Journal: Cell Death & Disease

Article Title: CBX8 exhibits oncogenic properties and serves as a prognostic factor in hepatocellular carcinoma

doi: 10.1038/s41419-018-1288-0

Figure Lengend Snippet: a Supervised hierarchical clustering of the differentially expressed genes after CBX8 silencing in SMMC-7721 cells. b BMP4 mRNA expression was measured by qRT-PCR and BMP4 protein expression was measured by western blotting in HCC cells with CBX8 overexpression or knockdown. c H3K4me3, H3K9me3, and H3k27me3 was measured by western blotting. d Presentation of five regions relative to the BMP4 transcriptional start site used as primers to test histone occupied abundance. e BMP4 is one of the genes that bind on CBX8 protein by ChIP-seq. f The relative methylation of the BMP4 promoter in HCC tissues. Representative results are shown between adjacent liver tissues and HCC tissues, and the comparison of the relative methylation of the BMP4 promoter between HCC tissues with or without metastasis. ** p < 0.01. CBX8 Chromobox homolog 8, BMP4 bone morphogenetic protein 4, mRNA messenger RNA, qRT-PCR quantitative reverse transcription-PCR, ChIP-seq chromatin immunoprecipitation-sequencing

Article Snippet: pBabe.puro retroviral constructs containing human CBX8 and BMP4 cDNAs and pSuper.retro.puro retroviral constructs containing short hairpin RNA (shRNA) against human CBX8 were prepared by GenePharma, Shanghai, China (shCBX8#1, 5′-GCAUGGAAUACCUCGUGAATT-3′; shCBX8#2, 5′-CGAGUUUCGAAGUGACUCATT-3′; shCBX8#3, 5′-CUUCGAAACAUGGGUUUGUTT-3′).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Knockdown, ChIP-sequencing, Methylation, Comparison, Reverse Transcription

Journal: Cell Death & Disease

Article Title: CBX8 exhibits oncogenic properties and serves as a prognostic factor in hepatocellular carcinoma

doi: 10.1038/s41419-018-1288-0

Figure Lengend Snippet: a Expression of (p-) Smad1/5/8 were measured by western blotting. b Expression of (p-) ERK and (p-) JNK were measured by western blotting. c – e Expression of BMP4, (p-) ERK, (p-) JNK, (p-) Smad1/5/8, E-cadherin, N-cadherin, Vimentin, Slug, and Snail were measured by western blotting. f BMP4 expression was positively correlated with CBX8 expression in HCC tissues. g Schematic illustration of the proposed function of CBX8 in HCC metastasis and stemness. CBX8 Chromobox homolog 8, BMP4 bone morphogenetic protein 4, EMT epithelial–mesenchymal transition, MAPK mitogen-activated protein kinase, p- phosphorylated, ERK extracellular signal-regulated kinase, JNK c-JUN N-terminal kinase, HCC hepatocellular carcinoma

Article Snippet: pBabe.puro retroviral constructs containing human CBX8 and BMP4 cDNAs and pSuper.retro.puro retroviral constructs containing short hairpin RNA (shRNA) against human CBX8 were prepared by GenePharma, Shanghai, China (shCBX8#1, 5′-GCAUGGAAUACCUCGUGAATT-3′; shCBX8#2, 5′-CGAGUUUCGAAGUGACUCATT-3′; shCBX8#3, 5′-CUUCGAAACAUGGGUUUGUTT-3′).

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet:

Article Snippet: L1210 cells (obtained from ATCC) were transfected using mammalian shRNA knockdown lentiviral vectors obtained from VectorBuilder Inc Each construct contained an shRNA sequence under a U6 promoter, as well as EGFP and Puro linked by T2A for selection.

Techniques: Generated, Transfection, Construct, shRNA, Isolation, Activity Assay, Software

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of characteristic genes in vivo (A) Relative expression levels of DDR-related genes in control and sepsis groups (n=4 in each group). (B) Relative expression levels of ARL4C in control, sepsis, sepsis+Ad-shNC, and sepsis+Ad-shARL4C group (n=8 in each group). (C) Expression levels of inflammatory factors (IL-1β, TNF-α, IL-10, and IL-18) in the peripheral blood of rat with sepsis+Ad-shNC and sepsis+Ad-shARL4C (n=8 in each group). (D) Survival status of rats in each group (n=10 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vivo, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of ARL4C in vitro . (A) Flow cytometry detected the apoptosis rate in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group) (B) Flow cytometry detected the ROS production in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group), *** p < 0.001, **** p < 0.001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vitro, Flow Cytometry, Control

Journal: Journal of Central South University Medical Sciences

Article Title: CYP2J2 - produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPAR γ- dependent manner

doi: 10.11817/j.issn.1672-7347.2021.210413

Figure Lengend Snippet: Sequences of primers used in this study

Article Snippet: Lentivirus vector transfection Lentivirus-constructed short hairpin RNA (shRNA) interference for CYP2J2: control-shRNA and CYP2J2 shRNA were purchased from GenePharma (Suzhou, China).

Techniques: Sequencing

Journal: Journal of Central South University Medical Sciences

Article Title: CYP2J2 - produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPAR γ- dependent manner

doi: 10.11817/j.issn.1672-7347.2021.210413

Figure Lengend Snippet: Information of antibodies used in this study

Article Snippet: Lentivirus vector transfection Lentivirus-constructed short hairpin RNA (shRNA) interference for CYP2J2: control-shRNA and CYP2J2 shRNA were purchased from GenePharma (Suzhou, China).

Techniques:

Journal: Journal of Central South University Medical Sciences

Article Title: CYP2J2 - produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPAR γ- dependent manner

doi: 10.11817/j.issn.1672-7347.2021.210413

Figure Lengend Snippet: A: Gene expressions of CYP2J2 in PDAC and para-carcinoma tissue were detected with real-time PCR. B-C: Protein expressions of CYP2J2 in PDAC and para-carcinoma tissue were detected with Western blotting. D: ELISA was used to evaluate the levels of 8,9-DHET in PDAC and para-carcinoma tissues. n=9, **P<0.01 and ***P<0.001.

Article Snippet: Lentivirus vector transfection Lentivirus-constructed short hairpin RNA (shRNA) interference for CYP2J2: control-shRNA and CYP2J2 shRNA were purchased from GenePharma (Suzhou, China).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Central South University Medical Sciences

Article Title: CYP2J2 - produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPAR γ- dependent manner

doi: 10.11817/j.issn.1672-7347.2021.210413

Figure Lengend Snippet: PANC-1 cells were infected with CYP2J2 shRNA or control shRNA mediated by lentivirus vector. A: Three days later, the CYP2J2 gene expression was detected with real-time PCR. B-C: Protein level of CYP2J2 was evaluated with Western blotting. D: Concentration of 8,9-DHET in PANC-1 cells was detected by ELISA. Then PANC-1 cells were treated with erastin (10 μmol/L) for 12 h. E: Cell viability was detected with CCK-8. F: Concentration of Fe2+ in PANC-1 cells was detected. G: MDA content in PANC-1 cells was detected. H: Gene expression of ACSL4 was detected with real-time PCR. I-J: Expression of ACSL4 protein was detected with Western blotting. K-L: Gene expression of FPN and FSP1 was detected by real-time PCR. Data are presented as the mean ± standard deviation, n=3, *P<0.05, **P<0.01, and ***P<0.001.

Article Snippet: Lentivirus vector transfection Lentivirus-constructed short hairpin RNA (shRNA) interference for CYP2J2: control-shRNA and CYP2J2 shRNA were purchased from GenePharma (Suzhou, China).

Techniques: Infection, shRNA, Control, Plasmid Preparation, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Expressing, Standard Deviation

Journal: Journal of Central South University Medical Sciences

Article Title: CYP2J2 - produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPAR γ- dependent manner

doi: 10.11817/j.issn.1672-7347.2021.210413

Figure Lengend Snippet: CYPs/EETs are highly expressed in PDAC and EETs inhibit the erastin-induced ferroptosis in a PPARγ-dependent manner in PANC-1 cells. ARA: Arachidonic acid; CYP2J2: Cytochrome P4502J2; EETs: Epoxyeicosatrienoic acids; PPARγ: Peroxisome proliferation-activated receptor γ; GPX4: Glutathione peroxidase; PDAC: Pancreatic ductal adenocarcinoma.

Article Snippet: Lentivirus vector transfection Lentivirus-constructed short hairpin RNA (shRNA) interference for CYP2J2: control-shRNA and CYP2J2 shRNA were purchased from GenePharma (Suzhou, China).

Techniques:

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, b, Centrosome localization of Piezo2 (a) and Piezo1 (b) in C2C12 myoblast cells visualized by IF for Piezo1/2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue) in mitotic and interphase cells. c, d, Centrosome localization of Piezo2 (d) and Piezo1 (c) in IMCD3 and Neuro-2A cells visualized by IF in interphase cells as in (a-b). e-f, STORM imaging performed for fixed unsynchronized C2C12 cells, stained with Piezo2 (green) and γ-Tubulin (magenta). The STORM image (e) showed co-localization of Piezo2 and γ-Tubulin, suggesting Piezo2 localized to the pericentrosomal region, and the insets (f) of the centrosomes are shown with x3 zoom-in. g, Rosa26 (off target control), Piezo1 and 2 CRISPR-Cas9 polyclonal KO (pKO) of C2C12 cells at day 1 post selection imaged by IF as in (a-b), showing supernumerary centrosomes in mitotic cells. h, Quantitative analysis of supernumerary centrosomes in interphase and mitotic C2C12 pKO cells from the IF experiments in (g). i, Quantitative analysis of mitotic cell populations from the IF experiments in . In 3 independent experiments, 190-280 cells were scored for each category based on the mitotic stage. Pro: prometaphase, Meta: metaphase, Ana: anaphase, Telo: telophase, Cyto: cytokinesis, scored as per Methods. j, Piezo2 -/- myoblasts derived from newborn mice imaged by IF as in (a-b), showing supernumerary centrosomes in Piezo2 -/- myoblasts. k, Quantitative analysis of supernumerary centrosomes in interphase and mitotic cells in WT and Piezo2 -/- myoblasts from IF experiments in (j). m, Cell cycle analysis by flow cytometry for WT and Piezo2 -/- myoblasts. All images are maximum intensity Z projections. Centrosomes and centrosome-localized Piezo proteins are marked with white arrowheads. Scale bars are 5 μm for (a-d), 1 μm (e), and 10 μm (g, j). For (h, k), data are represented by mean ± SEM from three independently quantified experiments counting 50-200 cells each. Statistical significance between an experimental group and a control group was assessed by 2-tailed t-test with ***, ** and * denote p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Imaging, Staining, Control, CRISPR, Selection, Derivative Assay, Cell Cycle Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, b, A field view of interphase C2C12 cells, showing Piezo2 (a) and Piezo 1 (b) localization at the centrosomes. Cells were visualized by IF for Piezo2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue). c, Controls for the fixation method, and primary and secondary antibodies in IF. Top: C2C12 cells were fixed in PFA (instead of methanol) and stained using primary antibodies rabbit anti-Piezo2 and mouse anti-γ-Tubulin, and secondary antibodies goat anti-rabbit Alexa Fluor 488 (green) and goat anti-mouse Alexa Fluor 647 (magenta), as well as Hoechst dye for DNA (blue). Middle: C2C12 cells were stained similarly but with a rabbit IgG replacing rabbit anti-Piezo2. Bottom: C2C12 cells were stained only with secondary antibodies. d, Quantitative analysis of the percentage of C2C12 cells with co-localized centrosome and Piezo1 or 2 from IF images as in (a) and (b). e, f, Centrosome localization of Piezo1 (e) and Piezo2 (f) in IMCD3 cells visualized by IF at different cell cycle stages, imaged by IF as in (a) and (b). g, Snapshots from live imaging of C2C12 cells stably expressing Piezo1-GFP during the cell cycle (Supplementary Video 1). Centrosomes are marked with green arrowheads. h , Co-localization of Piezo1-GFP fluorescence (green) with anti-Piezo1 IF (magenta). Cells were also stained for DNA (Hoechst, blue). i, Localization of Piezo1-GFP fluorescence (green) at the γ-Tubulin (magenta)-marked centrosome by IF. All IF mages are maximum intensity Z projections. Centrosomes and centrosome-localized Piezo1 are marked with white arrowheads. All scale bars are 10 μm, except that the scale bar for (g) is 20 μm.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Staining, Imaging, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Centrosomal localization of Piezo1 ( left) and Piezo2 ( right ) in E14.5 WT mouse forelimb sections, imaged by IF for Piezo1/2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue). b, A thoracic dorsal root ganglion (DRG) section of a WT mouse at E17.5, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). Right panel shows a 3x zoom-in of the dotted region in the left panel. Piezo2 puncta consistent with a centrosomal localization are labeled with white arrowheads. Of note, Piezo2 positive puncta are often present in DRG neurons whether or not the cell bodies are also Piezo2 positive. c, A thoracic DRG section of a Piezo2 -/- KO mouse at E17.5, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). Piezo2 expression at DRG neuronal cell body or centrosome is largely undetectable . d, A lung bronchial section of a WT mouse at P0, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). The cells labeled in green represent scattered pulmonary neuroepithelial cell bodies (NEBs). e, A comparable lung bronchial section of a Piezo2 -/- KO mouse at P0, imaged by IF with anti-Piezo2 antibody and Hoechst dye (blue), showing absence of Piezo2 staining. f, A comparable lung bronchial section of a WT mouse at P0, stained with rabbit IgG (green) as primary antibody and Hoechst dye (blue). g, h, Thoracic DRG sections of WT mice (g) and Piezo2 -/- KO mice, shown at 2 different contrast levels (h) at E17.5, stained with anti-Piezo2 antibody (green), γ-Tubulin (magenta) and Hoechst dye (blue). White arrowheads identify γ-Tubulin-marked centrosomes (magenta) and/or Piezo2. All scale bars are 10 μm.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Staining, Labeling, Expressing

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: iSIM was performed for fixed unsynchronized C2C12 cells, stained with Piezo2 (green) and γ-Tubulin (magenta). The iSIM image (left) shows co-localization of Piezo2 and γ-Tubulin, and the insets of the centrosomes (right) are shown with x4 zoom-in.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Staining

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Piezo1 and Piezo2 protein levels in Piezo1 pKO, Piezo2 pKO, or Rosa26 pKO (control) of C2C12 cells analyzed by Western blots with GAPDH as the loading control. Densitometric analysis of Western blot bands of Piezo1 or 2 are shown. b, Piezo1 pKO ( left ) and Piezo2 pKO ( right ) interphase C2C12 cells at day 1 post-pKO selection, imaged by IF for Piezo1 or Piezo2 (green), γ-Tubulin (magenta) and DNA (Hoechst, blue). Centrosomes are marked with white arrows, and supernumerary centrosomes are seen in Piezo pKO cells, in comparison with Rosa26 pKO control. c, Piezo1 pKO ( middle row ), Piezo2 pKO ( bottom row ) and Rosa26 pKO ( top row ) of mitotic C2C12 cells at day 1 post-pKO selection, imaged by IF for α-Tubulin (green), γ-Tubulin (magenta) and DNA (Hoechst, blue). Centrosomes and lagging chromatin are marked with white arrows, and misaligned microtubules are seen in Piezo pKO cells, in comparison with the Rosa26 pKO control d, Quantitative real-time RT-PCR (qRT-PCR) of Piezo 1 or 2 transcripts upon small hairpin RNA (shRNA) KD of Piezo1 or Piezo2 in C2C12 cells using two and three different shRNAs for Piezo1 and Piezo2, respectively, in comparison with an off target shRNA against firefly luciferase mRNA as the control. e, Control KD, Piezo1 shRNA KD (one shRNA) or Piezo2 shRNA KD (two shRNAs) C2C12 cells at day 4 post-KD selection. The cells were imaged by IF for γ-Tubulin (red) and DNA (Hoechst, blue), showing supernumerary centrosomes upon Piezo1 or Piezo2 KD. f, Quantitative analysis of C2C12 cells with supernumerary centrosomes upon Piezo1 shRNA KD (one shRNA) or Piezo2 shRNA KD (two shRNAs) at day 4 post-KD selection. All scale bars are 10 μm. g, Off target, Piezo1 and 2 pKO C2C12 cells at day 1 post selection and 45 min after release of G2/M border synchronization with RO-3066. Cells were imaged by IF for α-tubulin (green), γ-tubulin (magenta) and DNA (Hoechst dye, blue), shown at prometaphase and cytokinesis. Quantitative analysis of mitotic cell populations from the IF experiments shown in . Statistical significance between an experimental group and the control group was assessed by 2-tailed t-test. ***, ** and * denote p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Control, Western Blot, Selection, Comparison, Quantitative RT-PCR, shRNA, Luciferase

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Validation of the NLS-GCaMP6 reporter stably expressed in C2C12 cells by ionomycin (1 μM), BAPTA-AM (10 μM), or Yoda1 (10 μM) treatment. b , Quantitative analysis of maximal signals for cells imaged in (a). AFU denotes arbitrary fluorescence units. c, Phase and fluorescence snapshots of C2C12 cells expressing GCaMP6 reporter at interphase and different stages of mitosis (green). Concentrated Ca 2+ signals that represent centrosome locations are marked by white arrowheads. d , Phase and fluorescence snapshots of C2C12 cells at interphase and different stages of mitosis, imaged live at 45 min after treatment with the Ca 2+ dye Fluo4-AM (9 μM, green). Concentrated Ca 2+ signals that represent centrosome locations are marked by white arrowheads. e, Quantitative analysis of maximal GCaMP6 signals at centrosomes as shown in , revealing reductions to 43% and 34% in comparison to Rosa26 pKO control values in Piezo1 and 2 pKO cells, respectively. f, GCaMP6 expression by anti-GFP IF (green) in NLS-GCaMP6 expressing C2C12 cells at day 1 post-KO selection of Rosa26 pKO, Piezo1 pKO or Piezo2 pKO. IF for γ-Tubulin (magenta) and Hoechst dye staining of DNA (blue) are also shown, exhibiting equal GCaMP6 expression regardless of Piezo1 or 2 pKO. g , Quantitative analysis of maximal Ca 2+ signal indicated by GCaMP6 fluorescence intensity at centrosomes and in the cytosol as shown in , showing that there was a similar increase or decrease of local Ca 2+ concentration upon Yoda1 activation or GsMTx4 inhibition relative to the untreated control value both in the cytosol and at the centrosomes. Images are maximum intensity Z projections and all scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments. Statistical significance between an experimental group and the control was assessed by 2-tailed t-test with *** and ** for p < 0.0001 and 0.001, respectively.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Biomarker Discovery, Stable Transfection, Fluorescence, Expressing, Comparison, Control, Selection, Staining, Concentration Assay, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, HNMR Spectra of caged-Yoda1 and hydroxy-Yoda1 synthesis. The compounds were synthesized by WuXi AppTec with purity determined by LC-MS of 98.2% and 78.6%, respectively. For hydroxy-Yoda1, the compound was clean based on the HNMR spectrum but might not be stable in LC-MS and therefore a lower purity was reported. b, A table describing the top hits from the yeast two-hybrid screen performed for Piezo2 CTD.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Synthesized, Liquid Chromatography with Mass Spectroscopy, Two Hybrid Screening

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Validation of the Snx5 interaction with the Piezo2 C-terminal domain (CTD, last 70 aa) using an alternative yeast two-hybrid assay. The Piezo2 CTD was fused to the LexA DNA binding domain and full-length Snx5 was fused to a B42 activation domain. Positivity is indicated by yeast turning dark blue in the presence of the LacZ reporter X-gal (+++) and by yeast growth in the absence of leucine. Negativity is indicated by yeast staying white (-) and no growth in the absence of leucine. Phox homology (PX) domain for lipid binding and Bin/Amphiphysin/Rvs (BAR) domain for membrane-curvature sensing are shown. b, Western blot analysis of Ni-NTA pulldown of human Snx5 by human Piezo2 CTD. Piezo2-CTD-His-Myc was co-expressed with GFP-Snx5 or GFP control in HEK293T cells. The His-tagged protein was precipitated using Ni-NTA beads (lane 1&2) or as a control with NTA beads without Ni (lane 3). Lanes 4 and 5 are cell lysates. Precipitated Piezo2-His-Myc was detected with anti-Myc antibody and GFP-hSnx5 was detected with anti-GFP antibody. c, IF images of C2C12 cells (top panel) and Neuro-2A cells (bottom panel) stained for Piezo2 (green), Snx5 (olive), ɣ-Tubulin (magenta) and DNA (blue). Co-localization among Piezo2, Snx5 and ɣ-Tubulin is shown by arrowheads and all scale bars are 10 μm. d, Western blot analysis of fractionation of isolated centrosomes. Centrosomes isolated from C2C12 cells were fractionated on a sucrose gradient, and blotted with the following antibodies: anti-Piezo1, anti-Piezo2, anti-γ-Tubulin, anti-Pericentrin and anti-Snx5.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Biomarker Discovery, Y2H Assay, Binding Assay, Activation Assay, Membrane, Western Blot, Control, Staining, Fractionation, Isolation

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a-b, IF of C2C12 cells, fixed after 3h cold treatment, 2 h treatment with Parthenolide or Taxol and stained with Piezo1 (a) or Piezo2 (b), ⍺-Tubulin (green) and Hoechst (Blue). Untreated cells were used as a control. Centrosomes are marked with white arrowheads. c , Western blots of C2C12 cells at different cell cycle stages using anti-Piezo1, Piezo2, PLK1 and GAPDH (loading control) antibodies. While Piezo2 is highly expressed at S and G2, Piezo1 expression is relatively constant. d , Cell cycle analysis by PI staining for C2C12 cells treated with PLK1 inhibitor BI-6727. Optimal concentration for BI-6727 treatment in C2C12 was determined as 100 nM. Images are maximum intensity Z projections and all scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments. Statistical significance between an experimental group and the control was assessed by 2-tailed t-test with ***, ** and * for p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: shRNA MISSION TRC-Mm 2.0 (Mouse) RNA interference lentiviruses targeting mouse Piezo2 were obtained through the Broad Institute.

Techniques: Staining, Control, Western Blot, Expressing, Cell Cycle Assay, Concentration Assay

Journal: PLoS Pathogens

Article Title: New insights into the role of endosomal proteins for African swine fever virus infection

doi: 10.1371/journal.ppat.1009784

Figure Lengend Snippet: (A) GFP was immunoprecipitated in lysates from HEK293T cells transfected with EGFP, EGFP E248R or EGFP E199L. Representative immunoblot analysis of cell lysates (I) and GFP-immunoprecipitates (IP) using GFP, NPC1, NPC2, Lamp1, Lamp2 and PIKfyve antibodies. GAPDH was used as control. (n = 4 independent experiments, and Figs). (B) Schematic representation of NPC1 WT and specific recombinant constructions of individual domains shown in C and D. (C, D) HA was immunoprecipitated in lysates from HEK293T cells co-transfected with HA E248R (C) or HA E199L (D) with NPC1 Flag and FLAG NPC1 domains. Representative immunoblot analysis of total lysates (I) and HA-immunoprecipitates (IP) using Flag and HA antibody. Alpha-tubulin (C) or HSP90 (D) were used as control. “NPC1 dom.” refers to individual domains of NPC1 (n = 3 independent experiments, and Figs).

Article Snippet: Lentiviral vectors containing shRNAs to interfere Npc1 (shNPC1) and Npc2 (shNPC2) were purchased from Merk (Darmstadt, Germany).

Techniques: Immunoprecipitation, Transfection, Western Blot, Control, Recombinant

Journal: PLoS Pathogens

Article Title: New insights into the role of endosomal proteins for African swine fever virus infection

doi: 10.1371/journal.ppat.1009784

Figure Lengend Snippet: (A) NPC1, NPC2 and tubulin (loading control) were detected by western blot in Vero cells transduced with SCR (scramble) and shNPC2 (34 and 36) lentiviral particles. Quantification of NPC1 and NPC2 bands were corrected to tubulin data and then normalized to SCR values. (B) Percentage of B54GFP ASFV infected cells (1 pfu/ml) at 16 hpi in Vero cells shown in (A). Percentages were normalized to values in SCR cells. (C) Quantification of ASFV replication in SCR and shNPC2-36 transduced Vero cells by qPCR. Graphs represent mean±sem from three independent experiments. Statistically significant differences are indicated by asterisks (***p < 0.001, **p < 0.01, *p < 0.05).

Article Snippet: Lentiviral vectors containing shRNAs to interfere Npc1 (shNPC1) and Npc2 (shNPC2) were purchased from Merk (Darmstadt, Germany).

Techniques: Control, Western Blot, Transduction, Infection