Journal: Oncotarget

Article Title: Abrogation of PIK3CA or PIK3R1 reduces proliferation, migration, and invasion in glioblastoma multiforme cells

doi:

Figure Lengend Snippet: A, Cells were plated on fibronectin or laminin and were imaged every 5 minutes for 24 hours. Average velocity and distance traveled were measured at 6, 8, 12, 16, and 24 hours, and trends were similar for each time point (graph represents the 16 hour time point). In both D54 and SKMG26 cells, PIK3CA knockdown did not result in a statistically significant reduced migration rate on fibronectin. PIK3R1 knockdown reduced migration rates in both lines. D54 lines did not adhere to laminin-coated surfaces. In SKMG26 lines on laminin, both PIK3CA and PIK3R1 knockdown significantly reduced the average rate of cell migration, with the combined knockdown resulting in the greatest decrease in velocity. * represents a p-value ≤ 0.05, and compares a given result to that of the parental line. B, Cells were plated at 2.5x10 5 or 5x10 5 cells/well on BD Biocoat Matrigel Invasion Assay inserts. At 24 or 72 hours, migrated (control) and invaded (experimental) cells were stained and counted. Percent invasion is provided for each cell line. Both PIK3CA and PIK3R1 knockdown resulted in a reduced percent invasion as compared to parental lines and controls. * represents a p-value ≤ 0.05, and compares a given result to that of the parental line.

Article Snippet: Cells were plated in triplicate at 10,000 cells/well in 48-well plates coated with laminin or fibronectin (Becton Dickson).

Techniques: Knockdown, Migration, Invasion Assay, Control, Staining

Journal: bioRxiv

Article Title: Laminin alpha 5 is Necessary for Mammary Epithelial Growth and Function by Maintaining Luminal Epithelial Cell Identity

doi: 10.1101/2019.12.27.889451

Figure Lengend Snippet: a) Representative images of RNA in situ hybridization performed with probes for Lama1 , 3 , 4 and 5 on TEBs of 7-week old pubertal mammary glands. Scale bar 50 μm. b) Representative images of RNA in situ hybridization of mammary glands from E13.5 or E18.5 pregnant mice performed with probes for Lama1 , and 5 on TEBs. Scale bar 50 μm. c) Representative images of Lama5 RNA in situ hybridization (ISH) coupled with either K8 or K14 antibody staining. Scale bars 20 μm. d) Quantification of percentage of K14 or K8 positive cells showing either Lama1 or Lama5 expression. Data show mean ± SD. Two-tailed student’s t-test was used to compare groups. e) Schematic showing Laminin α -chain expression in the TEB and mature ducts of mouse mammary gland. Laminin α -chains expressed in the basal cell layer (pink) are depicted in light red and α -chains expressed in the luminal layer (light blue) are depicted in violet. Stromally expressed laminins are depicted in brown.

Article Snippet: For quantification of cell growth cells were seeded on Laminin coated multiwell plates and placed in Cell-IQ (Chip-Man Tecnologies) cell culture platform.

Techniques: RNA In Situ Hybridization, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Laminin alpha 5 is Necessary for Mammary Epithelial Growth and Function by Maintaining Luminal Epithelial Cell Identity

doi: 10.1101/2019.12.27.889451

Figure Lengend Snippet: a) Schematic showing the experiment outline of tamoxifen induction of K8-CreERT2 prior to pregnancy. Representative images of carmine alum-stained #4 mammary glands of 17.5 days post coitum (dpc) pregnant Lama fl/fl ;- and Lama fl/fl ;K8-CreERT2 animals. Scale bar 10 mm. b) Representative HE images of dpc 17.5 pregnant Lama5 +/+ ;K8-CreERT2 and Lama5 fl/fl ;K8-CreERT2 mice. Scale bar 200 μm. c) Representative immunofluorescence images of dpc 17.5 pregnant Lama5 +/+ ;K8-CreERT2 and Lama5 fl/fl ;K8-CreERT2 glands immunostained with Pan-laminin and E-cadherin or K8 and K14 antibodies. Scale bar 50 μm. d) Representative immunofluorescence images of postpartum day 2 (PP2) mammary glands of Lama fl/fl ;- and Lama5 fl/fl ;K8-CreERT2 mice immunostained with K14 or Pan-laminin and E-cadherin antibodies. Scale bar 50 μm. e) HE staining of PP2 mammary glands of Lama5 fl/fl ;- and Lama5 fl/fl ;K8-CreERT2 mice. f) Quantification of alveolar diameter of mice in e. Data show mean ± SD. Two-tailed student’s t-test was used to compare groups. g) Representative immunofluorescence images of p-STAT5a immunostaining in PP2 mammary glands of Lama fl/fl ;- and Lama5 fl/fl ;K8-CreERT2 , scale bar 50 μm, and quantification of the percentage of positive cells. Data show mean ± SD. h) qRT-PCR analysis of Csn3 , B-cas and Wap expression compared to GAPDH in PP2 lactating Lama fl/fl ;- and Lama5 fl/fl ;K8-CreERT2 mice. Two-tailed student’s t-test was used to compare indicated groups. i) Mean body weight of pups during the first 14 days post partum nursed by Lama5 +/+ , Lama5 fl/+ or Lama5 fl/fl ;K8-CreERT2 mothers. Two-tailed student’s t-test was used to compare Lama5 +/+ ;K8-CreERT2 and Lama5 fl/fl ;K8-CreERT2 groups at day 14.

Article Snippet: For quantification of cell growth cells were seeded on Laminin coated multiwell plates and placed in Cell-IQ (Chip-Man Tecnologies) cell culture platform.

Techniques: Staining, Immunofluorescence, Two Tailed Test, Immunostaining, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Laminin alpha 5 is Necessary for Mammary Epithelial Growth and Function by Maintaining Luminal Epithelial Cell Identity

doi: 10.1101/2019.12.27.889451

Figure Lengend Snippet: a) Schematic showing the outline of the laminin preadhesion assay, in which primary MMECs or HMECs are cultured on 2D LM-111 or LM-521 matrix for 48h. b) Representative immunofluorescence images of FACS sorted luminal and basal MMECs grown on indicated laminins and immunostained with α -tubulin. Scale bar 50 μm. c) Immunofluorescence images of α -tubulin and scanning electron (SEM) micrographs of HMECs grown on the indicated laminins. Scale bars 20 μm. d) GSEA plots showing correlation between the signature of HMECs grown on LM-521 and previously reported gene sets either downregulated in basal MECs (upper panel) or upregulated in basal MECs (lower panel). e) qRT-PCR analysis of KRT8 and SEMA3B expression compared to GAPDH in HMECs grown on indicated laminins for 48h and qRT-PCR analysis of Krt8 and Krt14 expression compared to GAPDH in basal (CD29 hi /CD24 + ) and luminal (CD29 low /CD24 + ) MECs pre-adhered on LM-111 or LM-521 for 48h. Data show mean ± SD. Two-tailed student’s t-test was used to compare indicated groups. f) Mammosphere or aggregate forming frequency of FACS sorted luminal and basal primary MMECs pre-adhered on the indicated laminins 14 days earlier. Data show mean ± SD from independent experiments. g) Schematic showing laminin pre-adhesion assay outline, in which FACS sorted primary MMECs are used for fat pad reconstitution assay either without treatment or after 48 hour of culturing on LM-111 or LM-521 matrix. h) Limiting dilution fat pad reconstitution of basal (CD29 hi /CD24 + ) and luminal (CD29 low /CD24 + ) MECs pre-adhered on LM-111 or LM-521 or untreated controls. Filling of the circles represents the extent of reconstitution (0, 25, 50, 75 or 100%) and each circle represents one gland.

Article Snippet: For quantification of cell growth cells were seeded on Laminin coated multiwell plates and placed in Cell-IQ (Chip-Man Tecnologies) cell culture platform.

Techniques: Cell Culture, Immunofluorescence, Quantitative RT-PCR, Expressing, Two Tailed Test, Cell Adhesion Assay, Reconstitution Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: High throughput phenotypic assay for compounds that influence mitochondrial health using iPSC derived human neurons

doi: 10.1177/24725552211000671

Figure Lengend Snippet: A) i3Neuron differentiation. At day 0, 10X image of iPSCs that were dissociated into single cells and plated in 15cm matrigel coated dishes for differentiation into i3Neurons. B) 10X images of i3Neurons days 1–3 (inset images are at 40X). Doxycycline was added to the media to induce the expression of neurogenin-2, encoded by a stable genomic insertion under the control of a TetOn promoter. By day 3, neuritic extensions were observed (see 40X insert image d3). On day 3, i3Neurons were dissociated as single cells for re-plating or for cryopreservation. C) 10X images of i3Neurons at cell days 5 through 10. These images show i3Neurons plated in PLO laminin coated 384 well plates at 15,000 cells/well. The i3Neurons make extensive networks over these 6 days.

Article Snippet: In all succeeding experiments with i3Neurons, each plate is derived from an individual cryovial of previously differentiated cells and can therefore be considered to be independent of other plates used. i3Neuron lentiviral MOI titration assay i3Neurons, now considered to be at cell day 4, were revived and plated in cortical neuron media: 1X B27 supplement, 10ng/mL BDNF (PeproTech, Rocky Hill, NJ, USA), 10ng/mL NT-3 (PeproTech), 1ug/mL laminin with a BrainPhysTM Neuronal Medium base (StemCell Technologies, Vancouver, Canada) in 384 well plates coated with PLO and laminin (Aurora Microplates, Whitefish, MT, USA). i3Neurons were plated at either 7,500 or 15,000 cells per well in 40μL of cortical neuron media.

Techniques: Expressing, Control

Journal: Molecular Therapy

Article Title: Gentamicin Induces Laminin 332 and Improves Wound Healing in Junctional Epidermolysis Bullosa Patients with Nonsense Mutations

doi: 10.1016/j.ymthe.2020.03.006

Figure Lengend Snippet: Topical Gentamicin Did Not Induce Anti-laminin 332 Antibodies (A) Sera were obtained from GS-JEB patients before treatment and 1 and 3 months after topical gentamicin treatment. These samples, along with appropriate normal human serum (NHS) and positive control sera, were subjected to ELISA on laminin 332-coated plates. Note that none of the patients’ sera exhibited any anti-laminin 332 antibodies at any time points during the study. Data represent the mean ± SD (n = 6). (B) Skin biopsies from the gentamicin-treated sites were obtained 1 and 3 months after treatment and subjected to direct IF using a FITC-conjugated goat anti-human IgG antibody. Note that IgG deposits were not detected at the DEJ for any of the patient samples at 1 and 3 months after treatment. Scale bar, 50 μm.

Article Snippet: The production of serum circulating anti-laminin 332 antibodies was assessed by ELISA using recombinant laminin 332-coated ELISA plates, as previously described, using immobilized recombinant human laminin 332 (BioLamina).

Techniques: Positive Control, Enzyme-linked Immunosorbent Assay