Journal: Translational Cancer Research

Article Title: Propofol regulates the progression of hepatocellular carcinoma via the POLR2L/TGF-β signaling pathway

doi: 10.21037/tcr-23-2066

Figure Lengend Snippet: Effects of POLR2L on proliferation, invasion and migration of HCC cell lines. (A) The expression of POLR2L in HCC cell lines and normal cells was detected by qRT-PCR and WB assays. (B,C) Detection of POLR2L knockdown efficiency of siRNA in SNU-387 and MHCC-97H cells. (D,E) The proliferation ability of SNU-387 and MHCC-97H cells after POLR2L knockdown was detected by CCK-8 methods. (F-I) Transwell assay was used to detect the invasion and migration ability of SNU-387 and MHCC-97H cells after POLR2L knockdown. Magnification ×50, staining with DAPI. *, P<0.05; **, P<0.01. NC, negative control; OD, optical density; HCC, hepatocellular carcinoma; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; WB, Western blotting; siRNA, small interfering RNA; CCK-8, cell counting kit-8; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: GeneChem Co., Ltd. (Shanghai, China) designed and produced the small interfering RNA (siRNA) targeting POLR2L (si-POLR2L #1 and si-POLR2L #2) and a nontargeting control siRNA (si-NC).

Techniques: Migration, Expressing, Quantitative RT-PCR, Knockdown, CCK-8 Assay, Transwell Assay, Staining, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Cell Counting

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of the levels of ncRNAs in different models of HD.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Transfection

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of protein and miRNA interactions of ncRNAs Meg3, Neat1, and Xist.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Expression of genes in HD associated with Huntington’s disease pathway (KEGG: 05016 and PANTHER: {"type":"entrez-protein","attrs":{"text":"P00029","term_id":"124076962","term_text":"P00029"}} P00029 ) and coded for protein interacting partners of NEAT1 or MEG3.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Expressing

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of co-expressed genes of MEG3, NEAT1, and XIST.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: MEG3, NEAT1 and XIST interacting protein enriched with HD pathway.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Transcription factors that bind within 5 Kb upstream sequences of NEAT1, MEG3, and XIST.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Sequencing

Journal: Kidney international

Article Title: TLR9 activation induces aberrant IgA glycosylation via APRIL- and IL-6-mediated pathways in IgA nephropathy

doi: 10.1016/j.kint.2019.08.022

Figure Lengend Snippet: (A) CpG-ODN supplementation enhanced production of IL-6. CpG-ODN enhanced gene expression of APRIL. Western-blot analysis demonstrated that CpG-ODN increased protein levels of APRIL. Results were evaluated densitometrically. Stimulation with CpG-ODN induced production of IgA and Gd-IgA1. Bars represent the mean±SEM. *P<0.05, **P<0.01. (B) APRIL siRNA knock-down reduced IL-6-induced overproduction of IgA and Gd-IgA1. *P<0.05, **P<0.01. (C) IgA1-producing cells stimulated with recombinant APRIL produced more IgA and Gd-IgA1. (D) IgA1-secreting cells were incubated with anti-IL-6 antibody after CpG-ODN stimulation. The enhanced production of IgA and Gd-IgA1 induced by CpG-ODN was reduced by anti-IL-6 antibody and by siRNA knock-down of APRIL. PBS, Phosphate Buffered Saline.

Article Snippet: Human IgA1-secreting cells were cultured in 24-well culture plates and transfected with APRIL -specific siRNA (GS8741, Qiagen) or siRNA non-targeting control (GS10673, Qiagen) using the transfection protocol according to the manufacturer’s instructions.

Techniques: Gene Expression, Western Blot, Knockdown, Recombinant, Produced, Incubation, Saline

Journal: The Journal of Biological Chemistry

Article Title: CYYR1 promotes the degradation of the E3 ubiquitin ligase WWP1 and is associated with favorable prognosis in breast cancer

doi: 10.1016/j.jbc.2024.107601

Figure Lengend Snippet: CYYR1 is ubiquitinated by WWP1 and WWP2 at lysine K154. A and B , CYYR1 is ubiquitinated by WWP1 and WWP2 at lysine K154. HEK293 cells were transfected with HA-CYYR1-WT or HA-CYYR1-K154R either alone or with His-Ubiquitin (His-Ub) and Flag-tagged WWP1 or WWP2 (WT) or their catalytically inactive mutants (CA) and treated with MG132 for 4 h before lysis. Cell lysates were pulled-down with the ubiquitin pan Selector affinity resin and analyzed by Western blotting with the indicated antibodies. Western blotting on TCL is shown as a transfection control. C , CYYR1-K154R binds to WWP1 and WWP2. Cell lysates from HEK293 cells transfected with GFP-CYYR1 or GFP-CYYR1-K154R were immunoprecipitated with the GFP-trap affinity resin and analyzed by Western blotting as indicated. D , MDA-MB-468 cells were transfected with a nontargeted siRNA control (siNT) or two independent siRNA (#1 and #2) targeting WWP1 or WWP2. Seventy-two hours posttransfection, cell lysates were analyzed by Western blotting. Quantifications of the CYYR1 intensity relative to GAPDH intensity in each condition normalized to the siNT control condition and p -values were calculated using one-way ANOVA followed by Dunnett’s test (n = 3). ∗ on anti-WWP2 Western blot indicates nonspecific band. CYYR1, cysteine and tyrosine-rich protein 1; HA, hemagglutinin; TCL, total cell lysate; WWP1/2, WW domain-containing protein 1/2.

Article Snippet: SiRNA targeting CYYR1 (#1: SI04152981, #2: SI04283825), WWP1 (#1: SI04287689, #2: SI04293030), WWP2 (#1: SI03095344, #2: SI04952626), and ANKRD13A (#1: SI04139800, #2: SI04320218), and the nontargeting siRNA control siNT (1027281) were purchased from Qiagen.

Techniques: Transfection, Ubiquitin Proteomics, Lysis, Western Blot, Control, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: CYYR1 promotes the degradation of the E3 ubiquitin ligase WWP1 and is associated with favorable prognosis in breast cancer

doi: 10.1016/j.jbc.2024.107601

Figure Lengend Snippet: CYYR1 regulates WWP1 autoubiquitination and protein level. A , CYYR1 increases WWP1 autoubiquitination. HEK293 cells transfected with His-Ub and Flag-WWP1-WT or Flag-WWP1-CA either alone or with different HA-CYYR1 constructs as indicated. Protein cell lysates were pulled-down with the ubiquitin pan Selector affinity resin and analyzed by Western blotting. B , CYYR1 decreases WWP1 protein level. Cell lysates form HEK293 cells transfected with Flag-WWP1-WT or Flag-WWP1-CA either alone or with different constructs for HA-CYYR1 were analyzed by Western blotting. C , CYYR1 depletion increases WWP1 protein level. Cell lysates from MDA-MB-468 cells transfected with a nontargeting siRNA control (siNT) or two independent siRNA (#1 or #2) targeting CYYR1 were analyzed by Western blotting using the indicated antibodies. Quantifications of the WWP1 intensity relative to GAPDH in each condition were normalized to the siNT control condition, and p -values were calculated with a one-way ANOVA followed by Dunnett’s test (n = 5). D , CYYR1 decreases endogenous WWP1 protein level. MDA-MB-231 cells expressing doxycycline (Dox)-inducible CYYR1-WT or CYYR1-ΔPPxY (TO-CYYR1-WT or TO-CYYR1-ΔPPxY clones #1 and #2) were treated with Dox for 24 h before analysis of the cell lysates by Western blotting. Low and high exposure of the anti-CYYR1 Western blot are shown to highlight CYYR1 expression leakiness in absence of Dox. Quantifications of the WWP1 intensity relative to GAPDH in each condition were normalized to the Ctrl condition and p -values were calculated with one-way ANOVA followed by Dunnett’s test (n = 3). CYYR1, cysteine and tyrosine-rich protein 1; Dox, doxycycline; WWP1, WW domain-containing protein 1.

Article Snippet: SiRNA targeting CYYR1 (#1: SI04152981, #2: SI04283825), WWP1 (#1: SI04287689, #2: SI04293030), WWP2 (#1: SI03095344, #2: SI04952626), and ANKRD13A (#1: SI04139800, #2: SI04320218), and the nontargeting siRNA control siNT (1027281) were purchased from Qiagen.

Techniques: Transfection, Construct, Ubiquitin Proteomics, Western Blot, Control, Expressing, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: CYYR1 promotes the degradation of the E3 ubiquitin ligase WWP1 and is associated with favorable prognosis in breast cancer

doi: 10.1016/j.jbc.2024.107601

Figure Lengend Snippet: ANKRD13A is involved in CYYR1-induced WWP1 degradation. A , CYYR1-interactome. Cell lysates from HEK293 cells transfected with GFP or GFP-CYYR1 were purified on GFP-Trap affinity resin and analyzed by quantitative label-free mass spectrometry. Volcano plot showing differentially expressed proteins significantly enriched in GFP-CYYR1 compared to GFP ( green line correspond to fold change ≥ 2, and red line to p -value ≤ 0.05, n = 5) that display at least three peptides in each of the five replicate experiments. Proteins that display no common peptides in the GFP condition (infinite ratio) are represented on the left of the graph with their distribution based on the number of peptides identified/100aa. B , CYYR1 interacts with ANKRD13A. Cell lysates from HEK293 cells transfected with GFP or GFP-CYYR1 were purified with GFP-Trap affinity resin and analyzed by Western blotting with the indicated antibodies. C , WWP1 interacts with ANKRD13A in the presence of CYYR1. HEK293 cells were transfected with Flag-WWP1 or Flag-WWP1-CA and HA-CYYR1-WT or HA-CYYR1-ΔPPxY with or without Flag-ANKRD13A. Cell lysates were immunoprecipitated with anti-ANKRD13A antibody and immunoblotted with the indicated antibodies. D , schematic representation of the ANKRD13A constructs. AR and UIM domains are shown. Lower panel: ANKRD13A binds WWP1 through its UIM domains. HEK293 cells were transfected with HA-CYYR1, Flag-ANKRD13A-WT, or deletion mutants and GFP or GFP-WWP1-WT as indicated. Cell lysates were immunoprecipitated with the GFP-trap affinity resin and immunoblotted with the corresponding antibodies. E , schematic representation of the ANKRD13A, B , C , and D . Lower panel : WWP1 binds all members of the ANKRD13 family except ANKRD13C. Cell lysates from HEK293 cells were transfected with HA-CYYR1 and Flag-tagged ANKRD13 family proteins, and GFP or GFP-WWP1-WT were immunoprecipitated with the GFP-trap affinity resin and analyzed by Western blotting with the indicated antibodies. F , ANKRD13A depletion attenuates the degradation of WWP1 induced by CYYR1. MDA-MB-231 TO-CYYR1-WT clone #1 were transfected with control siRNA (siNT) or two independent siRNA targeting ANKRD13A. Seventy-two hours post transfection, cells were treated with 500 pg/ml Dox for 24 h before lysis and Western blotting analysis with the indicated antibodies. Quantifications of the WWP1 intensity relative to GAPDH in each + Dox condition were normalized to the -Dox condition and p -values were calculated by performing a paired t test, ∗ p < 0.05 (n = 3). ANKRD13A, ANKyrin repeat domain-containing protein 13 A; AR, ankyrin-repeat; CYYR1, cysteine and tyrosine-rich protein 1; Dox, doxycycline; UIM, ubiquitin-interacting motif; WWP1, WW domain-containing protein 1.

Article Snippet: SiRNA targeting CYYR1 (#1: SI04152981, #2: SI04283825), WWP1 (#1: SI04287689, #2: SI04293030), WWP2 (#1: SI03095344, #2: SI04952626), and ANKRD13A (#1: SI04139800, #2: SI04320218), and the nontargeting siRNA control siNT (1027281) were purchased from Qiagen.

Techniques: Transfection, Purification, Mass Spectrometry, Western Blot, Immunoprecipitation, Construct, Control, Lysis, Ubiquitin Proteomics