Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 locates at ERα binding sites, and its binding is enhanced by E2. (A) De novo DNA motif analysis at AGO1-associated genomic regions. (B) Enrichment of TF binding at AGO1-associated regions in serum-maintained MCF-7 cells, divided according to whether they are also bound by ERα (left panel) or not (right panel). ChIP-seq peak overlaps are shown for AGO1(1), PR (2), FOXA1 (2), TEAD4 (1), CEBPB (1), and JUND (1), where the number of replicates is shown between parentheses. (C) TF overlap with ERα-bound regions (in E2-treated cells), expressed as fold enrichment over randomized regions. (D) Heat maps of ChIP-seq signal centered (±1 kb) on ERα binding site midpoints for ERα (+E2), AGO1 (−E2), and AGO1 (+E2) regions. Regions are ordered from high to low ERα signal. (E) AGO1 reads per million mapped reads (RPM) around (±1 kb). AGO1 binding sites are defined by pooling AGO1 peaks from both treatments (−E2 and +E2). (F) UCSC genome browser screenshots showing four loci comprising ERα enhancers and neighboring genes that are up-regulated upon E2 treatment. Tracks correspond to RefSeq genes, AGO1 ChIP-seq, ERα (+E2) ChIP-seq, and GRO-seq plus and minus strands −E2 and +E2, all from MCF-7 cells.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, ChIP-sequencing

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: Relative to : AGO1 locates at ERα binding sites, and its binding is enhanced by E2. (A) Analysis of known DNA binding motifs of TFs expressed in MCF-7 cells at AGO1-associated genomic regions in serum-maintained cells. (B) Enrichment of TF binding at AGO1-associated regions in serum-maintained MCF7 cells. (C) Schematic representation of the hormone treatment experiments time line. (D) Table showing the overlap between the number of genomic sites (peaks) bound by ERα and by AGO1, considering each replicate separately and the common peaks. (E) Correlation heat map between AGO1 ChIP-seq samples and replicates based on read counts data on peak sets. (F) MA plot (gene expression ratios depicting fold changes in AGO1 binding affinity between untreated and treated samples. (G and H) MCF-7 cells were hormone starved and then treated with vehicle or E2 for 1 h. (G) ERα binding to the enhancers was assessed by ChIP-qPCR. Data are represented as mean ± SD ( n = 3). IP, immunoprecipitation. (H) AGO1 binding to the enhancers was assessed by ChIP-qPCR. Data are represented as mean ± SD ( n = 3; *, P < 0.05; **, P < 0.01; two-tailed Student’s t test).

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, ChIP-sequencing, Gene Expression, ChIP-qPCR, Immunoprecipitation, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 locates at ERα binding sites, and its binding is enhanced by E2 without changes in its subcellular localization. (A–D) MCF-7 cells were hormone starved and then treated with vehicle or E2 for 1 h. (A) AGO1 levels in starved (−E2) and E2-treated (+E2) cells were tested by Western blot (WB) from whole-cell extracts (WCE; lanes 1–4). AGO1 levels were also assessed under these same conditions but transfected with a specific siRNA against AGO1 (lanes 5–8). (B) AGO1 and levels were assessed by Western blot in nuclear (NE) and cytoplasmic extracts (CE) from starved (−E2) and E2-treated MCF-7 cells. (C) AGO1 subcellular localization was assessed by immunofluorescence followed by confocal microscopy. The box plot corresponds to nucleus/cytoplasm integrated intensity ratios ( n = 100 cells; two-tailed Student’s t test). (D) Panels show representative images from C. Scale bars represent 10 µm.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, Western Blot, Transfection, Immunofluorescence, Confocal Microscopy, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: Relative to and . ERα binding intensity positively correlates with AGO1 binding, and AGO1 participates in the transcriptional activation of E2- responsive genes controlled by ERα enhancers. (A) Enrichment of the indicated chromatin states, obtained from E2-treated MCF-7 cells, over ERα binding sites divided into ERα + /AGO1 + and ERα + /AGO1 − . (B) . Heat maps of ChIP-seq signal centered (±3 kb) on ERα binding site midpoints for ERα (+E2), AGO1 (−E2), and AGO1 (+E2). ERα regions were divided according to whether they overlap with AGO1 (ERα + /AGO1 + ) or not (ERα + /AGO1 − ), and within each group, they were ordered from high to low ERα signal. (C–E) MCF-7 cells were transfected with an siControl or siAGO1. 24 h later, they were hormone starved, and 72 h later, they were treated with E2 or vehicle for the indicated time. (C) E2-dependent AGO1 recruitment to the enhancers upon AGO1 knockdown was controlled by ChIP-qPCR. Data are represented as mean ± SD ( n = 3; *, P < 0.05; **, P < 0.01; two-tailed Student’s t test). (D) RNA was extracted, and the indicated mRNA levels were analyzed by RT-qPCR. (E) Same as D, but a different siRNA against AGO1 was used. Values in D and E represent mean ± SE from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ANOVA and Tukey post-hoc test). In A and B, two different siRNAs against AGO1 were used.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, Activation Assay, ChIP-sequencing, Transfection, Knockdown, ChIP-qPCR, Two Tailed Test, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: ERα binding to chromatin depends on AGO1. (A) ERα (+E2) ChIP-seq normalized read counts over ERα + /AGO1 + and ERα + /AGO1 − regions. (B–D) MCF-7 cells were transfected with an siControl or siAGO1. 24 h later, they were hormone starved, and 72 h later, they were treated with E2 or vehicle for 1 h. (B) AGO1 knockdown efficiency was tested by Western blot (WB) from whole-cell extracts (WCE). (C) E2-dependent ERα recruitment to ERα + /AGO1 + regions was analyzed by ChIP-qPCR. (D) E2-dependent ERα recruitment to ERα + /AGO1 − regions was analyzed by ChIP-qPCR. Values in C and D represent mean ± SE with n = 3 (i.e., three independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ANOVA and Tukey post-hoc test. ns, not significant; IP, immunoprecipitation.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, ChIP-sequencing, Transfection, Knockdown, Western Blot, ChIP-qPCR, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 physically interacts with ERα in an E2-dependent and RNA-independent manner. (A) MCF-7 cells were hormone starved and treated with vehicle or E2 for 1 h. Whole-cell extracts (WCE) were treated or not with RNase A, and then they were immunoprecipitated (IP) with control or anti-AGO1 antibody. AGO1 and ERα presence in inputs and immunoprecipitates was analyzed by Western blot (WB). (B) Representative agarose gel showing RNase treatment efficiency.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Immunoprecipitation, Control, Western Blot, Agarose Gel Electrophoresis

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 participates in the transcriptional activation of E2-responsive genes controlled by ERα enhancers by promoting three steps of ERα enhancer activation. (A) GRO-seq read count fold change between −E2 and +E2 treatments over ERα + /AGO1 + and ERα + /AGO1 − regions. (B–H) MCF-7 cells were transfected with an siControl or siAGO1. 24 h later, they were hormone starved, and 72 h later, they were treated with E2 or vehicle for 1 h. (B) Total RNA was extracted, and AGO1 knockdown was assessed by RT-qPCR. (C) Nuclear RNA was extracted, and the indicated eRNA levels were analyzed by RT-qPCR. (D) Total RNA was extracted, and the indicated pre-mRNA levels corresponding to E2-regulated genes were analyzed by RT-qPCR. (E) Total RNA was extracted, and the indicated pre-mRNA levels corresponding to non–E2-regulated genes were analyzed by RT-qPCR. (F) Frequency of chromatin loops formation between the indicated restriction fragments was analyzed by 3C. The anchor region, with which all of the interactions were tested, comprises an ERα + /AGO1 + region. AGO1 ChIP-seq and ERα ChIP-seq tracks are shown along the position of the proximal E2-regulated genes. (G) Same as F, but here the anchor region comprises an ERα + /AGO1 − region. (H) Same as G, but here the anchor region comprises a non–E2-regulated enhancer region. Values in B–E represent mean ± SE from three independent experiments (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ANOVA and Tukey post-hoc test). Values in F–H represent mean ± SE from three independent experiments (*, P < 0.05; **, P < 0.01; two-tailed Student’s t test).

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Activation Assay, Transfection, Knockdown, Quantitative RT-PCR, ChIP-sequencing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 functional role at ERα transcriptional enhancers does not depend on small RNA binding. (A) Schematic representation of AGO1 protein domains showing the position of the point mutation in the MID domain that impairs small RNA binding. (B–D) MCF-7 cells were transfected with the indicated siRNAs, and AGO1 expression was rescued by the indicated constructs before hormone starvation and E2 treatment. (B) AGO1 knockdown and rescue levels were assessed by Western blot. RPB1 corresponds to the housekeeping control. (C) The levels of the indicated pre-mRNA corresponding to E2-regulated genes were assessed by RT-qPCR. (D) The levels of the indicated eRNA corresponding to an E2-regulated eRNA were assessed by RT-qPCR. Values in C and D represent mean ± SE from three independent experiments (*, P < 0.05; two-tailed Student’s t test). ns, not significant; WCE, whole-cell extracts.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Functional Assay, RNA Binding Assay, Mutagenesis, Transfection, Expressing, Construct, Knockdown, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: E2F4’s cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6

doi: 10.1091/mbc.E21-01-0039

Figure Lengend Snippet: E2F4 associates with Deup1 and SAS6. (A, B) Western blots with α-E2F4 or α-Flag antibodies of input lysates and (A) α-E2F4 or (B) α-Flag immunoprecipitates from 293FT cells overexpressing E2F4 and/or Flag-Deup1. (C, D) Western blots with α-E2F4 or α-SAS6 antibodies of input lysates and (C) α-E2F4 or (D) α-SAS6 immunoprecipitates from 293FT cells overexpressing E2F4 and/or SAS6. Mouse IgG was used as a negative control. For (C), an arrow shows the coimmunoprecipitated SAS6 band, HC denotes the IgG heavy chain, and * denotes a nonspecific band. (E) Western blots with α-E2F4 or α-SAS6 antibodies of input lysate and α-E2F4 immunoprecipitates from 293FT cells verified endogenous interaction of E2F4 and SAS6.

Article Snippet: C-terminal truncation mutants of double-tagged Deup1 constructs (Flag-Deup1 1–491 -HA, Flag-Deup1 1–461 -HA, and Flag-Deup1 1–432 -HA) were generated using standard PCR-based cloning techniques. pENTR-age-Hs-SAS6 was a kind gift from Pierre Gonczy (Addgene 46381).

Techniques: Western Blot, Negative Control

Journal: Molecular Biology of the Cell

Article Title: E2F4’s cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6

doi: 10.1091/mbc.E21-01-0039

Figure Lengend Snippet: E2F4 association is mediated by the N-terminal domains of both Deup1 and SAS6. (A) Schematic representation of Flag-tagged Deup1 deletion mutants and their determined E2F4 association. (B, C) Cell lysates containing E2F4 and the indicated Flag-tagged Deup1 mutants were subjected to Western blotting with α-Flag (upper panel) or α-E2F4 (lower panel) antibodies before (Input) or after IP with α-Flag antibodies. (D) Schematic representation of Flag-tagged SAS6 mutants and their determined E2F4 association. (E) Cell lysates containing E2F4 and Flag-tagged SAS6 mutants were subjected to Western blotting with α-Flag or α-E2F4 antibodies before (Input) or after IP with α-Flag antibodies. (F) Cell lysates overexpressing HA-E2F4 and Flag-tagged WT or point mutants (I62T or F131D) of SAS6 were subjected to Western blotting with α-HA or α-Flag antibodies before (Input) or after IP with α-HA antibodies, showing that Flag-SAS6 F131D is unable to associate with E2F4. Numbers indicate amino acid positions. For C and E, * denotes the IgG heavy and light chains, respectively.

Article Snippet: C-terminal truncation mutants of double-tagged Deup1 constructs (Flag-Deup1 1–491 -HA, Flag-Deup1 1–461 -HA, and Flag-Deup1 1–432 -HA) were generated using standard PCR-based cloning techniques. pENTR-age-Hs-SAS6 was a kind gift from Pierre Gonczy (Addgene 46381).

Techniques: Western Blot

Journal: Molecular Biology of the Cell

Article Title: E2F4’s cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6

doi: 10.1091/mbc.E21-01-0039

Figure Lengend Snippet: Deup1 and SAS6 association is mediated by the N-terminal domain of E2F4. (A) Schematic representation of HA-tagged E2F4 mutants and their determined association with Deup1 or SAS6. (B, C) Cell lysates containing the indicated HA-tagged E2F4 mutants alone or with Flag-Deup1, either before (Input) or after IP with α-Flag antibodies, were subjected to Western blotting with (B) α-Flag or α-HA antibodies, or (C) α-Deup1 or α-E2F4 antibodies, as indicated. (D, E) Cell lysates from cells expressing SAS6 and the indicated HA-tagged E2F4 mutants were subjected to Western blotting with (D) α-HA or α-SAS6 antibodies, or (E) α-Flag or α-E2F4 antibodies, before (Input) or after IP with either (D) α-HA or (E) α-Flag antibodies. Numbers indicate amino acid positions and Δ denotes the residues deleted from the full-length protein. For B, * denotes the IgG light chain.

Article Snippet: C-terminal truncation mutants of double-tagged Deup1 constructs (Flag-Deup1 1–491 -HA, Flag-Deup1 1–461 -HA, and Flag-Deup1 1–432 -HA) were generated using standard PCR-based cloning techniques. pENTR-age-Hs-SAS6 was a kind gift from Pierre Gonczy (Addgene 46381).

Techniques: Western Blot, Expressing

Journal: Molecular Biology of the Cell

Article Title: E2F4’s cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6

doi: 10.1091/mbc.E21-01-0039

Figure Lengend Snippet: Association of Deup1 and SAS6 with other E2F family members. (A) Schematic representation of the full-length E2F4, E2F5, and E2F1 and their determined Deup1 and SAS6 binding abilities. (B) Cell lysates containing the indicated HA-tagged E2F species with or without Flag-Deup1 were subjected to Western blotting with the indicated α-Flag and α-HA antibodies before (Input) or after IP of Flag-Deup1. (C) Cell lysates overexpressing indicated HA-tagged E2F species with or without SAS6 were subjected to Western blotting with the indicated α-HA or α-SAS6 antibodies before (Input) or after IP with α-HA antibodies.

Article Snippet: C-terminal truncation mutants of double-tagged Deup1 constructs (Flag-Deup1 1–491 -HA, Flag-Deup1 1–461 -HA, and Flag-Deup1 1–432 -HA) were generated using standard PCR-based cloning techniques. pENTR-age-Hs-SAS6 was a kind gift from Pierre Gonczy (Addgene 46381).

Techniques: Binding Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: E2F4’s cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6

doi: 10.1091/mbc.E21-01-0039

Figure Lengend Snippet: Efficient Deup1 and SAS6 binding requires a short motif (E2F4 48–53 ) that is conserved in E2F4 and E2F5 but not other E2Fs. (A) Schematic representation of E2F4 1–197 , E2F1 121–301 and the various E2F4/E2F1 chimeras. Red lines show the positions of the M1 and M2 mutations. (B) Cell lysates containing HA-tagged E2F4/E2F1 chimeras (HA-E2F4/1), alone or with Flag-Deup1 were subjected to Western blotting with the indicated α-Deup1 or α-HA antibodies before (Input) or after IP with α-Flag antibodies. (C) Amino acid alignments of E2F4, E2F5 and E2F1 with the red boxes indicating the M1 and M2 motifs, which were swapped from E2F4 to E2F1 sequences in the M1 and M2 mutants. (D) Crystal structure of the E2F4 DBD illustrating the exterior surface position of the M1 site (in red). The M2 mutation site is C-terminal to the structured DBD region and was unresolved in the crystal structure ( Zheng et al. , 1999 ). (E–G) Cell lysates with the indicated WT, M1, M2 or M1+2 versions of (F, G) HA-tagged full-length E2F4 or (E) HA-E2F4 1–197 , expressed alone or with (E, F) Flag-Deup1 or (G) Flag-SAS6, were subjected to Western blotting with the indicated (E, F) α-Deup1, (G) α-Flag, (E) α-HA, and (F, G) α-E2F4 antibodies before (Input) or after IP with α-Flag antibodies. For F and G, HC denotes the heavy chain of IgG.

Article Snippet: C-terminal truncation mutants of double-tagged Deup1 constructs (Flag-Deup1 1–491 -HA, Flag-Deup1 1–461 -HA, and Flag-Deup1 1–432 -HA) were generated using standard PCR-based cloning techniques. pENTR-age-Hs-SAS6 was a kind gift from Pierre Gonczy (Addgene 46381).

Techniques: Binding Assay, Western Blot, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Structural Complementarity of Toll/Interleukin-1 Receptor Domains in Toll-like Receptors and the Adaptors Mal and MyD88

doi: 10.1074/jbc.m301742200

Figure Lengend Snippet: FIG. 4. Wild type and P/H mutant forms of Mal and MyD88 bind to TLR4. a, HEK293 cells (1 106) stably expressing TLR4 were transfected with 4 g of AU1-MyD88 or AU1-MyD88 P/H. Lysates were immunoprecipitated (IP) with a polyclonal antibody to TLR4. b, lysates from HEK293 cells transfected with the MyD88 constructs were incubated with GST-tagged TLR4. c, HEK293 cells were co-transfected with FLAG-tagged TLR4 and HA-Mal or HA-Mal P/H. Lysates were immunoprecipitated with a monoclonal antibody to the FLAG epitope. d, HEK293 cells transfected with wild-type and mutant Mal were incubated with GST-TLR4. The resulting complexes were analyzed by SDS-PAGE and Western blotting. IB, immunoblotted; WCE, whole cell extract.

Article Snippet: The polyclonal antibody against the HA epitope tag (Y-11) was obtained from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Construct, Incubation, FLAG-tag, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Structural Complementarity of Toll/Interleukin-1 Receptor Domains in Toll-like Receptors and the Adaptors Mal and MyD88

doi: 10.1074/jbc.m301742200

Figure Lengend Snippet: FIG. 5. Homodimerization and heterodimerization of the adaptors is not influenced by the Pro/His mutation. a, results of GRAMM docking of Mal (left) and MyD88 (right) TIR domains. Structural features representing the conserved boxes of the TIR domains are shown in green (box 1), blue (box 2, BB-loop), and purple (box 3, not present in Mal). The side chain of the semi-conserved proline residue in the BB-loop is colored purple. b, association of Mal with MyD88. GST-Mal (upper arrow) and His-Myd88 (lower arrow) were co-expressed in E. coli BL21(DE3) cells (lane 1 (uninduced) and lanes 2–3 (induced with isopropyl-1-thio--D-galactopyranoside)). Cleaved GST-Mal (lower arrow) and His-MyD88 (upper arrow) co-elute after thrombin cleavage on a glutathione-Sepharose resin (lanes 4–7). In c–e, HEK293 cells (1 106) were transfected with 4 g of the isolated wild type and mutant TIR domains of Mal and/or MyD88 as indicated. c, homodimerization and heterodimerization of full-length Mal was studied in a GST pull-down experiment with GST-Mal and HA-tagged Mal, Mal P/H or Myd88 P/H. d, MyD88 TIR domain homodimerization was studied using AU1- and Myc-tagged TIR-domain constructs of the wild type and P/H form of MyD88. Lysates were immunoprecipitated with a monoclonal antibody to the AU1 epitope. e, heterodimerization of the TIR domains of Mal and MyD88 and their P/H forms was studied using HA-tagged Mal and Myc-tagged MyD88. Lysates were immunoprecipitated with a polyclonal antibody to the HA tag.

Article Snippet: The polyclonal antibody against the HA epitope tag (Y-11) was obtained from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Residue, Transfection, Isolation, Construct, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A , B Quantification and representation of LKB1 and m6A modulators in KL relative to K lung cancer patients. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. C Spearman correlation analysis of ALKBH5 expression with the KRAS mutant lung cancer pathological characters. D Venn diagram showing the differentially expressed genes (DEGs) of LKB1 , m6A modulators, and readers between KL and K lung cancer. E Kaplan–Meier survival curve of patients with high and low ALKBH5 mRNA expression from the TCGA dataset. F Global m6A level regulation by LKB1 in lung cancer cell lines. G Western blot showing the LKB1 and ALKBH5 protein expression. H Co-immunofluorescence staining for LKB1 and ALKBH5 in A549 cells. Arrows show LKB1 positive and ALKBH5 negative cells. I LKB1 and m6A co-staining by LKB1 and/or ALKBH5 overexpression in A549 cells. Arrows show cells of LKB1 positive and m6A with strong (Second panel) or weak (Forth panel) intensity. Data were mean ± SD in F ( n = 5). * P < 0.05 (Student’s t -test). Si-CN, SiRNA-A; OE-CN, OE-c-Flag pcDNA3; OE, overexpression. KRAS Mut ; LKB1 Loss (KL); KRAS Mut ; LKB1 Wt (K).

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Expressing, Mutagenesis, Western Blot, Immunofluorescence, Staining, Over Expression

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A Western blot analysis shown that LKB1 and ALKBH5 were successfully knocked down in H1792 cells and overexpressed in A549 cells. B , C The measurement of global m6A by ELISA assay in H1792 ( B ) or A549 ( C ) cells, respectively. The colony formation ability ( D ) and cell migration ( E ) were reduced at both of basal level and LKB1 silenced level by ALKBH5 knockdown in H1792 cells, respectively. The reversible effect was found at both of basal level and LKB1 overexpressed level by ALKBH5 transfected in A549 cells using colony formation ( F ) and transwell assay ( G ). Data were mean ± SD ( n = 5) and were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparison test for B – G . * P < 0.05. Bar = 5 mm in D and F ; Bar = 50 µm in E and G .

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Knockdown, Transfection, Transwell Assay, Comparison

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A 5mC DNA methylation was detected by ELISA assay. Data as mean ± SD ( n = 5), * P < 0.05 (Student’s t -test). B – D Representation and quantification of ALKBH5 expression at protein ( B , C ) and mRNA levels ( D ) in response to 5-aza treatment in A549 and H1792 cells for 72 h. Error bars, SD ( n = 4 for WB; n = 5 for qRT-PCR), * P < 0.05 as compared their corresponding controls (2-way ANOVA with Bonferroni multiple comparison post hoc test). E CTCF peak located in the CpGs Island of human ALKBH5 gene promoter. Bottom panel: the Methprimer histogram of CpG islands (CpGI, red box) and CpG dinucleotides (red vertical lines) in the regulatory region of ALKBH5 . F MeDIP assay showing the decrease of 5mC enrichment on the ALKBH5 promoter containing CTCF motif fragment by treated with 5-aza and LKB1 overexpression. G , H Representative bisulfite sequencing of four clones ( G ) and quantification of DNA methylation ( H ) of ALKBH5 promoter containing CTCF motif. n = 4 in F and H , mean ± SD, * P < 0.05 vs. A549 DMSO by one-way ANOVA followed by Tukey’s test in F and H . Si-CN, Si-RNA-A. OE-CN, OE-c-Flag pcDNA3. OE overexpression.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Comparison, Methylated DNA Immunoprecipitation, Over Expression, Methylation Sequencing, Clone Assay

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A – C Representative images and quantification of CTCF and ALKBH5 protein expression by WB and qRT-PCR in A549 cells with LKB1 overexpression and/or CTCF knockdown for 48 h. Error bars, SD ( n = 4 for WB; n = 5 for qRT-PCR), * P < 0.05 vs. Si-CN or OE-CN (one-way ANOVA with Bonferroni multiple comparison post hoc test). D Luciferase reporter assay of A549 cells transfected with pGL3-basic constructs containing serial LKBH5 promoters or deletion of CTCF peak fragment. n = 8/group, mean ± SD. * P < 0.05 by one-way ANOVA followed by Tukey’s test. E Reduction of ALKBH5 Luc:-1168 bp wild-type (WT) construct activities by treatment of 5-aza or LKB1 overexpression in A549 cells, but not for ALKBH5 Luc:-1168 bp deletion (Del). Data as mean ± SD ( n = 5), * P < 0.05 (Student’s t -test). F ChIP-qPCR showing that the ALKBH5 -CTCF peak region was occupied by the suppressor of CTCF and activators of histone modulators in A549 cells. G – J ChIP-qPCR analyses of 5-aza treated or LKB1 overexpressed A549 cells. n = 5/group, mean ± SD. * P < 0.05 vs. IgG in F , vs. A549-DMSO in G – J by one-way ANOVA followed by Tukey’s test.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Comparison, Luciferase, Reporter Assay, Transfection, Construct, ChIP-qPCR

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A , B Representative western blotting and quantification of SOX2, SMAD7, MYC, and ALKBH5 protein. Data as mean ± SD ( n = 4). C Mutation of m6A site or ALKBH5 overexpression released the SOX2, SMAD7 , and MYC gene posttranscriptional repression by LKB1 in A549 cells ( n = 4). D m6A-RIP analysis demonstrated that SOX2, SMAD7 , and MYC were subjected to ALKBH5-mediated m6A modifications ( n = 5). E YTHDF2-RIP-qPCR shown that YTHDF2 could occupy m6A sites of SOX2, SMAD7 , and MYC , which was mediated by ALKBH5 ( n = 4). F qRT-PCR was performed to indicate that knockdown of YTHDF2 significantly upregulated SOX2, SMAD7 , and MYC gene mRNA expression at basal level, LKB1 overexpressed or ALKBH5 silenced A549 cells ( n = 6). Data as mean ± SD. * P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for B – F . Si-CN, Si-RNA-A. OE-CN, OE-c-Flag pcDNA3. OE overexpression.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Western Blot, Mutagenesis, Over Expression, Quantitative RT-PCR, Knockdown, Expressing, Comparison

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: A Representative immunostaining images and quantification of 5mC DNA in lung cancer patients with KW and KM. Boxes and whiskers represent the 10th to 90th percentiles, respectively; the median is the central line in each box. Bar = 50 µm. B , C High levels of ALKBH5 DNA methylation and mRNA expression in cases of LKB1 loss compared with that of LKB1 positive expression in KM patients. D – F The correlation of ALKBH5 protein with the global m6A modification ( D ), m6A levels of SOX2, SMAD7 , and MYC genes ( E , F ) in KM and KW groups. G – I Negative correlations of the m6A enrichment with mRNA expressions of SOX2 , SMAD7 , and MYC in KM group. J Representative immunostaining images and quantification of SOX2 , SMAD7 , and MYC in KM group with LKB1 loss or not. K The positive correlations of ALKBH5 with SOX2, SMAD7 , and MYC protein expression in KM group. Data as mean ± SD. KRAS mutation (KM). KRAS wild-type (KW). * P < 0.05 by one-way ANOVA followed by Bonferroni multiple comparison post hoc test for A , B , and C . Student’s t- test for J .

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Immunostaining, DNA Methylation Assay, Expressing, Modification, Mutagenesis, Comparison

Journal: Cell Death & Disease

Article Title: Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

doi: 10.1038/s41419-021-03793-7

Figure Lengend Snippet: Loss of LKB1-induced DNA hypermethylation, which prevents CTCF binding on the ALKBH5 gene promoter, maintains ALKBH5 expression, and further represses global RNA methylation. Oncogenic SMAD7, SOX2 , and MYC are crucial targets of m6A meditated by LKB1 deficiency, and are involved in KRAS mutation lung cancer progression.

Article Snippet: We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 24 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.

Techniques: Binding Assay, Expressing, Methylation, Mutagenesis