Journal: Nature Communications

Article Title: Autophagy deficiency promotes triple-negative breast cancer resistance to T cell-mediated cytotoxicity by blocking tenascin-C degradation

doi: 10.1038/s41467-020-17395-y

Figure Lengend Snippet: a The Top10 KEGG pathways enriched for commonly upregulated proteins in MDA-MB-231-Atg5KO#4 cells and MEF-Atg5 −/− cells compared to control cells. b Immunoassay of extracts of the indicated MDA-MB-231 cells and MEF cells. c The indicated MDA-MB-231 cells were co-cultured with CD3/CD28- activated human T-lymphocyte cells. Upper, representative dot plots of the cleavage of caspase-3 in tumour cells measured by flow cytometry. Bottom, percentage of the cleaved caspase-3 in tumour cells ( n = 3 biological independent samples). d The effect of TNC knockout in 4T1-Atg5KO cells using CRISPR-Cas9 technology. e Tumour growth of indicated mouse 4T1-Atg5KO#1 cells in BALB/c mice ( n = 5 mice per group). Tumour volumes were calculated (left), and tumour weights from experiment on autopsy on day 27 (right). f FACS analysis of CD45 + CD4 + , CD45 + CD8 + , and IFNγ + in CD45 + CD4 + T and CD45 + CD8 + T-cell populations from the isolated TILs in ( e ) ( n = 5 mice per group, right). Representative dot plots from a representative mouse for each group (left). Error bars represent mean ± SEM. The P value in c was determined by one-way ANOVA with Tukey’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in e , f was determined by a two-tailed unpaired Student’s t test. NS no significance. All data are representative of three independent experiments.

Article Snippet: To establish autophagy-deficient cell models, Atg5 Double Nickase Plasmids (h/m), Atg7 Double Nickase Plasmids (h/m) and Beclin1 Double Nickase Plasmid (m) were purchased from Santa Cruz Co. All of these plasmids were transfection-ready purified DNA plasmids.

Techniques: Control, Cell Culture, Flow Cytometry, Knock-Out, CRISPR, Isolation, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, IRE1α KO cells reconstituted with IRE1α–HA were processed to obtain purified MAM fractions followed by western blot analysis of indicated proteins (n = 3 independent experiments). H, homogenate; C, cytosol; Cr, crude mitochondria; M, MAMs; P, pure mitochondria; Cyt c, cytochrome c; CNX, calnexin. b, Liver extracts were processed to obtain subcellular fractions enriched for MAMs and analysed by western blot (n = 9 independent experiments). c,d, IRE1α KO cells reconstituted with IRE1α–HA or mock control were simultaneously imaged for calcium signals in the cytosol (Fura2; c) and mitochondria with Rhod2 (d). Left, the Fura2 ratio (c) and mean Rhod2 intensity (d) of normalized data before and after ATP is added; arrow, 100 μM ATP. Right, the data for the maximum peak are shown (total cells analysed: mock, n = 116 cells; IRE1α–HA, n = 138 cells). e,f, Similar experiments for Fura2 (e) and Rhod2 (f) were performed in CRISPR control and IRE1α KO cells (total cells analysed: control, n = 129 cells; IRE1α KO, n = 117 cells). WT, wild type. g, Indicated cell lines were processed for western blot analyses to monitor the levels of indicated proteins (n = 4 independent experiments). h, IRE1α null and control cells were imaged for calcium levels in mitochondria by transiently expressing CEPIA2mt mitochondrial calcium probe (left) after addition of 50 μM M3M3FBS (arrow), (Mito red; Mitochondrila Ds-Cherry control). Scale bars, 10 μm. Right, maximum CEPIA2mt intensity for every cell analysed (mock, n = 14 cells; IRE1α–HA, n = 14 cells). i, Maximum peaks from Fura2/Rhod2 measurements from samples described in c and d were calculated using nonlinear regression analyses to determine the correlation constant (K) and s.e.m. (mock, K = 0.199 ± 0.009; IRE1α–HA, K = 0.231 ± 0.01). j, Cells were imaged for calcium levels in the ER after loading with Mag-Fluo4 in permeabilized cells followed by stimulation with InsP3R (n = 5 independent experiments; left). Middle, percentage activity for InsP3R for each condition normalized to maximum release (ionomycin). Right, the first derivative was calculated. Data in c–f,h–j are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-tests except for j; right, which was one-tailed. Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Purification, Western Blot, CRISPR, Expressing, Activity Assay, Two Tailed Test, One-tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Ern1 and Ern1ΔK liver samples were processed for metabolomics studies (n = 4 animals per group). The heat map for the metabolites indicates significantly different metabolite levels in each experimental animal. b, Pathway analysis and statistical significance (two-tailed Student’s t-test) for the metabolites shown in a. c, The affected pathways and main hits from a are indicated. Altered metabolites and their associated pathways are indicated using the same colour code (coloured dots, size stands for P value as in b)) in a–c. d, Whisker and dot plots of the indicated metabolites of the TCA, indicating median and quartiles derived from samples in a (n = 4 animals per group) levels represent the log2 of the normalized area in a.u. e, Schematic of the TCA cycle. Metabolites with increased or decreased levels in Ern1 and Ern1ΔK samples are indicated by arrows. f, Glucose tolerance test in Ern1 control and Ern1ΔK mice (left). Right, data represent the area under the curve (AUC) for the whole glucose tolerance test (n = 4 animals per group). g, Proposed model: IRE1α expressed at MAMs docks the InsP3Rs at the mitochondrial–ER contact sites—possibly through a physical interaction, which may enhance InsP3R channel activity. The presence of IRE1α at MAMs favours calcium transfer into the mitochondria and bursts in ATP production. IRE1α deficiency leads to a metabolic stress condition that is characterized by the constitutive activation of AMPK, enhanced compensatory autophagy and altered mitochondrial morphology. Data are mean ± s.e.m. Statistical differences were detected with one-tailed (d) or two-tailed Student’s t-tests (f) or two-way ANOVA (f). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Two Tailed Test, Whisker Assay, Derivative Assay, Activity Assay, Activation Assay, One-tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, IRE1α KO cells that were reconstituted with either IRE1α–HA or an empty vector (mock) were imaged for TMRM signals before and after addition of 1 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (left). Scale bar, 20 μm. Right, mean TMRM intensity normalized to IRE1α–HA cells (n = 6 independent experiments). b, CRISPR control and IRE1α KO cells were analysed as described in a (n = 4 independent experiments). c,d, Percentage of ATP of the indicated cells using a luminescence assay (n = 18 biologically independent samples). e,f, ATP levels were measured in the indicated cell lines using the AT01 mitochondrial (yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP)) FRET probe FRET labeling stands for 440 nm excitation emmited in YFP channel. White numbers indicate regions of interest (left). Right, quantification of YFP/CFP ratio excited at 440 nm (mock, n = 52 cells; IRE1α–HA, n = 58 cells; control, n = 145 cells; IRE1α KO, n = 151 cells). Scale bars, 10 μm and 2 μm. g,h, The indicated cell lines were analysed for oxygen consumption rate (OCR). O, 1 μM oligomycin, F, 0.5 μM FCCP; A/R = 1 μM antimycin/rotenone (n = 4 independent experiments). i, pAMPK was analysed in the indicated cells using western blots (left) and normalized to total AMPK levels (right; n = 6 independent experiments). j, Determination of LC3-II levels in the indicated cell lines using western blots (left), followed by quantification normalizing to actin (right; n = 6 independent experiments). k, TEM-derived morphological parameters of mitochondria were obtained from indicated cells. Scale bar, 4 μm (left). Right, the data represent the area in μm2 and circularity (mock, n = 52 cells; IRE1α–HA, n = 58 cells). l, Cells were stained for ERp72 and TOM20 by indirect immunofluorescence (left) followed by colocalization quantification (right; Mander’s index: mock, n = 33 cells; IRE1α–HA, n = 40 cells; Pearson’s index: mock, n = 68 cells; IRE1α–HA, n = 78 cells). Scale bar, 20 μm and 5 μm. m, The indicated cells were imaged using TEM to visualize MAMs (pointed with red arrows) (left) using two quantification methods (right; mock, n = 38 contacts; IRE1α–HA, n = 30 contacts). Scale bars, 500 nm. Data in a–m are mean ± s.e.m. Statistical differences detected with one-tailed (k) or two-tailed unpaired Student’s t-tests. A Wilcoxon signed-rank test was applied in a–d and paired Student’s t-tests were applied in h,i,m (right panel). Source data for statistical analyses are provided in Supplementary table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Plasmid Preparation, CRISPR, Luminescence Assay, Labeling, Western Blot, Derivative Assay, Staining, Immunofluorescence, One-tailed Test, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Indicated cells were processed to obtain subcellular fractions and analysed using western blots. Right, quantification of the MAM fractions for the indicated proteins (InsP3R1, n = 4; InsP3R1, n = 8 independent experiments). b,c, Cells described in a were stained with a PLA (red) and DAPI (Blue) using anti-InsP3R1 (b) or anti-InsP3R3 (c) antibodies paired with anti-VDAC1 antibodies. Scale bars, 20 μm (left). Right, quantification of the number of positive PLA dots per cell (n = 3 independent experiments). d, Cells were imaged using TEM (left) to calculate ER to mitochondrial width (right; n = 3 independent experiments; mock, n = 46 contacts; IRE1α–HA, n = 30 contacts). Scale bars, 200 nm. e, CRISPR control and IRE1α KO cells were were imaged using TEM (left) to calculate ER to mitochondrial width (right; n = 3 independent experiments; CRISPR control, n = 27 contacts; CRISPR IRE1α KO, n = 47 contacts). Scale bars, 200 nm. f, The same cells as described in a were transiently transfected with SPLICSL to visualize MAMs with a width of 40–50 nm (left). Nuclei were stained with DAPI. Scale bar, 25 μm. Right, quantification of SPLICSL signal as dots per cell (n = 5 independent experiments; total cells analysed: mock, n = 41 cells; IRE1α–HA, n = 38 cells). g, Schematic representation and representative TEM images of indicated cells transiently expressing either a AKAP1 (34–63)-linker 9x-mRFP (9xL) construct or a control linker construct. Scale bar, 500 nm (top). MAM width was determined by TEM (bottom; mock control linker, n = 14 contacts; IRE1α–HA control linker, n = 12 contacts; mock 9xL, n = 15 contacts). h, Cells described in g were stained with PLA (green) and DAPI (blue) to measure the close proximity between InsP3R1 and VDAC1 proteins (left) in mRFP positive cells. Right, the number of dot counts per cell was quantified (n = 4 independent experiments) Scale bars, 20 μm. Data in a–h are mean ± s.e.m. Statistical differences were detected using one-way ANOVA and Tukey post-tests for multiple comparison (g,h), two-tailed Student’s t-tests (b–f) or Wilcoxon signed-rank test (a). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Western Blot, Staining, CRISPR, Transfection, Expressing, Construct, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Schematic of IRE1α structure and the mutants analysed (left) (TM; transmembrane domain). Right, the indicated cell lines were treated with 0.1 μg ml−1 tunicamycin for 4 h and then Xbp1 mRNA splicing was evaluated using PCR analysis. The agarose gel image was sliced to eliminate irrelevant lanes. Xbp1u, unspliced Xbp1s, spliced (n = 2 independent experiments). b, Calcium levels in the cytosol after ATP stimulation were analysed in IRE1α KO cells reconstituted with the indicated constructs. Arrow, 100 μM ATP (left; Fura2; n = 4 independent experiments; total cells analysed: mock, n = 131 cells; IRE1α–HA, n = 149 cells; IRE1α-P830L–HA, n = 120 cells; IRE1α-ΔC–HA, n = 97 cells). The maximum peak for the normalized Fura2 ratio was measured (middle). The same cells were imaged simultaneously with Rhod2 to measure mitochondrial calcium uptake. Arrow, 100 μM ATP (right two panels). c–e, HEK293T cells were transiently transfected with the indicated constructs and immunoprecipitation (IP) was performed using anti-HA antibodies. Western blot (WB) analysis was performed for the indicated proteins in immunoprecipitations and total input (c, n = 3 independent experiments; d,e are representative of two independent experiments). f, The indicated MEF cell lines were processed for immunoprecipitation using anti-HA antibodies. Western blot analysis was performed for the indicated proteins in immunoprecipitations and total input. g, Cells described in f were stained for PLA (red) and DAPI (blue) using anti-InsP3R1 antibodies paired with anti-HA antibodies and analysed by confocal microscopy. Scale bar, 20 μm (left). Right, the number of dots per cell were quantified (n = 3 independent experiments). h, Schematic of InsP3R1 domains used to generate recombinant proteins and perform in vitro pull-down assays (left; the asterisk indicates that residues 167–169 and 267 are relevant for channel function). Right, in vitro pull-down assay for purified GST-fused domains of InsP3R1 with recombinant IRE1α cytosolic portion (IRE1α-ΔN) followed by western blot analysis (D1, domain 1; D2, domain 2; D3, domain 3; n = 3 independent experiments). Data in b and g are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-test (g) or ANOVA with Tukey multiple comparison test (b). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Agarose Gel Electrophoresis, Construct, Transfection, Immunoprecipitation, Western Blot, Staining, Confocal Microscopy, Recombinant, In Vitro, Pull Down Assay, Purification, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Strategy to generate CRa particles using the synergistic activator mediators and sgRNA complex. b, IRE1α KO cells were generated that stably express either a CRa that targets the InsP3R1 promoter or a control vector. Representative western blot analysis of the indicated proteins was performed to confirm InsP3R1 upregulation (n = 10 independent experiments). c, The cells described in a were stained with a PLA (red) DAPI (blue) using anti- InsP3R1 and anti-VDAC1 antibodies, and were analysed by confocal microscopy. Scale bar, 20 μm (left). Right, the number of dots per cell were quantified (n = 6 independent experiments). d, CRa-InsP3R1 or CRacontrol cells were imaged with Rhod2 to measure mitochondrial calcium uptake. Arrow, stimulation using 50 μM M3M3FBS (left). Right, the maximum peak for normalized Rhod2 was calculated (total cells analysed: CRa-InsP3R1, n = 46 cells; CRa-control, n = 42 cells). e, CRa-InsP3R1 or CRa-control cells were imaged for mitochondrial membrane potential after TMRM staining (left). Arrow, stimulation with 1 μM FCCP; AU, arbitrary units. Right, normalized TMRM intensity (n = 4 independent experiments). f, pAMPK and total AMPK levels were determined in CRa-InsP3R1 or CRa-control cells using western blot (left). Right, quantification of the pAMPK/AMPK ratio (n = 7 independent experiments). g, The indicated cells were lysed and ATP levels were determined using a luminescence assay (n = 13 biologically independent experiments). h, IRE1α KO cells were generated that stably express either a CRa that targets the InsP3R3 promoter or a control vector. Representative western blot analysis of the indicated proteins was performed to confirm InsP3R3 upregulation (n = 5 independent experiments). i, The indicated cells were imaged with Rhod2. Arrow, stimulation with 50 μM M3M3FBS (left). Right, the maximum peak for the normalized Rhod2 was calculated (total cells analysed: CRa-InsP3R1, n = 132 cells; CRa-control, n = 112 cells). j, ATP levels were determined in the indicated cells using a luminescence assay (n = 25 biologically independent experiments). Data in b–j are mean ± s.e.m. Statistical differences were detected with unpaired Student’s t-tests (c,d,g,i,j) or Wilcoxon signed-rank test (b,e,f,h). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Generated, Stable Transfection, Plasmid Preparation, Western Blot, Staining, Confocal Microscopy, Luminescence Assay

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Schematic of Ern1 structure (the gene encoding IRE1α) (TM; transmembrane domain) and the strategy to delete the kinase domain (Ern1ΔK; left). Middle, livers from Ern1 control and Ern1ΔK mice were processed for western blot analysis to measure the levels of indicated proteins (n = 3 independent experiments). Right, mice were intraperitoneally injected with 1 mg kg−1 of tunicamycin or vehicle for 6 h. Xbp1s mRNA splicing was evaluated by RT–PCR analysis of cDNA obtained from total liver extracts. b–f, Ern1 and Ern1ΔK livers were processed for TEM analysis (b) to determine morphological parameters including mitochondrial area (arrows indicate MAMs, scale bar, 500 nm) (c), MAM width (d), mitochondrial circularity (e) and MAM length (f; n = 4 animals per group). g, Ern1 and Ern1ΔK MAM fractions were processed for quantitative mass spectrometry analysis (see Methods). The volcano plot shows all of the detected proteins (grey) and those that are known to be present at MAMs (red dots; n = 3 animals per group). h, A summary of statistically significant hits observed in the proteomic screening of MAMs. i,j, Ern1 and Ern1ΔK liver samples were processed to obtain subcellular fractions, and were analysed by western blot for the indicated proteins (i). Quantification of protein expression was performed for the indicated proteins by normalizing to calnexin (CNX; j; Ern1, n = 9 animals; Ern1ΔK, n = 7 animals). k, The protein content (in mg) from liver MAM fractions (left) or pure mitochondria (right) was quantified and normalized by total liver extract (in g) to obtain the percentage of MAM proteins in the liver (Ern1, n = 6 animals; Ern1ΔK, n = 4 animals). l, Liver homogenates from Ern1 and Ern1ΔK were immunoprecipitated for IRE1α and analysed for the indicated proteins by western blot. Ab, antibody (representative of three independent experiments). Data in c–f,j and k are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-tests (c,d). For j,k one-tailed Student’s t-tests were applied. Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Western Blot, Injection, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Expressing, Immunoprecipitation, Two Tailed Test, One-tailed Test

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: CD44 mediates shear stress mechanotransduction in an in vitro blood-brain barrier model through small GTPases RhoA and Rac1

doi: 10.1096/fj.202100822RR

Figure Lengend Snippet: Expression of CD44 regulates barrier formation. (A) Western blot of CD44 and beta-actin in CRISPR-modified cells. The ratio of intensities of CD44 and beta-actin signals normalized to scrambled levels. *Indicates p < .05 compared to scrambled and upregulated conditions. (B) Permeability coefficients of the channels seeded with transfected cells after 4 days of culture * denotes p < .05 compared to all conditions. (C) TEER measurements taken over the course of 4 days for different conditions, * indicates p < .05 compared to Day 1 timepoint for all conditions. (D,E) Fluorescent images of channels stained with DAPI (blue), anti-ZO-1 (red) (isolated in ii), and anti-adducin-γ (isolated in iii) for two conditions: scrambled control cells in collagen/HA hydrogels exposed to flow (D) and knockout cells in collagen/HA hydrogels exposed to flow (E). Scale: 50 μm. (F) Relative intensity (RQ) of RhoA activation in channels measured with ELISA. (G) Relative intensity (RQ) of Rac1 activation in channels measured with ELISA. *Indicates p < .05

Article Snippet: Commercially available CRISPR plasmids (Santa Cruz) were used to alter the expression of CD44 within the hCMEC/D3 cells prior to introduction into the 3D BBB model. To knockout CD44 in the cells, two plasmids encoding a D10A mutated Cas9 nuclease and a CD44-specific 20 nucleotide guide RNA were transfected into cells (Santa Cruz, sc-400209-NIC).

Techniques: Expressing, Western Blot, CRISPR, Modification, Permeability, Transfection, Staining, Isolation, Control, Knock-Out, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: Immunoblots confirming the absence of SHIP2 protein in independent ATDC5 (a) and Saos-2 clones (b). Wild-type cells plus two clones of ATDC5 (c) and SaOs-2 (d) genetically-depleted for SHIP2 differentiated and then stained with Alizarin Red S. ATDC5 (e) or SaOs-2 (f) cells differentiated in presence of DMSO (vehicle) or SHIP2 inhibitor AS1949490 (0-10 μM) and stained for mineralization with Alizarin Red S. Histograms represent the quantification of the cell staining after extraction. Data is representative of 3 individual experiments (3 wells per experiment). * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Western Blot, Clone Assay, Staining, Extraction

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Differentiated ATDC5 were stained for proteoglycans using Alcian Blue. Histograms represent the quantification of the cell staining after extraction. (b) Relative expression of hypertrophy and terminal differentiation markers (c) and matrix related genes in ATDC5-neg#1 cells. (d) Relative expression of osteoblast differentiation markers in SaOs-2 neg#1 cells. Histograms represent the difference in expression compared to WT cells (b, c, d). Data is representative of 3 individual experiments (3 wells per experiment). *p<0.05, **p<0.01, ***p<0.001. WT vs SHIP2-neg.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Staining, Extraction, Expressing

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Immuno-detection and quantification of MMP13 protein and (b) detection of MMP13 activity by collagen zymography in conditioned media of ATDC5 WT and SHIP2-negative ATDC5 cells. Data is representative of two individual experiments.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Activity Assay, Zymography

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) ATDC5 cells differentiated in presence of DMSO (VEH) or 1.5 μM of AS1949490 (top panels) and SHIP2-negative ATDC5 cells (bottom panels) were immunostained for PI(3,4,5)P3. Arrows indicate focal adhesion-like structures. (b) Immunodetection of Akt and pAKT (S473) protein in SHIP2-negative ATDC5 cells. Scale bars represent 0.01mm.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Immunodetection

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Wild-type and SHIP2-negative ATDC5 (top panels) and SaOs-2 (bottom panels) cells immunostained for vinculin. Arrows indicate focal adhesion-like structures. Table shows the number of focal adhesions per cell and their surface. (b) Cell adhesion assay showing increased ATDC5 and SaOs-2 attachment 30 min after seeding cells on fibronectin * p <0.05, ** p <0.01, *** p <0.001 for wild-type vs SHIP2-deleted cells. Scale bars represent 0.01 mm.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Cell Adhesion Assay

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Alizarin Red staining of ATDC5 and SaOs-2 WT cells vs SHIP2-negative cells differentiated in presence of 0.3μM of the actin polymerization inhibitor, cytochalasin D. (b) Alizarin Red staining of ATDC5 and SaOs-2 WT cells vs SHIP2-negative ATDC5 cells differentiated in presence of 15 μM of the ROCK pathway inhibitor, Y-27632. (c) Alcian Blue staining of ATDC5 WT cells vs SHIP2-negative cells differentiated in presence of 15 μM of the ROCK pathway inhibitor, Y-27632. Histograms represent the quantification of the cell staining after extraction. *,‡ p <0.05, **,‡‡ p <0.01, ***,‡‡‡ p <0.001. * WT vs SHIP2-neg. ‡ vehicle vs treatment.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Staining, Extraction

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: In the absence of SHIP2, PI(3,4,5)P3 accumulates, activates ROCK which stimulates the production of ECM through the phosphorylation of Sox9 in chondrocytes. Increased ROCK activity also leads to stabilization of the actin cytoskeleton and inhibition of MV budding and ECM mineralization.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Phospho-proteomics, Activity Assay, Inhibition

Journal: Nature Communications

Article Title: iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

doi: 10.1038/s41467-021-23902-6

Figure Lengend Snippet: a qPCR analysis of mRNA levels of iPLA2β in the MCF-7, U2OS, A375, and H1299 cells treated with 0.2 μg/mL doxorubicin or 10 μM Nutlin for 24 h. b qPCR analysis of mRNA levels of iPLA2β in the U2OS CRISPR control versus p53 −/− cells treated with 10 μM Nutlin for 24 h. c Schematic representation of the promoter region in the human iPLA2β gene. The p53-binding sites upstream of the first exon are indicated as responsive elements (RE). TSS, transcription start site. d ChIP-qPCR was performed in H1299 cells transfected with empty vector or p53. p values were calculated using two-sided unpaired Student’s t -test. Detailed statistical tests are described in the ‘Methods’. p = 0.564 for RE1; p = 0.0000000196 for RE2; p = 0.000118 for RE3; and p = 0.00316 for TIGAR. e H1299 cells were transfected with empty vector, wild-type p53 or mutants (R175H, R273H, and R248W), and iPLA2β mRNA levels were analyzed by qRT-PCR. a , b , d , e Error bars are mean ± s.d., n = 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: To obtain iPLA2β, CRISPR-cas9-knockout U2OS cells were generated by transfecting iPLA2β double nickase plasmid (sc-402107-NIC; Santa Cruz).

Techniques: CRISPR, Binding Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

doi: 10.1038/s41467-021-23902-6

Figure Lengend Snippet: a Western blot analysis of extracts of U2OS cells with different time of 10 μM Nutlin treatment. The experiments were repeated twice, independently, with similar results. b qPCR analysis of mRNA levels of iPLA2β for the above cells. c qPCR analysis of mRNA levels of SLC7A11 for the same cells as ( a ). d Western blot analysis of extracts of U2OS cells with different time of 10 μM Nutlin + TBH treatment. The experiments were repeated twice, independently, with similar results. e qPCR analysis of mRNA levels of iPLA2β for the same cells as ( d ). f qPCR analysis of mRNA levels of SLC7A11 for the same cells as ( d ). g Western blot analysis of extracts of HCT116 cells with low dose (lanes 1, 2) or high dose of doxorubicin (lanes 3, 4) as indicated for 30 h. The experiments were repeated twice, independently, with similar results. b , c , e , f Error bars are mean ± s.d., n = 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: To obtain iPLA2β, CRISPR-cas9-knockout U2OS cells were generated by transfecting iPLA2β double nickase plasmid (sc-402107-NIC; Santa Cruz).

Techniques: Western Blot

Journal: Nature Communications

Article Title: iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

doi: 10.1038/s41467-021-23902-6

Figure Lengend Snippet: a Western blot analysis of extracts of A375 CRISPR control (lanes 1, 2) versus p53 −/− cells (lanes 3, 4) treated with control RNAi (lanes 1, 3) or iPLA2β RNAi (lanes 2, 4) by the antibodies to iPLA2β, p53, p21, or actin. The experiments were repeated twice, independently, with similar results. b Quantification of ROS-induced ferroptotic cell death. The same cells in ( a ) were pre-incubated with 10 μM Nutlin for 24 h, then treated with 120 μM TBH and 10 μM Nutlin (error bars, s.d. from three independent samples). c Quantification of ferroptotic cell death-mediated by ROS. After iPLA2β RNAi treatment, MCF-7 cells were pre-incubated with 10 μM Nutlin for 24 h, then treated with 80 μM TBH and 10 μM Nutlin (error bars, s.d. from three independent samples). d Western blot analysis of extracts of A375 CRISPR control (lanes 1, 2), p53 −/− cells (lanes 3, 4), iPLA2β −/− cells (lanes 5, 6), or p53 −/− ; iPLA2β −/− cells treated with Nutlin 10 μM for 24 h (lanes 2, 4, 6, 8) versus control (lanes 1,3, 5, 7) by the antibodies to iPLA2β, p53, p21, or actin. The experiments were repeated twice, independently, with similar results. e Quantification of cell death in the same cells as ( d ) with additional of 150 μm TBH as indicated. Error bars are mean ± s.d., n = 3 biologically independent experiments. f Quantification of ROS-mediated ferroptotic cell death. U2OS CRISPR control versus iPLA2β −/− cells pre-incubated with Nutlin (10 μM) for 24 h were treated with TBH (250 μM), Nutlin (10 μM) and Ferr-1 (2 μM) as indicated (error bars, s.d. from three independent replicates). Source data are provided as a Source Data file.

Article Snippet: To obtain iPLA2β, CRISPR-cas9-knockout U2OS cells were generated by transfecting iPLA2β double nickase plasmid (sc-402107-NIC; Santa Cruz).

Techniques: Western Blot, CRISPR, Incubation

Journal: Nature Communications

Article Title: iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

doi: 10.1038/s41467-021-23902-6

Figure Lengend Snippet: a Xenograft tumors from A375 CRISPR control, iPLA2β −/− , p53 −/− or p53 −/− ; iPLA2β −/− cells as indicated. b Tumor weights were determined from ( a ) (error bars, from eight tumors). The experiments were repeated twice independently with similar results and representative data were shown. p = 0.00049 for control versus iPLA2β −/− ; p = 0.469 for p53 −/− versus iPLA2β −/− ; p53 −/− . c qPCR of Ptgs2 mRNA from tumors harvested in ( a ). p = 0.00349 for control versus iPLA2β −/− . d A549 cells (Lanes 1, 2), p53 −/− cells (lanes 3, 4), iPLA2β −/− cells (lanes 5, 6), or p53 −/− ; iPLA2β −/− cells (lanes 7, 8) treated with Nutlin 10 µM for 24 h (lanes 2, 4, 6, 8) versus control (lanes 1, 3, 5, 7) by the antibodies to iPLA2β, p53, p21or actin. The experiments were repeated twice, independently, with similar results. e Xenograft tumors from A549 CRISPR control, iPLA2β −/− , p53 −/− or p53 −/− ; iPLA2β −/− cells as indicated. f Tumor weights were determined from ( e ) (error bars, from eight tumors). The experiments were repeated twice independently with similar results and representative data were shown. p = 0.00253 for A549 versus iPLA2β −/− ; p = 0.686 for p53 −/− versus iPLA2β −/− ; p53 −/− . g qPCR of Ptgs2 mRNA from tumors harvested in ( e ). p = 0.00000727 for A549 versus iPLA2β −/− . b , f Error bars are mean ± SEM. c , g Error bars are mean ± s.d., b – c , f – g n = 8 biologically independent samples. All p values were calculated using two-tailed unpaired Student’s t -test. Detailed statistical tests are described in the ‘Methods’. Source data are provided as a Source Data file.

Article Snippet: To obtain iPLA2β, CRISPR-cas9-knockout U2OS cells were generated by transfecting iPLA2β double nickase plasmid (sc-402107-NIC; Santa Cruz).

Techniques: CRISPR, Two Tailed Test

Journal: Nature Communications

Article Title: iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

doi: 10.1038/s41467-021-23902-6

Figure Lengend Snippet: a Western blot analysis of U2OS ACSL4 −/− ; GPX4 −/− cells transfected with AlOX12 or iPLA2β vector. The experiments were repeated twice, independently, with similar results. b Lipid peroxidation levels of U2OS ACSL4 −/− ; GPX4 −/− cells transfected with ALOX12 or iPLA2β vector were treated with TBH (300 μM) and Ferr-1 (2 μM) as indicated for 8 h. c Quantification of lipid peroxidation levels as shown in ( b ). Error bars are mean ± s.d., n = 3 independent experiments. d Western blot analysis of U2OS ACSL4 −/− ; GPX4 −/− cells transfected with iPLA2β or iPLA2γ vector. The experiments were repeated twice, independently, with similar results. e Quantification of Lipid peroxidation levels. U2OS ACSL4 −/− ; GPX4 −/− cells with overexpression of iPLA2β or iPLA2γ were pre-incubated with Nutlin (10 μM) for 12 h, then treated with TBH (300 μM) and Nutlin (10 μM) as indicated for 8 h. f Quantification of cell death. U2OS ACSL4 −/− ; GPX4 −/− cells with overexpression of iPLA2β or iPLA2γ were pre-incubated with Nutlin (10 μM) for 24 h, then treated with TBH (250 μM) and Nutlin (10 μM) as indicated. g Relative content of oxPE (18:0/22:4)sn2 in H1299 cells transfected with alox12, or/and iPLA2β (PE, phosphatidylethanolamines). p values were calculated using two-tailed unpaired Student’s t -test. Detailed statistical tests are described in the ‘Methods’. h Relative content of oxPE (18:0/20:4)sn2 in H1299 cells transfected ALOX12, or/and iPLA2β. i Quantification of cell death in H1299 cells transfected with vector, p53, iPLA2β WT or G517C, and additional TBH treatment. e – i Error bars are mean ± s.d., n = 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: To obtain iPLA2β, CRISPR-cas9-knockout U2OS cells were generated by transfecting iPLA2β double nickase plasmid (sc-402107-NIC; Santa Cruz).

Techniques: Western Blot, Transfection, Plasmid Preparation, Over Expression, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4

doi: 10.1038/s41467-021-23902-6

Figure Lengend Snippet: a Western blot analysis of extracts of U2OS CRISPR control (lanes 1, 2) or FSP1 −/− cells (lanes 3, 4) with overexpression of iPLA2β-V5 (lanes 2, 4) versus empty vector (lanes 1, 3) by the antibodies to FSP1, V5, or actin. The experiments were repeated twice, independently, with similar results. b Quantification of cell death in the same cells as ( a ) treated with 250 μM TBH after pretreatment with Nutlin 10 μM for 24 h. Error bars are mean ± s.d., n = 3 biologically independent experiments. c A model for the roles of ALOX12 and iPLA2β in regulating p53-mediated ferroptosis. d Kaplan-Meier survival curve was generated for overall survival by stratifying patient samples with kidney renal clear cell carcinoma (TCGA, PanCancer Atlas) from cBioPortal based on iPLA2β expression levels. High-expression group was defined as mRNA expression z -score > 1 (PLA2G6: EXP > 1). e Kaplan-Meier survival curve was generated for overall survival by stratifying patient samples with acute myeloid leukemia (TCGA, PanCancer Atlas) from cBioportal based on iPLA2β expression levels. d , e p values were calculated using two-sided unpaired Student’s t -test. Detailed statistical tests are described in the ‘Methods’. Source data are provided as a Source Data file.

Article Snippet: To obtain iPLA2β, CRISPR-cas9-knockout U2OS cells were generated by transfecting iPLA2β double nickase plasmid (sc-402107-NIC; Santa Cruz).

Techniques: Western Blot, CRISPR, Over Expression, Plasmid Preparation, Generated, Expressing

Journal: Molecular cancer research : MCR

Article Title: HILPDA regulates lipid metabolism, lipid droplet abundance, and response to microenvironmental stress in solid tumors.

doi: 10.1158/1541-7786.MCR-18-1343

Figure Lengend Snippet: HILPDA dependent lipid droplet formation. A) Hilpda WT and KO MEFs were kept in complete media (CM), incubated in 1% O2 for 72h or supplemented with 60μM oleate/linoleate/BSA complexes for 24h (FA), and lipid droplets were stained with Nile Red. B) HILPDA protein levels in WT and KO MEFs treated as in A. C) Quantification of LDs per cell in WT and KO MEFs in complete media or supplemented with FA for 24h. D) LD diameter distribution in WT and KO MEFs. Floating bars: minimum to maximum, vertical line at the median. E) Restoration of HILPDA expression in KO MEFs. Cells were transfected with either pLenti-Hilpda-C-Myc-DDK-IRES-neo (KO+Hilpda) or empty vector (KO) and stable transfectants were selected by G418 resistance. Black arrow: endogenous HILPDA, gray arrow: HILPDA-myc-Flag. Asterisk: non specific. F) Nile Red staining of KO MEFs and pool of reintroduced clones following treatment with oleate/linoleate for 24h. G) Genetic manipulations in HCT116 cells. Knockout lines were generated by the nickase CRISPR technology and four independent KO clones after puromycin resistance selection were pooled together. H) Quantification of LD per cell in complete media and after FA loading. I) TLC of hexane/isopropanol extracted lipids separated in cyclohexane:diethyl acetate:acetic acid and stained with primuline. Lipid class standards: CE cholesteryl esters, TAG: triglycerols, FA: fatty acid. ****p<0.0001, N.S. not significant by one way ANOVA and Sidak post hoc test.

Article Snippet: The double nickase CRISPR plasmid set targeting human HILPDA was from Santa Cruz (sc-403510-NIC)

Techniques: Incubation, Staining, Expressing, Transfection, Plasmid Preparation, Clone Assay, Knock-Out, Generated, CRISPR, Selection

Journal: BMC Cancer

Article Title: Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

doi: 10.1186/s12885-019-5771-5

Figure Lengend Snippet: CPEB2A expression in various un-manipulated and manipulated cell lines. a COX-2 , miR-655, miR-526b and CPEB2A were quantified in different (COX-2 disparate) breast cancer cell lines, relative to MCF7 cells. Cell lines (MDA-MB-231 and MCF7-COX-2) with high COX-2 and miRNAs expression showed low expression of CPEB2A (measured with an A/E probe). b Western blots for CPEB2 protein (about 60 kDa, presumably CPEB2 A, identified with an isoform nonselective antibody) revealed a similar trend, high expression of CPEB2 in poorly malignant, low COX-2 expressing MCF7 and T47D cells, and low expression in high COX-2 expressing and metastatic MDA-MB-231 and Hs578T cells. c , d CPEB2 knockout (KO) through a double nickase CRISPR plasmid in MCF10A cells resulted in downregulation of CPEB2 mRNA (shown with RT-PCR) and protein (shown with Western blot). CPEB2 protein expression was knocked out with 80% efficiency. e , f . SiRNA-mediated knockdown of CPEB2 mRNA using a pool of siRNAs in MCF7 cells shows 75% downregulation. Data presented as mean of 3 replicates ± SEM. (*) indicates p < 0.05, (**) p < 0.001, (***) p < 0.0002

Article Snippet: Total CPEB2 was knocked out in MCF10A cells with a CRISPR double nickase plasmid (Santa Cruz, Dallas, TX, USA) targeting exon 1 of the gene, that combines a Cas9 nickase mutant with pairs of guide RNAs to introduce targeted double-strand breaks, ensuring a high knockout specificity [ ].

Techniques: Expressing, Western Blot, Knock-Out, CRISPR, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

doi: 10.1186/s12885-019-5771-5

Figure Lengend Snippet: Increased spheroid formation by CPEB2-KO and CPEB2-KD cells. a Representative images of spheroids (Scale = 60 μm) and ( b ) spheroid forming efficiency (SFE) of WT and CPEB2KO MCF10A cells grown on ultra-low attachment plates for 4 days. SFE is computed as the number of spheroids (> 60 μm) divided by total number of cells plated. CPEB2KO cells showed 5.12-fold increase in SFE ( p < 0.001). c Dot plot of spheroid size (Mann-Whitney Test for statistical significance) showing increased average diameter (WT = 70.92 μm, CPEB2KO = 91.42 μm; p < 0.0005), indicating enhanced spheroid growth rate. d Images of spheroids and ( e ) SFE in Mock and CBEB2-KD MCF7 cells, showing an increase in CPEB2-KD cells. h Representative Western blot and ( g ) quantification (Mean of 3 ± SEM) of β- Catenin protein expression. f qRT-PCR (Mean of 3 ± SEM) for downstream genes of β-Catenin pathway. β-Catenin was increased 1.29 fold ( p = 0.048) in CPEB2KO cells, with increases in downstream target genes CCND1 (3.49 fold, p = 0.039), and AXIN1 (1.298 fold, p = 0.034). No significant change was observed in AXIN2 (1.73 fold change, p = 0.10) or Myc (0.76 fold change, p = 0.051)

Article Snippet: Total CPEB2 was knocked out in MCF10A cells with a CRISPR double nickase plasmid (Santa Cruz, Dallas, TX, USA) targeting exon 1 of the gene, that combines a Cas9 nickase mutant with pairs of guide RNAs to introduce targeted double-strand breaks, ensuring a high knockout specificity [ ].

Techniques: MANN-WHITNEY, Western Blot, Expressing, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

doi: 10.1186/s12885-019-5771-5

Figure Lengend Snippet: SLC markers in Mock and CPEB2KD MCF7 spheroids. Immunofluorescence images for SLC markers ( a ) SOX2, ( b ) NANOG and ( c ) ALDH1 in green, nuclei stained blue with DAPI in spheroids, (scale = 50 μm). Quantitative data for SLC markers, ( d ) for SOX2, ( e ) for NANOG and ( f ) ALDH1 showing an increase in markers bearing cells in CPEB2-KD spheroids compared to MCF7-Scramble spheroids. Data presented as mean of 3 replicates ± SEM (*) indicates p < 0.05

Article Snippet: Total CPEB2 was knocked out in MCF10A cells with a CRISPR double nickase plasmid (Santa Cruz, Dallas, TX, USA) targeting exon 1 of the gene, that combines a Cas9 nickase mutant with pairs of guide RNAs to introduce targeted double-strand breaks, ensuring a high knockout specificity [ ].

Techniques: Immunofluorescence, Staining

Journal: BMC Cancer

Article Title: Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

doi: 10.1186/s12885-019-5771-5

Figure Lengend Snippet: Expression of CPEB2 isoforms A and B in human breast cancer tissues and non- tumor breast tissues. Expression of CPEB2 isoforms A (measured with an A/E probe) and B (measured with a B/D probe) mRNAs were analyzed by qPCR in105 breast cancer tissues (6 samples failing to amplify expression of GAPDH or CPEB2A were removed from the study) and in 20 control (histologically tumor-free) breast tissues. a The control non-tumor tissues expressed relatively higher expression of isoform A, and lower expression of isoform B than tumor tissues, when normalized for GAPDH . They are plotted as A/B ratios of the delta Ct ± SEM. * indicates p < 0.05. b We measured CPEB2A (with an A/E probe) and CPEB2B (with a B/D probe) mRNA in various tumor subsets, ER+, ER-, PR+, PR-, HER2+, HER2-, ER/PR/HER2- normalized to GAPDH. Data presented as delta Ct ± SEM. No significant difference in the expression of A vs B was noted in any subset. c A schema for molecular partners in CPEB2 regulation and action in breast cancer. COX-2 via EP4 activation upregulates two oncogenic miRNAs miR-526b and miR-655, both of which target CPEB2. Tumor suppressor functions of CPEB2 (resulting from the isoform A) are mediated by multiple partners: translational regulation of p53, HIF-1α and Twist-1 mRNAs. In addition CPEB2 appears to suppress COX-2/EP4 expression by hitherto unknown mechanisms

Article Snippet: Total CPEB2 was knocked out in MCF10A cells with a CRISPR double nickase plasmid (Santa Cruz, Dallas, TX, USA) targeting exon 1 of the gene, that combines a Cas9 nickase mutant with pairs of guide RNAs to introduce targeted double-strand breaks, ensuring a high knockout specificity [ ].

Techniques: Expressing, Activation Assay

Journal: Cancers

Article Title: Forchlorfenuron-Induced Mitochondrial Respiration Inhibition and Metabolic Shifts in Endometrial Cancer

doi: 10.3390/cancers16050976

Figure Lengend Snippet: Effect of septin depletion in MFE296 cells. ( A ) Transient knockdown of septin-2 and -7 by siRNAs. ( B ) Septin-7 knockout using the CRISPR/Cas9 system. Cells were transfected with control or septin-7 double nickase plasmid. Following puromycin selection, polyclonal cells were treated with DMSO or FCF (100 μM, 6 h) and subject to immunoblotting with indicated antibodies. ( C ) Effect of FCF on OCR and ECAR in septin depleted cells. Wild-type MFE296 or single cell derived septin-7 knockout was transfected with siRNAs as indicated. After 48 h, cells were split into 96-well plates (for Seahorse analysis) or 6-well plates (for immunoblotting) and allowed to adhere overnight. Afterwards, expression of septins was analyzed by immunoblotting with specific antibodies to each septins (left), or OCR and ECAR were monitored following DMSO or FCF (100 µM) treatment. y -axis: changes after DMSO or FCF injection (right). (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)

Article Snippet: To establish a stable knockout of septin-7, MFE296 cells were transfected with septin-7 Double Nickase plasmid (Santa Cruz Biotechnology, sc-401655) and were selected under puromycin pressure (1.0 μg/mL) for 1–2 weeks.

Techniques: Knockdown, Knock-Out, CRISPR, Transfection, Control, Plasmid Preparation, Selection, Western Blot, Derivative Assay, Expressing, Injection

Journal: Frontiers in Cellular Neuroscience

Article Title: Leptin-dependent neurotoxicity via induction of apoptosis in adult rat neurogenic cells

doi: 10.3389/fncel.2015.00350

Figure Lengend Snippet: List of primary antibodies and details of immunohistochemical procedures used .

Article Snippet: Doublecortin (DCX) , Goat polyclonal, Santa-Cruz , Immature/migrating neurons , 1:200.

Techniques: Immunohistochemical staining