Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Validation of CRISPR activation system in Aedes cells using multicistronic plasmid vectors

doi: 10.3389/fbioe.2023.1142415

Figure Lengend Snippet: Efficiency of T2A peptides in Aedes cells. (A) Schematic diagram showing the design of dual fluorescent reporter cassettes to evaluate T2A (plasmid pJVG-T2A) and dT2A (plasmid pJVG-dT2A) activity. (B-D) Fluorescence micrograph of the cells transfected with plasmids as shown on the left of the panels. The untransfected cells (B-B”) were used to determine the baseline for imaging mCherry and eGFP channels, respectively. (E) Western blotting analysis of the extracts prepared from the transfected cells showing presence of processed mCherry and eGFP proteins. The blot was probed with anti-mCherry antibody (left panel) and anti-GFP antibody (right panel). Both blots were subsequently developed using anti-actin antibody. The arrow marks the position of the unprocessed mCherry-eGFP fusion protein.

Article Snippet: Post-transfer, the blot was cut into two and blocked using 5% BSA-TBST solution for 30 min at room temperature followed by incubation with rabbit anti-GFP antibody (1:1000, CST #2956S) and rabbit anti-mCherry antibody (1:1000, Millipore #AB356482) separately.

Techniques: Plasmid Preparation, Activity Assay, Fluorescence, Transfection, Imaging, Western Blot

Journal: bioRxiv

Article Title: Overexpressed Malat1 Drives Metastasis through Inflammatory Reprogramming of Lung Adenocarcinoma Microenvironment

doi: 10.1101/2023.03.20.533534

Figure Lengend Snippet: (A) Representative H&E and GFP IHC staining of liver and lymph node (LN) metastasis (Met) of the long-term experiment of KPC animals infected with mA1 dRNA; (B) Quantification of metastasis of the mice analyzed in (A) (n=10 mice per group, unpaired t test); (C) Survival curves of the animals shown in (A). Log-rank test was used for statistical analysis. Presence of ethical endpoint criteria were used as endpoint; (D) IHC staining for the metastasis markers Nkx2.1 and Hmga2 of KPC animals analyzed in (A); (E) Quantification of Nkx2.1 staining in images in (D) (n>20 tumors, unpaired t-test); (F) Contingency analysis showing the percentage of positive tumors for Hmga2 immunostaining (Chi-square 28.68, P<0.001, n=79 tumors); (G) IHC staining for Hmga2 marker in the KC the cohort; (H) Contingency analysis showing the percentage of positive tumors for Hmga2 immunostaining in KC cohort (Fisher exact test P=0.504, n=157 tumors).

Article Snippet: Afterwards, tissues were incubated with primary antibodies: Hmga2 (BioChek, 59170AP; 1:500), GFP (Cell Signaling Technology, 2555S; 1:2000), α-Sma (ThermoScientific, 14-9760-82; 1:000), Cd68 (Abcam, ab125212; 1:100), Ki67 (D3B5, Rabbit mAb Mouse Preferred IHC Formulated; 1:500), pHH3 (Cell Signaling Technology, 9701S; 1:500), and Cdh1 (Cell Signaling, 24E10, 1:1000) at 4°C overnight.

Techniques: Immunohistochemistry, Infection, Staining, Immunostaining, Marker

Journal: Nature Communications

Article Title: Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish

doi: 10.1038/s41467-023-37026-6

Figure Lengend Snippet: a Strategy for the generation of Ara and Arb KI medaka strains. We designed a gRNA targeting ara intron 8 and arb intron 7 (the last intron). In the donor plasmids, we cloned a genomic fragment beginning from the end of exon 8 of ara and exon 7 of arb and ending just before the stop codon in the last exon, exon 9 of ara , and exon 8 (shown as black closed boxes with ‘E8’) of arb , where the 3xFLAG sequences (shown as blue boxes) and a P2A (2A peptide from porcine teschovirus-1)-mClover3 cassette (shown as green boxes) was placed in the frame. Thus, endogenous Ara and Arb were expressed as FLAG fusion proteins. Both AR-FLAG and P2A-mClover3 were expected to be expressed under the control of the endogenous Ar promoter. To generate KI medaka, sgRNA (for genome digestion in the final intron), donor plasmid, and Cas9 mRNA were co-injected into one-cell-stage medaka embryos. After injection, concurrent cleavage of the targeted genomic locus and the donor plasmid resulted in the integration of donor plasmid DNA containing 3xFLAG-T2A-mClover3 by non-homologous end joining (NHEJ). The scheme shows the forward integration of 3xFLAG-T2A-mClover3. b Expression of mClover3 in adult males of the two knock-in medaka strains, ara FLAG-2A-mClover3 (Ara-KI) and arb FLAG-2A-mClover3 (Arb-KI). White, grey, and red arrows indicate the regions adjacent to pectoral, dorsal, and anal fins, respectively (Ara-KI). White, grey, and red arrows indicate the pectoral, dorsal, and anal fins, respectively (Arb-KI). c Immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in longitudinal sections of papillary processes of the anal fin (6 μm thickness). The merged images represent red fluorescence for immunostaining of FLAG (anti-DDDDK-tag mouse mAb monoclonal antibody), green fluorescence for immunostaining of mClover3 (anti-GFP D5.1XP rabbit mAb monoclonal antibody), and blue fluorescence for nuclear staining by DAPI. The medaka Arb-FLAG, but not Ara-FLAG, translocated into the nuclei of cells located in the distal tip of a bone nodule of papillary processes (marked by white arrows). d Representative micrographs of Masson/trichrome staining and immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in adjacent sections of the urogenital region (8 μm thickness). Nuclear localisation of Ara-FLAG and Arb-FLAG was observed in the medulla ventral to the sperm duct, where ar DKO exhibited hyperplasia. e, f Representative micrographs showing immunohistochemical detection of FLAG-tagged endogenous Ara and Arb, and DAPI counterstaining in the brain (12 μm thickness). f shows a higher magnification of the POA. Nuclear localisation of both Ara-FLAG and Arb-FLAG was observed in the POA. n = 6 for Ara-KI and Arb-KI males, respectively.

Article Snippet: Briefly, after antigen retrieval by incubating slides in 0.1 mM citrate buffer (pH 6.0) in an autoclave (121 °C) for 1 min, the sections were blocked for 1 h using 1xPBS containing 0.1% Tween 20, 2% BSA, and 2% foetal bovine serum, and incubated with anti-DDDDK-tag (FLAG) mouse mAb monoclonal antibody (M185-3L, MBL, Nagoya, Japan, 1:300 dilution) and anti-GFP D5.1XP rabbit mAb monoclonal antibody having cross-reactivity to the mClover3 (#2956, Cell Signaling, Danvers, MA, USA, 1:300 dilution) overnight at 4 °C.

Techniques: Clone Assay, Plasmid Preparation, Injection, Non-Homologous End Joining, Expressing, Knock-In, Immunohistochemical staining, Fluorescence, Immunostaining, Staining

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Chlamydomonas expressing KAP-GFP. Indicated are the measured areas of KAP-GFP localization: the two basal bodies and cilia. Scale bar is 5 μm. (B) Super plot quantification of basal body fluorescence in (A) after a 2-h treatment with BCI at the indicated concentrations. Error bars are the mean with the 95% confidence interval for the averages from the trials. P = 0.5126, which was determined by an ordinary one-way ANOVA. (C) Ciliary length plotted against ciliary fluorescence for cells in DMSO (orange circles) or 30 μM BCI (purple) after a 2-h treatment (n = 40, N = 1). Simple linear regression was used to generate linear regression lines and comparisons. For DMSO, y = 0.1171x + 0.4305 and r 2 = 0.05676. For BCI, y = 0.1174x + 0.4051 and r 2 = 0.2636. Comparing slopes, F = 4.048e-006 (1, 76) and P = 0.9984. Comparing intercepts, F = 0.005429 (1, 77) and P = 0.9415. (D) Comparison of total ciliary fluorescence shown in (C). P -values were determined using a t test. (E) Comparisons of ciliary fluorescence in DMSO or BCI per micrometer of cilia in (C). P -values were determined using a t test. (F) Example kymographs collected from total internal reflection fluorescent microscopy of KAP-GFP movement in cilia in cells treated with DMSO (top) or 30 μM BCI (bottom) for 2 h. Vertical scale bars are 4 μm. Horizontal scale bars are 2 s. (G, H, I) KAP-GFP dynamics quantified from the kymographs (n = 20, N = 1). Error bars are the mean with the 95% confidence interval (n = 20, N = 1). P -values were calculated from a two-tailed unpaired t test for pairwise comparisons. (G) Frequency of KAP-GFP trains measured as the total amount of trains counted over the total amount of time the kymograph was collected. For DMSO, n = 40 (1 and 2 h). For BCI, n = 30 (1 h) and 34 (2 h). (H) Velocity of KAP-GFP trains measured as the distance traveled in μm over time in seconds. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). (I) Relative injection size of KAP-GFP trains measured as the relative total fluorescent intensity of each train relative to the maximum measurement. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). P -values were determined using a t test.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Expressing, Fluorescence, Microscopy, Two Tailed Test, Injection

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Immunofluorescent images of KAP-GFP cells during regeneration in either DMSO or 30 μM BCI. Scale bars are 5 μm. Red arrows point to basal bodies, which are the object of quantification in (D). (B) Western blot for KAP-GFP expression in regenerating cells in either DMSO (D) or 30 μM BCI (B). Total protein was measured with amido black. (C) Quantification of (B). Error bars are the mean with SD for three independent experiments. P -values were calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (D) Quantification of KAP-GFP fluorescence at the basal bodies in (A). Error bars are the mean with the 95% confidence interval for the averages from three independent trials (n = 30, N = 3). The P -value was calculated using an unpaired t test with Welch’s correction.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing, Fluorescence

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Western blot of KAP-GFP expression compared with total protein shown with amido black. Cells were treated for 2 h with either 0.5% DMSO, 20 μM BCI, 50 μM MG132, or both 50 μM MG132 and 20 μM BCI. (A, B) Quantification of (A). Error bars are SD of the mean (N = 3). The P -value was determined by an unpaired t test between BCI and BCI + MG132.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: BCI inhibits phosphate removal from MAPK. This ultimately alters or inhibits ciliogenesis, KAP-GFP dynamics in cilia, KAP-GFP protein synthesis, NPHP4 protein localization at the transition zone, membrane trafficking, and microtubule organization.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques:

Journal: Nature Communications

Article Title: Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A

doi: 10.1038/s41467-023-36693-9

Figure Lengend Snippet: A Sites of CIP2A-B56α cross-links (in red; PDB: 6NTS) mapped in relation to indicated LxxIxE-binding groove of B56α. Residues indicated in light purple constitutes the LxxIxE-binding region of B56α between amino acids 212-271. Residues in dark purple indicate amino acids involved in PP2A-A interaction as explained in Fig. . B CIP2A(1-560)V5 out-competes prototypical LxxIxE motif target BubR1(LxxIxE peptide 647-720) from its direct association with B56α. Source data are provided as a Source Data file. C Quantification of GST pull-down data from ( B ) shown as a mean + S.E.M from N = 3 biological repeats. Two-sided t-test. D B56α competition assay using GST-BubR1(647-720) alone, or in combination with CIP2A N-terminal head peptide (aa. 18-40). Source data are provided as a Source Data file. E Quantification for data from D shown as a mean + S.E.M from N = 4 biological repeats. Two-sided t-test. F In vitro binding assay using purified recombinant GST-tagged CIP2A(1-560) and B56α WT or B56 variants Y215Q and R222E, which contain mutations that prevent interaction with LxxIxE groove substrate proteins . Source data are provided as a Source Data file. G Quantification of data from ( F ) shown as mean + S.E.M from N = 3 biological repeats. H GFP-tagged B56α WT or indicated triple mutant were expressed in HEK-293-T cells. The amount of PP2A-A subunit bound to GFP-tagged B56α upon GFP trap pull-down was quantified by anti-PP2A-A immunoblotting. Shown are the mean values + S.E.M of the ratios of the quantified anti-PP2A-A signal versus the quantified anti-GFP signal, relative to the B56α WT (set at 100 %) from N = 3 biological replicates. A two-sided one-sample t-test. I Triple B56α mutant (K181A/K217A/K227A) exhibits loss of K227 (B56)-D117(PP2A-A) salt bridge. Overlay of PP2A-B56α (PDBID 6NTS: PP2A-A, yellow; B56α, beige) and PP2A-B56γ (PDBID 2IAE: PP2A-a, light cyan, B56γ, cyan), with PP2A-A D177 and B56α/B56γ K227/K202 residues shown as sticks and labelled. H-bond interactions are shown using dotted lines. Image was generated using Pymol.

Article Snippet: The following antibodies were used: anti-V5 (monoclonal mouse (mM) Ab (E10/V4RR), Thermo Fisher Scientific, MA5-15253; 1:5000), anti-V5 (mM, Thermo Fischer Scientific, R960-25; 1:2000), anti-CIP2A (mM Ab(2G10-3B5), sc-80659, 1:1000), anti-GST (polyclonal rabbit (pR Ab), Thermo Fisher Scientific CAB4169; 1:10,000), anti-GST (pR Ab, Cell Signalling 2622 S; 1:10,000), anti-GST (mM Ab (B-14), sc-138; 1:10,000), anti-PR65 (pR Ab (H-300), sc-15355; 1:1000 and 1:5000), anti-B56α (mM Ab (23), sc-136045; 1:500 and 1:5000), anti-β-Actin (mM Ab (C4), sc-47778; 1:5000 and 1:10,000), anti-B56γ (mM Ab (E6), 374380; 1:500), all from Santa Cruz Biotechnology), anti-c-Myc (mM Ab (9E10), sc-40, 1:1000), anti-PP2Ac (pR Ab, Cell Signalling 2038S; 1:1000 and 1:5000), anti-GAPDH (mM Ab (6C5), 5G4-6C5 Hytest, 1:10,000), anti-PR65 (clone C5.3D10, 1:1000) and anti-PP2Ac (clone F2.6A10, 1:500), both generously supplied by Dr. S. Dilworth at Middlesex University, London, UK), anti-GFP (pRb Ab, 2555 S, Cell Signalling; 1:1000), anti-cleaved PARP (mM Ab(E51), ab32064 abcam; 1:1000), anti-Phospho-MEK1/2 (Ser217/221) (pR Ab, 9121S Cell Signallings; 1:1000), anti-phospho Vimentin (Ser39) (pR Ab, 13614 Cell Signalling; 1:1000), anti-HA tag (mRb Ab (C29F4), 3724 Cell Signalling; 1:200 and 1:1000).

Techniques: Binding Assay, Competitive Binding Assay, In Vitro, Purification, Recombinant, Mutagenesis, Western Blot, Generated