Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 1 The pedigree of the fam- ily with the JAK2 L604F muta- tion. The patient and her sister have the L604F mutation (blue) in a homozygous state, and her parents have heterozygous L604F. The germline L604F probably arises from a common ancestor pair (encircled in blue). The presence of the mutation in the nontested family members is presumed (light blue) as a highly likely way to yield the results obtained in the tested members

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Mutagenesis

Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 3 Effect of endogenous JAK2 mutations in HeLa cells. Western blot analysis of HeLa cells with V617F or L604F JAK2 mutations introduced by CRISPR. The expected gene modification was confirmed by sequencing. Eight independent harvests were performed for each modified subline and the wild type (WT) parental line. The cell lysates were analyzed in groups – each western-blot membrane contained 4 WT samples and 4 samples from one mutated subline. The measured band intensities were normal- ized to ACTIN and related to the mean value from WT samples included in the given membrane (100%). Top: repre- sentative examples of JAK2 and pJAK2 Tyr1007/1008 signals. ACTIN was used as the loading control. Bottom: means ± SD of the relative band intensities from 8 independent samples for each JAK2 variant. The differences between modified sublines and the WT control were evaluated by unpaired Student´s t test (** p < 0.01, *** p < 0.001)

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Western Blot, CRISPR, Modification, Sequencing, Membrane, Control, Variant Assay

Journal: Bio-protocol

Article Title: Sensitive and Adaptable Turn-On Maturation (ATOM) Fluorescent Biosensors for Detecting Subcellular Localization of Protein Targets in Cells

doi: 10.21769/BioProtoc.5239

Figure Lengend Snippet: HEK 293T cells were transfected with one plasmid encoding y-ATOM mCh and a second plasmid expressing either decoy target (hRAS) or correct target (mCherry containing the Y70A mutation that rendered it nonfluorescent). y-ATOM mCh binds equally well to mCherry and the Y70A variant; the latter was used here to avoid interfering with the fluorescence of the Alexa Fluor594-labeled antibody (item 1 in Reagents) that recognized the mClover domain of the biosensor. Cells were stained with this antibody to quantitate the total expression of the biosensor. (A) Raw, two-channel images were analyzed using the SegmentAlone.ijm macro in Fiji. (B) The antibody channel was selected for identifying cells. (C) The same mask was then applied to the biosensor channel to measure biosensor signal intensity. Scale bar = 100 μm. Raw image files in Zeiss Vision Image format (mCherryNeg.zvi and mCherryPos.zvi) are included as Dataset S2.

Article Snippet: PCMV-hRAS (G12V) for expressing hRAS G12V in mammalian cells (Addgene plasmid # 209705; http://n2t.net/addgene:209705 ; RRID: Addgene_209705) Reagents 1.

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Variant Assay, Fluorescence, Labeling, Staining

Journal: Bio-protocol

Article Title: Sensitive and Adaptable Turn-On Maturation (ATOM) Fluorescent Biosensors for Detecting Subcellular Localization of Protein Targets in Cells

doi: 10.21769/BioProtoc.5239

Figure Lengend Snippet: Fluorescence intensities of HEK 293T cells expressing y-ATOM mCh and either the correct ligand (mCherry Y70A, circles) or a decoy ligand (hRAS, crosses) were measured according to step I3, using the images from Figure 6. Data points from the ATOM biosensor channel are magenta and data points from the antibody (Ab) channel are cyan. Turn-on ratios are indicated. Error bars correspond to the standard deviation. Data were obtained from a single transfection, and each symbol represents one cell (n = 131 for samples with correct ligand, n = 199 for samples with decoy ligand). The mean intensities were quantified using 12-bit images and are as follows: 316 ± 133 (ATOM signal, correct ligand) and 12 ± 5 (ATOM signal, decoy ligand), 466 ± 125 (Ab signal, correct ligand) and 261 ± 13 (Ab signal, decoy ligand). The GraphPad Prism file of cell fluorescence intensities is included as Dataset S3.

Article Snippet: PCMV-hRAS (G12V) for expressing hRAS G12V in mammalian cells (Addgene plasmid # 209705; http://n2t.net/addgene:209705 ; RRID: Addgene_209705) Reagents 1.

Techniques: Fluorescence, Expressing, Standard Deviation, Transfection

Journal: Nature Communications

Article Title: CD44 connects autophagy decline and ageing in the vascular endothelium

doi: 10.1038/s41467-023-41346-y

Figure Lengend Snippet: a Fluorescence analysis of the colocalization of CD44ICD with endogenous STAT3 and core proteins of the PtdIns3K complexes in HUVECs transduced with Ev or CD44ICD. Nuclei were stained with DAPI. Bar = 10 μm. The line scanned profiles at the right of confocal images show the distribution of fluorescence for each channel in the white line in the corresponding confocal images. b Reactivity of CD44ICD or STAT3 with PtdIns3K complex proteins, and STAT3 with CD44ICD in a yeast two-hybrid system. Yeast strain AH109 was co-transformed with a bait plasmid, BD-CD44ICD or BD-STAT3, and a prey plasmid, pGADT7-PtdIns3K complex proteins (AD-ATG14L, AD-BECN1, AD-UVRAG, AD-PIK3C3, and AD-PIK3R4) or pGADT7-CD44ICD, which encodes PtdIns3K complex proteins or CD44ICD fused to the Gal4 activation domain. Co-transformation of BD-Lam/AD-T and BD-P53/AD-T was used as negative and positive controls, respectively. c BiFC analysis of CD44ICD-STAT3 and STAT3-PIK3R4 interactions. HUVECs were transfected with indicated combinations of constructs. Co-transfection of VN-Jun and VC-Fos was used as positive control. Bar = 30 μm. d , e GST-pull-down of recombinant STAT3 with a GST-CD44ICD fusion protein ( d ) or a GST-P150 domain fusion protein ( e ). f The binding mode of full-sequence structures of STAT3 and CD44ICD. Colors indicate: STAT3, green; CD44ICD, red. The key residues interacting between STAT3 and CD44ICD are indicated as gray and yellow, respectively. Hydrogen bond is described by pink dash lines. g Western blots showing co-IP of CD44ICD_ΔN35 with endogenous STAT3 and core proteins of the PtdIns3K complexes in HUVECs transduced with Ev or CD44ICD_ΔN35 (DDK tag). Lysates, whole cell lysates; IP, immunoprecipitates. h Proposed schematic diagram of CD44ICD-mediated autophagy decline. CD44ICD suppresses the levels of PIK3C3 and PIK3R4 and the kinase activity of PIK3C3, activates STAT3, and disrupts the assembly of the PtdIns3K complex by interacting with STAT3. Three biologically independent experiments. Source data are provided as a Source data file.

Article Snippet: For the BiFC assay, the sequences of CD44ICD, PIK3R4 and STAT3 were cloned and inserted into both VN155 and VC155 (Addgene, MA, USA).

Techniques: Fluorescence, Transduction, Staining, Transformation Assay, Plasmid Preparation, Activation Assay, Transfection, Construct, Cotransfection, Positive Control, Recombinant, Binding Assay, Sequencing, Western Blot, Co-Immunoprecipitation Assay, Activity Assay

Journal: Nature cell biology

Article Title: Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression.

doi: 10.1038/s41556-021-00820-9

Figure Lengend Snippet: Fig. 3 | Tumour-cell-derived GABA induces β-catenin signalling to support proliferation. a, Growth curves of GAD1/Gad1-knockdown or control H520, HT29 and MC38 cell lines. n = 3 per group. b, Analysis of cell numbers in GAD1-knockdown or control H520 (left) and HT29 (right) cell lines (n = 3 per group) in the absence or presence of GABA for 6 d. c, Tumour growth of GAD1-knockdown or control subcutaneous H520 tumours in nude mice. n = 5 per group. Intratumoural injection of GABA was initiated on day 13. d, β-Catenin target gene expression in subcutaneous Gad1-knockdown or control MC38 tumours. n = 3 per group. e, Pearson correlation of GAD1 expression (left) and GABA levels (right) with β-catenin expression in 89 samples from patients with COAD. f, β-Catenin and cyclin D1 protein expression in the indicated GAD1-knockdown or control cancer cell lines. g, mRNA expression of the indicated genes in GAD1-knockdown or control H520 (left) and HT29 (right) cell lines. n = 3 per group. h, β-Catenin and cyclin D1 protein expression in H520 cells incubated with vehicle or 3-MPA (5 μM) for 48 h, followed by treatment with GABA as indicated. i, GAD1, β-catenin and cyclin D1 protein expression in GAD1-knockdown or control H1650 (left) and HT29 (right) cell lines, followed by GABA (50 μM) treatment for 48 h. j,k, Mutant β-catenin(S33Y) or empty vectors (EV) were expressed in GAD1-knockdown or control H520 cells. The indicated proteins were analysed by western blot (j) and cell numbers were examined on day 6 after cells were seeded (k). n = 3 per group. β-Actin was used as a loading control in the western blot analysis. The western blot experiments shown in f and h–j were repeated independently at least twice with similar results. n indicates the number of biological (a–d and k) or technical (g) replicates. Data are mean ± s.d. P values were determined using two-tailed Student’s t-tests (a, b, g and k) and two-way analysis of variance (ANOVA) (c). The correlation coefficient (r) and P values in e were determined using two-tailed Pearson correlation analysis. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. The exact P values are provided in the source data.

Article Snippet: The pcw107-β-catenin (S33Y) vector (Addgene, 64615) and pcw107 empty vector (Addgene, 62511) were gifts from D. Sabatini, J. Doench and K. Wood49. pRRLSIN-β-catenin (∆GSK) (Addgene, 24312) was a gift from R. Nusse50. pRRLSIN empty vector (Addgene, 12252), PAX2 (Addgene, 12260) and VSV-G (Addgene, 12259) were gifts from D. Trono.

Techniques: Derivative Assay, Knockdown, Control, Injection, Targeted Gene Expression, Expressing, Incubation, Mutagenesis, Western Blot, Two Tailed Test