Journal: bioRxiv

Article Title: Argonaute Binding within 3’-Untranslated Regions Does Not Predict Gene Repression

doi: 10.1101/2020.02.25.956623

Figure Lengend Snippet: miRNA expression profile in cancel cells based on Cancer Cell Line Encyclopedia (CCLE). ( A ) Relative expression or miRNAs cancer cell lines. ( B ) Relative expression of the most highly expressed miRNAs in HCT116 cells and other cell lines. For ( A ) and ( B ), The top lines represent expression of miRNAs in HCT116 cells and show similar trends for miRNA expression from higher expressed to less highly expressed. miRNAs ordered from HCT116 mostly to lowest expressed. Tissue color bar order from top to bottom (LARGE INTESTINE, AUTONOMIC GANGLIA, BILIARY TRACT, BONE, BREAST, CNS, ENDOMETRIUM, FIBROBLAST, HAEMATOPOIETIC AND LYMPHOID, KIDNEY, LIVER, LUNG, OESOPHAGUS, OVARY, PANCREAS, PLEURA, PROSTATE, SALIVARY GLAND, SKIN, SMALL, SOFT TISSUE, STOMACH, THYROID, UPPER AERODIGESTIVE TRACT, URINARY TRACT).

Article Snippet: Distribution of AGO2 binding clusters For eCLIP, we isolated RNA from the cytoplasmic fraction of HCT116 cells ( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: Argonaute Binding within 3’-Untranslated Regions Does Not Predict Gene Repression

doi: 10.1101/2020.02.25.956623

Figure Lengend Snippet: Comparison of miRNAs in HCT116 cells from eCLIP and RIP. ( A ) miRNA abundance in cytoplasm. ( C ) Top 25 most prevalent miRNAs in cytoplasm detected by AGO2-eCLIP-seq compared to detection of those miRNAs identified by Nanostring sequencing of HCT116 cells in published CCLE encyclopedia ( Ghandi et al. 2019 ).

Article Snippet: Distribution of AGO2 binding clusters For eCLIP, we isolated RNA from the cytoplasmic fraction of HCT116 cells ( ).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Argonaute Binding within 3’-Untranslated Regions Does Not Predict Gene Repression

doi: 10.1101/2020.02.25.956623

Figure Lengend Snippet: Characterization of CRISPR-derived HCT116AGO1/2/3 knockout cell lines. ( A-C ) Location of guide RNA and characterization of mutations for knocking out AGO1, AGO2, and AGO3. ( D ) Western blot validating the AGO1, AGO2, AGO1/2, and AGO1/2/3 knockout cell lines (n=3). (E ) HCT116 WT, AGO1, AGO2, AGO1/2, and AGO1/2/3 knockout cell lines were seeded at the same density, harvested, and analyzed for live cell count (n=3). ( F ) Scheme showing knockout of AGO variants, RNAseq analysis of gene expression changes relative to wild-type cells; and CLIP-sequencing with anti-AGO2 antibody to identify potential miRNA-AGO2 binding sites. See also Figure S1 .

Article Snippet: Distribution of AGO2 binding clusters For eCLIP, we isolated RNA from the cytoplasmic fraction of HCT116 cells ( ).

Techniques: CRISPR, Derivative Assay, Knock-Out, Western Blot, Cell Counting, Expressing, Cross-linking Immunoprecipitation, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Argonaute Binding within 3’-Untranslated Regions Does Not Predict Gene Repression

doi: 10.1101/2020.02.25.956623

Figure Lengend Snippet: Prediction of miRNAs targeting to 3’-UTR regions of studied genes. Red outline – labeled top 25 expressed miRNAs in HCT116 cells.

Article Snippet: Distribution of AGO2 binding clusters For eCLIP, we isolated RNA from the cytoplasmic fraction of HCT116 cells ( ).

Techniques: Labeling

Journal: Scientific Reports

Article Title: Acidification of Cytoplasm in Escherichia coli Provides a Strategy to Cope with Stress and Facilitates Development of Antibiotic Resistance

doi: 10.1038/s41598-020-66890-1

Figure Lengend Snippet: Measurement of the cytoplasmic pH (pH i ) of the WT and EV18 strains. ( A ) Development of the method for determination of the intracellular pH in E. coli by using the ratiometric pH sensor pHluorin. (1) E. coli cells transformed with the pGFPR01 plasmid grown in LB-K medium for 8 h (2 and 3) were diluted in MMA + glycerol as a carbon source and 0.2% of arabinose and incubated ON at 25 °C. (4) After spinning down and resuspension in 1 mL of MMA they were (5) transferred to a 96-well microtiter plate (OD 600 = 0.1). (6) After 1 h with constant shaking (37 °C) fluorescence was assessed (excitation at 410 and 470 nm with an emission at 510 nm). (7) pH i values were obtained by calculating the fluorescence ratio 410/470 nm and interpolating this value in the calibration curve. ( B ) Fluorescence spectra of the WT strain (380–480 nm, emission: 510 nm) generated after incubating the strain for 1 h in MMA + glycerol at different pHs (5.92–8.20) in the presence of 40 mM benzoic acid and 40 mM methylamine hydrochloride (collapsing compounds). ( C ) Calibration curve for the measurement of pHi of the BW25113 E. coli strain. WT + pGFPR01 strain was incubated in a plate reader for 1 h (37 °C, constant shaking) in the presence of MMA + glycerol prepared at different pHs and collapsing compounds. A Boltzmann sigmoid best-fit curve was applied to the extracted ratio data (410/470 nm) by using the GraphPad Prism 7 software. ( D ) Comparison of the cytoplasmic pH of the WT + pGFPR01 and EV18 + pGFPR01 strains incubated at different external pHs. Strains were incubated for 1 h (37 °C, constant shaking) in MMA + glycerol at the indicated pH. All the experiments were performed in triplicates. The error bars are not shown when smaller than the symbol.

Article Snippet: A Boltzmann sigmoid best-fit curve was generated by the GraphPad Prism 7 software and used for the calculation of the cytoplasmic pH.

Techniques: Transformation Assay, Plasmid Preparation, Incubation, Fluorescence, Generated, Software

Journal: Nature Communications

Article Title: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development

doi: 10.1038/s41467-018-04167-y

Figure Lengend Snippet: mTOR pathway repression is required for competition in vivo. a Chimeras generated by injecting 4n and 4n- Tsc2 −/− cells into wild-type blastocysts. Levels of pS6 were analysed at 5.0 dpc and 6.5 dpc, respectively. b Chimeras generated by injecting 4n ( n = 12) and 4n- Tsc2 −/− ( n = 4) cells into wild-type blastocysts. The white arrow indicates epiblast overgrowth of 4n- Tsc2 –/– cells in these chimeras. Levels of cell death were assessed by cleaved caspase-3 staining at 6.5 dpc and quantified ( c ). d Chimeras generated by injecting 4n-Cas9-GFP and 4n- Tsc2 −/− -GFP cells into wild-type blastocysts. Embryos were analysed for GFP expression to determine contribution of 4n cells. e 4n- Tsc2 −/− GFP cells were injected into wild-type blastocysts, embryos were collected at 9 dpc, and levels of 4n Tsc2 −/− GFP contribution were assessed by whole-mount immunofluorescence or by sectioning. Morphology of chimera is compared to wild-type control embryo. Scale bars are 50 μm for a , b and 100 μm for d , e . Error bars denote SEM ( c ). ** p

Article Snippet: Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions.

Techniques: In Vivo, Generated, Staining, Expressing, Injection, Immunofluorescence

Journal: Nature Communications

Article Title: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development

doi: 10.1038/s41467-018-04167-y

Figure Lengend Snippet: Repression of the mTOR pathway triggers cell death in differentiating ESCs. a Schematic of experimental setup whereby cells were cultured separately and together for two days in N2B27 before being treated with 100 μM caspase inhibitors for 48 h. Cells were then fixed and levels of pS6 S240/244 were assessed. b Levels of pS6 S240/244 were assessed by flow cytometry in wild-type and Bmpr1a −/− cells following 48 h treatment with DMSO or caspase inhibitors (100 uM), and c median fluorescence of Bmpr1a −/− cells was quantified. d Wild-type cells were cultured in N2B27 for 2 days, treated with mTOR inhibitor rapamycin for 24 h and cell count was assessed following treatment with and without caspase inhibitors. e Wild-type cells cultured in ESC media, to maintain pluripotency, and N2B27, to initiate differentiation, were treated with rapamycin for 6 h and levels of cell death were assessed by western blot analysis of cl. caspase-3 and cl. PARP. f Quantification of cleaved caspase-3 levels relative to β-actin. Error bars denote SEM. * p

Article Snippet: Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions.

Techniques: Cell Culture, Flow Cytometry, Cytometry, Fluorescence, Cell Counting, Western Blot

Journal: Nature Communications

Article Title: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development

doi: 10.1038/s41467-018-04167-y

Figure Lengend Snippet: Constitutive activation of the mTOR pathway in defective cells rescues their elimination during competition. a Fold change in Bmpr1a −/− and Bmpr1a −/− ; Tsc2 −/− (two independent clones) cell numbers between d0–d4 and percent of cleaved caspase-3 positive cells at day 4 cells when cultured alone (separate) or with wild-type cells in N2B27. b Fold change in wild-type cell numbers between d0-d4 when cultured separately or co-cultured with Bmpr1a −/− or Bmpr1a −/− ; Tsc2 −/− cells. c Fold change in 4n and 4n; Tsc2 −/− (two separate independent clones) cell numbers between d0–d4 and percent of cleaved of caspase-3 positive cells at day 4 when cultured separately or with wild-type cells in N2B27. d Fold change in wild-type cell numbers between d0–d4 when cultured separately or co-cultured with 4n or 4n; Tsc2 −/− cells. e Fold change in wild-type cell numbers between d0–d4 and percent of cleaved caspase-3 positive cells at day 4 when cultured separately or co-cultured with Cas9 or Tsc2 −/− cells (two independent clones) in N2B27. f Fold change in Tsc2 −/− cell numbers between d0–d5 when cultured separately or co-cultured with wild-type cells. n = 3 for all studies. Error bars denote SEM. * p

Article Snippet: Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions.

Techniques: Activation Assay, Cell Culture

Journal: Oncotarget

Article Title: Erbin exerts a protective effect against inflammatory bowel disease by suppressing autophagic cell death

doi: 10.18632/oncotarget.23925

Figure Lengend Snippet: Expressions of Erbin, LC3, ATG16L1, ATG2A and ATG7 in DSS-induced experimental colitis mice and IL-10 knockout mice (IL-10 -/- ) (A) Expressions of Erbin (red) and LC3 (green) in colorectum of DSS -treated GFP-LC3 +/- mice by immunofluorescent staining and confocal microscopy. The nuclei were stained with DAPI (blue). (B-C) Expressions of Erbin, LC3, ATG16L1, ATG2A and ATG7 by Western Blots (WB) (B), and immunohistochemical staining (IHC) (C). Scale bars = 50 μm (A and C).

Article Snippet: Transferred membranes were then blocked for 1 h at room temperature with 5% non-fat dried milk in 1× Tris-buffered saline and 0.1% Tween-20 before incubating with antibodies against Erbin (A303-762A; Bethyl), LC3B (3868; CST), ATG16L1 (8089; CST), ATG7 (8558; CST), ATG2A (15011; CST), Bax (bsm-33279; Bioss), Bcl-2 (bsm-33411; Bioss), and active Caspase3 (bsm-33199; Bioss) respectively.

Techniques: Mouse Assay, Knock-Out, Staining, Confocal Microscopy, Western Blot, Immunohistochemistry

Journal: Oncotarget

Article Title: Erbin exerts a protective effect against inflammatory bowel disease by suppressing autophagic cell death

doi: 10.18632/oncotarget.23925

Figure Lengend Snippet: Autophagic program of colorectum in experiment colitis mouse model after Erbin was deleted (A) LC3 (green) in colorectum of DSS -treated GFP-LC3 +/- mice by immunofluorescent staining and confocal microscopy. The nuclei were stained with DAPI (blue). (B) Ultrastructural changes of autophagic/lysosomal vacuoles (labelled as 1 and mitochondrial (labelled as 2) in colorectal mucosa of Erbin -/- mice treated with DSS. (C-D) Expression of Erbin, LC3, ATG16L1, ATG7, ATG2A of colorectum in DSS-induced wild type (WT) and Erbin -/- mice by Western Blots (WB) (C), and immunohistochemical staining (IHC) in IL-10 -/- mice (D). Scale bar, 1 μm and 0.5 μm (B). Scale bar, 50 μm (A and D).

Article Snippet: Transferred membranes were then blocked for 1 h at room temperature with 5% non-fat dried milk in 1× Tris-buffered saline and 0.1% Tween-20 before incubating with antibodies against Erbin (A303-762A; Bethyl), LC3B (3868; CST), ATG16L1 (8089; CST), ATG7 (8558; CST), ATG2A (15011; CST), Bax (bsm-33279; Bioss), Bcl-2 (bsm-33411; Bioss), and active Caspase3 (bsm-33199; Bioss) respectively.

Techniques: Mouse Assay, Staining, Confocal Microscopy, Expressing, Western Blot, Immunohistochemistry

Journal: PLoS Genetics

Article Title: Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

doi: 10.1371/journal.pgen.1008721

Figure Lengend Snippet: Endogenous SP-1277 isoform during mitosis. Endogenous SP-1277 protein was localised in dividing RPE-1 cells, during mitosis using the N-terminus antibody and counterstained using anti-acetylated ⍺-tubulin antibody (A), or counter stained with two mitotic markers PLK-1 (B) and CENP-E (C). (A) SP-1277 was detected in the nucleus as speckles at G0, which intensified at the onset of prophase, aligning along the kinetochore at metaphase, and anaphase, before returning to its G0 localisation pattern. (B) SP-1277 globules show co-localisation with the kinetochore marker PLK-1 at prophase, metaphase and anaphase. At cytokinesis the PLK-1 staining is primarily localized at midbody between the dividing cell which also co-stains for SP-1277. (C) SP-1277 show strong co-localisation with another kinetochore marker CENP-E at prophase, prometaphase and anaphase. At anaphase the colocalization primarily is around the separated chromosomes. Nuclei were stained using DAPI (blue).

Article Snippet: To investigate this further, we examined the localisation of SP-1277 in RPE-1 cells at different stages of cell division ( ).

Techniques: Staining, Marker

Journal: PLoS Genetics

Article Title: Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

doi: 10.1371/journal.pgen.1008721

Figure Lengend Snippet: Colocalization analysis of SP-1277 with CENP-E at prometaphase and anaphase. (A) At prometaphase SP-1277 (green) co-localises with CENPE-E (red) globules. To evaluate whether colocalization occurs, a line was drawn connecting different globules (B). The green and red intensity was measured using ImageJ in globules along the line and plotted (C). The data was analysed using Image J and Imaris softwares and showed colocalization of 84% (tM0.84) of CENP-E with SP-1277 and 75% (tM = 0.75) of SP-1277 with CENP-E. Fluorescence intensity data of two images were distributed linearly as shown in the scatterplots (Fig 8D 8E). The colocalization of SP-1277 with CENP-E in anaphase is shown in (F). The regions of interest showing highest colocalization are labelled as 1 and 2. The degree of colocalization of the two molecules was analysed using ImageJ (1a, and 1b for region 1) and (2a and 2b for region 2) and gave a colocalization of more than 80%.

Article Snippet: To investigate this further, we examined the localisation of SP-1277 in RPE-1 cells at different stages of cell division ( ).

Techniques: Fluorescence

Journal: PLoS Genetics

Article Title: Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

doi: 10.1371/journal.pgen.1008721

Figure Lengend Snippet: Characterisation of SP-1277 isoform. (A) Schematic representation of the domain structure of SP-1277 isoform. Light blue bar shows the epitope for the N-terminal antibodies and the red bar shows epitope for the C-terminal antibodies. The nuclear localisation signal in the terminal region SP-1277N 625 is shown by a hashed blue bar. The boundary of SP-1277-N 625 and SP-652 are shown two discontinuous lines. The SH3, DBL homology (DH) and PH domains are shown in SP-652. (B) Western blot analysis of the SPATA13 and its fragments analysed by N-terminus (ab122627) and anti-AcGFP antibodies. AcGFP fusion protein of SP-1277, SP-1277-N 625 and the SP-652 were expressed in HT1080 cells, total cell lysate was separated on 4–15% SDS polyacrylamide gel, transferred on nitrocellulose membrane and probed with different antibodies. Specific reactivity with protein bands is shown by asterisks. (C) Western blot of total RPE-1 lysate probed with the C-terminus (lane 1) and N-terminal (lane 2, ThermoFisher; lane 3, Abcam) antibodies. (D) Reactivity of N- and C-terminal antibodies with RPE-1 cells overexpressing SP-1277 compared with AcGFP control. Immuohistochemical reactivity of murine eye tissues (E, F) and human eye tissues (G) with the N-terminus antibody from Abcam (ab122627). CE = ciliary epithelium, CP = ciliary process, PP = pars plana, GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer, SM = sphincter muscle, DM = dilator muscle, PE = posterior muscle. Re = retina, Co = cornea, Le = lens.

Article Snippet: To investigate this further, we examined the localisation of SP-1277 in RPE-1 cells at different stages of cell division ( ).

Techniques: Western Blot

Journal: PLoS Genetics

Article Title: Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

doi: 10.1371/journal.pgen.1008721

Figure Lengend Snippet: SPATA13 mutations induced GEF activity in SP-1277. (A) RPE-1 cells were co-transfected with Flag-RAC-1 along with wildtype SP-1277, wildtype SP-652 or mutants (S292I, S473N, 478-480d, P964L) cloned in pLPCpuro_NAcGFP and after lysis the GTP-RAC-1 was immunoprecipitated with PAK-PBD beads and analysed on western blot using anti-Flag antibody. (B) Quantification of GEF activity of SP-1277 (wildtype and mutants). One way ANOVA was used to test the null hypothesis of no difference between GEF activity in mutants and wildtype, with there being a significant difference (p = 0.002).

Article Snippet: To investigate this further, we examined the localisation of SP-1277 in RPE-1 cells at different stages of cell division ( ).

Techniques: Activity Assay, Transfection, Clone Assay, Lysis, Immunoprecipitation, Western Blot

Journal: PLoS Genetics

Article Title: Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

doi: 10.1371/journal.pgen.1008721

Figure Lengend Snippet: Location and predicted effect of mutations in SPATA13 gene on protein conformation. (A) Structure of SPATA13 gene structure and location of 8 mutations in exon 2 and the 9 th mutation in exon 9. (B) Conservation of amino acids mutated in PACG in 9 different species. (C) Secondary structure prediction for the amino acid sequence of SPATA13 polypeptide. (D) Predicted tertiary structure of SP-1277 containing the 9bp (p. 478-480del) deletion. (E) Wildtype SP-1277 showing the location of all the missense variants.

Article Snippet: To investigate this further, we examined the localisation of SP-1277 in RPE-1 cells at different stages of cell division ( ).

Techniques: Mutagenesis, Sequencing

Journal: PLoS Genetics

Article Title: Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

doi: 10.1371/journal.pgen.1008721

Figure Lengend Snippet: Endogenous and ectopic expression of SPATA13 isoforms. Human RPE-1 cells were immunostained with SP-1277 (N-terminus antibody, A) and SP-652 (C-terminus antibody, B) and co-stained for endogenous F-actin (Alexa Fluor 594 conjugated phalloidin). (C) To detect nuclear actin, RPE-1 cells were transfected with nuclear actin chromobody (nAC) probe construct [ 55 ] and were counterstained with N-terminal specific SP-1277 antibody. RPE-1 cells were transiently transfected with AcGFP-tagged SP-1277 (D), and SP-1277-N 625 (E) respectively, both counterstained for F-actin. AcGFP-SP-1277 localised to nuclear speckles with a diffuse but variable cytoplasmic signal (S), AcGFP-SPATA13-N 625 (E) only showed nuclear staining. Nuclei were stained with DAPI (blue). Scale bar = 10 μM.

Article Snippet: To investigate this further, we examined the localisation of SP-1277 in RPE-1 cells at different stages of cell division ( ).

Techniques: Expressing, Staining, Transfection, Construct

Journal: PLoS Genetics

Article Title: Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

doi: 10.1371/journal.pgen.1008721

Figure Lengend Snippet: Expression of different SPATA13 transcripts in human cell lines and eye tissues. (A) Schematic representation of the transcripts of different isoforms of SPATA13. The two isoform SP-1277 and SP-652 isoforms are generated by alternative splicing of exons toward the N-terminal. The position of qPCR primers used to differentiate the 2 transcripts are also shown. (B) Expression of SPATA13 transcripts by qPCR in 17 different cell lines derived from human eye, breast, liver, skin, head and neck, cervix and kidney. (C) Expression of SP-1277 and SP-652 transcripts in human iris, ciliary epithelium, retinal pigment epithelium, retina, cornea and lens. The green bars represent SPATA13-Total whereas the blue bars represent SP-1277 and the red bars represent SP-652.

Article Snippet: To investigate this further, we examined the localisation of SP-1277 in RPE-1 cells at different stages of cell division ( ).

Techniques: Expressing, Generated, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Nodal involvement by marginal zone B-cell lymphoma harboring t(14;22)(q32;q11) involving immunoglobulin heavy chain and light chain lambda as the sole karyotypically recognizable abnormality in a patient with systemic lupus erythematosus

doi:

Figure Lengend Snippet: (A) A balanced reciprocal translocation between 14q32 (first arrow) and 22q11 (second arrow) was shown (G banding). (B, C) Dual color break-apart signals were observed for both IgH (B) and IgL (C), respectively.

Article Snippet: Translocation between the IgH gene at 14q32 and the IgL gene at 22q11 occurs rarely.

Techniques: Translocation Assay