay for detection of AGO2 M1-Ubi. b M1-Ubi of endogenous AGO2 was enhanced by LUBAC. 293T cells were co-transfected with components of LUBAC. Lysates were used for IP with anti-AGO2 antibody, followed by WB with a specific linear ubiquitin antibody 1E3 clone. c LUBAC catalyzed M1-Ubi of GST-AGO2 in vitro. Purified GST-AGO2 were incubated with lysates from 293T cells transfected with components of LUBAC, and followed by GST pull-down/WB assay with a specific linear ubiquitin antibody LUB9 clone. d LUBAC increased M1-Ubi of endogenous AGO2. Stable HeLa cells expressing HOIP and HOIL-1L were lysed for IP with anti-AGO2 antibody, and then determined M1-Ubi of AGO2 by WB with antibody 1E3 clone. e In vitro M1-Ubi assay for AGO2. Flag-AGO2 purified from 293T cells was co-incubated with purified ubiquitin and LUBAC proteins for in vitro M1-Ubi assay. f Knockdown of HOIP inhibited AGO2 M1-Ubi. Lysates from stable HeLa-shHOIP or -pLKO.1 cells were used for IP/WB. g AGO2 M1-Ubi was dependent on the catalytic activity of HOIP, but not that of HOIL-1L. 293T cells were co-transfected with indicated plasmids, and M1-Ubi of AGO2 was detected by IP/WB. h M1-Ubi of AGO2 was removed by OTULIN. 293T cells were co-transfected with indicated plasmids, and M1-Ubi of AGO2 was analyzed by M1-SUB pull-down/WB. i Knockdown of OTULIN increased AGO2 M1-Ubi. Stable HeLa-shOTULIN cells were transfected with indicated plasmids, and AGO2 M1-Ubi was analyzed by M1-SUB pull-down/WB. j OTULIN mutants (W96A, C129A) did not inhibit AGO2 M1-Ubi. OTULIN or indicated OTULIN mutants were transfected into 293T cells, respectively. M1-Ubi of AGO2 was analyzed by IP/WB. k K820 is a major site for M1-Ubi of AGO2. The M1-Ubi of AGO2-WT and indicated AGO2 mutants were detected by M1-SUB pull-down and WB. l Hypoxia-induced M1-Ubi of AGO2. 293T and HeLa cells were treated with hypoxia or CoCl 2 (300 μM) for indicated times, and AGO2 M1-Ubi was determined by IP/WB.
    Figure Legend Snippet: a LUBAC catalyzed M1-Ubi of AGO2. LUBAC related plasmids were transfected into 293T cells and subsequently performed by M1-SUB pull-down assay for detection of AGO2 M1-Ubi. b M1-Ubi of endogenous AGO2 was enhanced by LUBAC. 293T cells were co-transfected with components of LUBAC. Lysates were used for IP with anti-AGO2 antibody, followed by WB with a specific linear ubiquitin antibody 1E3 clone. c LUBAC catalyzed M1-Ubi of GST-AGO2 in vitro. Purified GST-AGO2 were incubated with lysates from 293T cells transfected with components of LUBAC, and followed by GST pull-down/WB assay with a specific linear ubiquitin antibody LUB9 clone. d LUBAC increased M1-Ubi of endogenous AGO2. Stable HeLa cells expressing HOIP and HOIL-1L were lysed for IP with anti-AGO2 antibody, and then determined M1-Ubi of AGO2 by WB with antibody 1E3 clone. e In vitro M1-Ubi assay for AGO2. Flag-AGO2 purified from 293T cells was co-incubated with purified ubiquitin and LUBAC proteins for in vitro M1-Ubi assay. f Knockdown of HOIP inhibited AGO2 M1-Ubi. Lysates from stable HeLa-shHOIP or -pLKO.1 cells were used for IP/WB. g AGO2 M1-Ubi was dependent on the catalytic activity of HOIP, but not that of HOIL-1L. 293T cells were co-transfected with indicated plasmids, and M1-Ubi of AGO2 was detected by IP/WB. h M1-Ubi of AGO2 was removed by OTULIN. 293T cells were co-transfected with indicated plasmids, and M1-Ubi of AGO2 was analyzed by M1-SUB pull-down/WB. i Knockdown of OTULIN increased AGO2 M1-Ubi. Stable HeLa-shOTULIN cells were transfected with indicated plasmids, and AGO2 M1-Ubi was analyzed by M1-SUB pull-down/WB. j OTULIN mutants (W96A, C129A) did not inhibit AGO2 M1-Ubi. OTULIN or indicated OTULIN mutants were transfected into 293T cells, respectively. M1-Ubi of AGO2 was analyzed by IP/WB. k K820 is a major site for M1-Ubi of AGO2. The M1-Ubi of AGO2-WT and indicated AGO2 mutants were detected by M1-SUB pull-down and WB. l Hypoxia-induced M1-Ubi of AGO2. 293T and HeLa cells were treated with hypoxia or CoCl 2 (300 μM) for indicated times, and AGO2 M1-Ubi was determined by IP/WB.

    Techniques Used: Transfection, Pull Down Assay, In Vitro, Purification, Incubation, Expressing, Activity Assay

    a , b AGO2/TNRC6A interacted with LUBAC. HeLa cells were lysed for co-IP with anti-HOIL-1L ( a ) or anti-TNRC6A ( b ) antibody, and then the interactions between AGO2/TNRC6A with HOIP and HOIL-1L were detected by WB with indicated antibodies. c , d LUBAC was co-localized with AGO2/TNRC6C of miRISC. GFP-AGO2 and HA-TNRC6C were co-transfected with mCherry-HOIP ( c ) or mCherry-HOIL-1L ( d ) into HeLa cells, the co-localization of GFP-AGO2 (green), TNRC6C (purple) with mCherry-HOIP (red) or mCherry-HOIL-1L (red) were observed by immunofluorescence staining. e – h Hypoxia increased the co-localization of HOIP and HOIL-1L to AGO2/TNRC6A miRISC. GFP-AGO2 was transfected into HeLa cells, the co-localization of GFP-AGO2 (green), TNRC6A (purple) with HOIP (red) or HOIL-1L (red) were observed by immunofluorescence staining. The scale of the magnified region (white frame) is 2 μm x 2 μm ( e , g ). Co-localization between GFP-AGO2/TNRC6A with HOIP or HOIL-1L were analyzed by Pearson’s correlation coefficient. Data were mean ± s.d., and P -values were determined by unpaired two-sided t-test ( f , h ).
    Figure Legend Snippet: a , b AGO2/TNRC6A interacted with LUBAC. HeLa cells were lysed for co-IP with anti-HOIL-1L ( a ) or anti-TNRC6A ( b ) antibody, and then the interactions between AGO2/TNRC6A with HOIP and HOIL-1L were detected by WB with indicated antibodies. c , d LUBAC was co-localized with AGO2/TNRC6C of miRISC. GFP-AGO2 and HA-TNRC6C were co-transfected with mCherry-HOIP ( c ) or mCherry-HOIL-1L ( d ) into HeLa cells, the co-localization of GFP-AGO2 (green), TNRC6C (purple) with mCherry-HOIP (red) or mCherry-HOIL-1L (red) were observed by immunofluorescence staining. e – h Hypoxia increased the co-localization of HOIP and HOIL-1L to AGO2/TNRC6A miRISC. GFP-AGO2 was transfected into HeLa cells, the co-localization of GFP-AGO2 (green), TNRC6A (purple) with HOIP (red) or HOIL-1L (red) were observed by immunofluorescence staining. The scale of the magnified region (white frame) is 2 μm x 2 μm ( e , g ). Co-localization between GFP-AGO2/TNRC6A with HOIP or HOIL-1L were analyzed by Pearson’s correlation coefficient. Data were mean ± s.d., and P -values were determined by unpaired two-sided t-test ( f , h ).

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Staining

    a HOIP/HOIL-1L, but not HOIP-C885A/HOIL-1L and HOIP-C916A/HOIL-1L reduced the inhibition of luciferase activity mediated by AGO2. b OTULIN recovered the reduced inhibition of luciferase activity by LUBAC. c , d Knockdown HOIP by siRNAs in 293T cell ( c ) or by shRNA in HeLa cell ( d ) increased the inhibition of luciferase activity mediated by AGO2. 293T cells ( a – c ) or HeLa-shHOIP cells ( d ) transfected with indicated plasmids were harvested for the psiCHECK2-4xlet-7a-BS of dual-luciferase assay. Dual-luciferase reporter assay data were mean ± s.e.m., n = 3 ( a , c , d ) or n = 4 ( b ) biologically independent experiments, and P -values were determined by unpaired two-sided t-test. e , f Knockdown of HOIP increased let-7a-targeted GFP protein and mRNA levels. HeLa cells were transfected with indicated plasmids and siRNAs, the GFP protein expression was detected by WB ( e ). 293T cell stably expressing GFP-4x-let-7a-BS was transfected with HOIP siRNAs, and then total RNAs were extracted for detection of GFP mRNA levels by qRT-PCR. Data were mean ± s.d., n = 4 biologically independent samples, and P -values were determined by unpaired two-sided t-test ( f ). g , h Knockdown of OTULIN decreased let-7a-targeted GFP and c-MYC decay. HeLa cells were transfected with indicated plasmids and siRNAs, the proteins GFP ( g ) and c-MYC ( h ) expression were measured by WB analysis.
    Figure Legend Snippet: a HOIP/HOIL-1L, but not HOIP-C885A/HOIL-1L and HOIP-C916A/HOIL-1L reduced the inhibition of luciferase activity mediated by AGO2. b OTULIN recovered the reduced inhibition of luciferase activity by LUBAC. c , d Knockdown HOIP by siRNAs in 293T cell ( c ) or by shRNA in HeLa cell ( d ) increased the inhibition of luciferase activity mediated by AGO2. 293T cells ( a – c ) or HeLa-shHOIP cells ( d ) transfected with indicated plasmids were harvested for the psiCHECK2-4xlet-7a-BS of dual-luciferase assay. Dual-luciferase reporter assay data were mean ± s.e.m., n = 3 ( a , c , d ) or n = 4 ( b ) biologically independent experiments, and P -values were determined by unpaired two-sided t-test. e , f Knockdown of HOIP increased let-7a-targeted GFP protein and mRNA levels. HeLa cells were transfected with indicated plasmids and siRNAs, the GFP protein expression was detected by WB ( e ). 293T cell stably expressing GFP-4x-let-7a-BS was transfected with HOIP siRNAs, and then total RNAs were extracted for detection of GFP mRNA levels by qRT-PCR. Data were mean ± s.d., n = 4 biologically independent samples, and P -values were determined by unpaired two-sided t-test ( f ). g , h Knockdown of OTULIN decreased let-7a-targeted GFP and c-MYC decay. HeLa cells were transfected with indicated plasmids and siRNAs, the proteins GFP ( g ) and c-MYC ( h ) expression were measured by WB analysis.

    Techniques Used: Inhibition, Luciferase, Activity Assay, shRNA, Transfection, Reporter Assay, Expressing, Stable Transfection, Quantitative RT-PCR

    a , b Scatter plots of miRNA expression profiles by miRNA-Seq in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L ( a ) and HeLa cells knocking down HOIP ( b ). c , d M1-Ubi of AGO2 inhibited recruitments of mRNAs. Cumulative fraction analyses for mRNA transcripts recruiting to AGO2 were conducted by using RIP-Seq data in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L ( c , n = 7014 mRNA transcripts) and HeLa cells knocking down HOIP ( d , n = 5788 mRNA transcripts). e , f Venn diagram analysis ( e ) and scatter plots analysis of mRNA transcripts recruited to AGO2 with more than 1.5-fold changes ( f ). g , h Several mRNAs bound to AGO2 were examined by RIP/qRT-PCR in above stable HeLa cells. i , j Cumulative fraction analyses for top 10 non-difference miRNA-targeted mRNA transcripts recruiting to AGO2 ( i , n = 2792 mRNA transcripts; j , n = 982 mRNA transcripts) were conducted by using RIP-Seq data in stable HeLa cells. k LUBAC inhibit the association of let-7-targeted HMGA2 and c-MYC mRNAs with AGO2. HMGA2 and c-MYC mRNAs bound to AGO2 were measured by RIP/qRT-PCR. The RIP efficiency were determined by WB and Northern blotting analysis. l – n Knockdown of HOIP augmented the associations of let-7a-targeted mRNAs to AGO2. HeLa-shHOIP cells transfected with indicated plasmids were used for target mRNA pull-down assay ( l ) and GST-MS2 pull-down assay ( m , n ). l RNA bound fraction (beads) and unbound fraction (supernatant) were detected by WB. m A schematic illustration of the GST-MS2 pull-down assay. n AGO2 associated RNAs through let-7a were assessed by GST-MS2 pull-down. o K820R of AGO2 augmented its recruiting with let-7-targeted mRNAs. The abundance of let-7a and let-7a targeted HMGA2/c-MYC mRNAs associated with AGO2 or mutants were determined by RIP/northern blotting and RIP/qRT-PCR, respectively. qRT-PCR data were mean ± s.d., n > = 3 biologically independent samples, and P -values were determined by unpaired two-sided t-test ( g , h , k , o ). In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P -values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis ( c , d , i , j ).
    Figure Legend Snippet: a , b Scatter plots of miRNA expression profiles by miRNA-Seq in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L ( a ) and HeLa cells knocking down HOIP ( b ). c , d M1-Ubi of AGO2 inhibited recruitments of mRNAs. Cumulative fraction analyses for mRNA transcripts recruiting to AGO2 were conducted by using RIP-Seq data in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L ( c , n = 7014 mRNA transcripts) and HeLa cells knocking down HOIP ( d , n = 5788 mRNA transcripts). e , f Venn diagram analysis ( e ) and scatter plots analysis of mRNA transcripts recruited to AGO2 with more than 1.5-fold changes ( f ). g , h Several mRNAs bound to AGO2 were examined by RIP/qRT-PCR in above stable HeLa cells. i , j Cumulative fraction analyses for top 10 non-difference miRNA-targeted mRNA transcripts recruiting to AGO2 ( i , n = 2792 mRNA transcripts; j , n = 982 mRNA transcripts) were conducted by using RIP-Seq data in stable HeLa cells. k LUBAC inhibit the association of let-7-targeted HMGA2 and c-MYC mRNAs with AGO2. HMGA2 and c-MYC mRNAs bound to AGO2 were measured by RIP/qRT-PCR. The RIP efficiency were determined by WB and Northern blotting analysis. l – n Knockdown of HOIP augmented the associations of let-7a-targeted mRNAs to AGO2. HeLa-shHOIP cells transfected with indicated plasmids were used for target mRNA pull-down assay ( l ) and GST-MS2 pull-down assay ( m , n ). l RNA bound fraction (beads) and unbound fraction (supernatant) were detected by WB. m A schematic illustration of the GST-MS2 pull-down assay. n AGO2 associated RNAs through let-7a were assessed by GST-MS2 pull-down. o K820R of AGO2 augmented its recruiting with let-7-targeted mRNAs. The abundance of let-7a and let-7a targeted HMGA2/c-MYC mRNAs associated with AGO2 or mutants were determined by RIP/northern blotting and RIP/qRT-PCR, respectively. qRT-PCR data were mean ± s.d., n > = 3 biologically independent samples, and P -values were determined by unpaired two-sided t-test ( g , h , k , o ). In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P -values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis ( c , d , i , j ).

    Techniques Used: Expressing, Quantitative RT-PCR, Northern Blot, Transfection, Pull Down Assay, MANN-WHITNEY

    a – c M1-Ubi of AGO2 promoted mRNA accumulation. Scatter plot of mRNA expression profiles in RNA-Seq ( a ); and cumulative fraction analyses for the abundance of AGO2 bound mRNA transcripts (which were covered by RIP-Seq) in RNA-Seq ( b , n = 1402 mRNAs), which further were categorized as short-, middle- and long-half-life ( c ), in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L. In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P -values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis ( b , c ). d Knockdown of HOIP accelerated the degradation of let-7a-targeted GFP-4xlet-7a-BS. HeLa cells stably knocking down of HOIP by shRNAs were transfected with GFP-4xlet-7a-BS for 24 h, and then cells were treated with 5 μg/ml Actinomycin D for indicated times. GFP mRNA levels were measured by qRT-PCR and normalized with β-actin mRNA. Data were mean ± s.d., n = 3 biologically independent samples, and P -values were determined by unpaired two-sided t-test. e Enriched Gene Ontology (GO) categories for genes with 2-fold up-regulated (red, left panel) and down-regulated (blue, right panel) mRNA transcripts when HOIP and HOIL-1L were overexpressed. P values for enrichment of the indicated GO category were computed by Metascape. f Ectopically expressed LUBAC increased lncRNA transcripts accumulation. Scatter plots of lncRNA expression profiles by RNA-Seq in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L or control vector.
    Figure Legend Snippet: a – c M1-Ubi of AGO2 promoted mRNA accumulation. Scatter plot of mRNA expression profiles in RNA-Seq ( a ); and cumulative fraction analyses for the abundance of AGO2 bound mRNA transcripts (which were covered by RIP-Seq) in RNA-Seq ( b , n = 1402 mRNAs), which further were categorized as short-, middle- and long-half-life ( c ), in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L. In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P -values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis ( b , c ). d Knockdown of HOIP accelerated the degradation of let-7a-targeted GFP-4xlet-7a-BS. HeLa cells stably knocking down of HOIP by shRNAs were transfected with GFP-4xlet-7a-BS for 24 h, and then cells were treated with 5 μg/ml Actinomycin D for indicated times. GFP mRNA levels were measured by qRT-PCR and normalized with β-actin mRNA. Data were mean ± s.d., n = 3 biologically independent samples, and P -values were determined by unpaired two-sided t-test. e Enriched Gene Ontology (GO) categories for genes with 2-fold up-regulated (red, left panel) and down-regulated (blue, right panel) mRNA transcripts when HOIP and HOIL-1L were overexpressed. P values for enrichment of the indicated GO category were computed by Metascape. f Ectopically expressed LUBAC increased lncRNA transcripts accumulation. Scatter plots of lncRNA expression profiles by RNA-Seq in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L or control vector.

    Techniques Used: Expressing, RNA Sequencing Assay, MANN-WHITNEY, Stable Transfection, Transfection, Quantitative RT-PCR, Plasmid Preparation

    a , b Analysis of all ( a ) and top 10 ( b ) non-difference miRNAs between normal tissues ( n = 91) and lung cancer tissues ( n = 999) from TCGA lung cancer miRNA-seq data. c The expression levels of the top 10 non-difference miRNA-targeted mRNAs were positively correlated (Pearson r = 0.7718, P value (two tailed) < 0.0001(****)) and the non-miRNA targets were slightly negative correlated (Pearson r = −0.07952, P value (two tailed) = 0.0108(*)) with hypoxia scores from TCGA lung cancer ( n = 1028) RNA-Seq data, respectively. d The expression levels of the top 10 non-difference miRNA targets and the non-miRNA targets in high-hypoxia lung cancer tissues were much higher and slightly lower than those in low-hypoxia lung cancer tissues, respectively. Clinical lung cancer samples were divided into two groups, low-hypoxia (hypoxia score < 0, n = 457) and high-hypoxia (hypoxia score > 0, n = 571), according to the hypoxia score. Data were mean ± s.d., and P -values were determined by unpaired two-sided t-test. e Mechanistic model summarizing how hypoxia in the tumor microenvironment was an incentive for mRNA accumulation by inducing AGO2 M1-Ubi. Briefly, Hypoxia induces HOIL-1L transcription and enhances LUBAC interacting with AGO2, thereby catalyzing M1-Ubi of AGO2, which can be conversely removed by OUTLIN. M1-Ubi of AGO2 suppresses miRNA-targeted mRNAs loading to AGO2, thus to inhibit the miRISC activity, consequently augment global-mRNA accumulation.
    Figure Legend Snippet: a , b Analysis of all ( a ) and top 10 ( b ) non-difference miRNAs between normal tissues ( n = 91) and lung cancer tissues ( n = 999) from TCGA lung cancer miRNA-seq data. c The expression levels of the top 10 non-difference miRNA-targeted mRNAs were positively correlated (Pearson r = 0.7718, P value (two tailed) < 0.0001(****)) and the non-miRNA targets were slightly negative correlated (Pearson r = −0.07952, P value (two tailed) = 0.0108(*)) with hypoxia scores from TCGA lung cancer ( n = 1028) RNA-Seq data, respectively. d The expression levels of the top 10 non-difference miRNA targets and the non-miRNA targets in high-hypoxia lung cancer tissues were much higher and slightly lower than those in low-hypoxia lung cancer tissues, respectively. Clinical lung cancer samples were divided into two groups, low-hypoxia (hypoxia score < 0, n = 457) and high-hypoxia (hypoxia score > 0, n = 571), according to the hypoxia score. Data were mean ± s.d., and P -values were determined by unpaired two-sided t-test. e Mechanistic model summarizing how hypoxia in the tumor microenvironment was an incentive for mRNA accumulation by inducing AGO2 M1-Ubi. Briefly, Hypoxia induces HOIL-1L transcription and enhances LUBAC interacting with AGO2, thereby catalyzing M1-Ubi of AGO2, which can be conversely removed by OUTLIN. M1-Ubi of AGO2 suppresses miRNA-targeted mRNAs loading to AGO2, thus to inhibit the miRISC activity, consequently augment global-mRNA accumulation.

    Techniques Used: Expressing, Two Tailed Test, RNA Sequencing Assay, Activity Assay

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