Journal: The Journal of cell biology

Article Title: The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover.

doi: 10.1083/jcb.201904165

Figure Lengend Snippet: Figure 2. APC-m4 alters actin dynamics at FAs. U2OS cells were depleted of endogenous APC and rescued with refractory APC constructs (APC-WT or APC-m4) along with plasmids ex- pressing GFP-actin and mCherry-zyxin. (A) FRAP analysis, in which ROI were selected where GFP- actin and mCherry-zyxin signals overlap (see orange box in cartoon). ROIs were then bleached and monitored for GFP-actin fluorescence re- covery. Graphs show mean recovery profiles normalized to zero after bleaching. Data aver- aged from three independent experiments (n = 30 ROIs from n = 15 cells per condition). (B) FRAP experiments as in A except that ROIs were se- lected along stress fibers at a distance (>5 µm) from FAs (see orange box in cartoon). Graphs show mean recovery profiles normalized to zero after bleaching. Data averaged from three inde- pendent experiments (n = 30 ROIs from n = 15 cells per condition). (C) Average time to 50% maximal recovery for experiments in A. Error bars, SEM. Statistical significance calculated by non- parametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (D) Average immobile fraction (does not recover in observation window) for experiments in A. Error bars, SEM. Statistical significance calculated by nonparametric Mann– Whitney two-tailed Student’s t test: *, P < 0.05. (E) Average time to 50% maximal recovery for experiments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann– Whitney two-tailed Student’s t test: n.s., not sig- nificant. (F) Average immobile fraction for ex- periments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann– Whitney two-tailed Student’s t test: n.s., not significant.

Article Snippet: For immunoprecipitations (Fig. 6 I and Fig. S5 E), cells were transfected as described above with plasmids expressing fulllength APC (WT or m4), GFP-LC3 (11546; Addgene), GFPLAMP1 (34831; Addgene), and/or GFP empty vector (54522; Addgene).

Techniques: Construct, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of cell biology

Article Title: The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover.

doi: 10.1083/jcb.201904165

Figure Lengend Snippet: Figure 3. APC-m4 decreases the levels and/or densities of key molecular components at FAs. All data are from MDA-MB-231 cells expressing APC constructs (APC-WT or APC-m4), using fixed or live cell imaging as indicated. (A) Representative immunostaining of endogenous active phospho-Src detected by confocal imaging. Scale bar, 40 µm. Green boxed regions correspond to zoom panels (right; scale bar, 10 µm), which highlight the localization of phospho- Src at the cell periphery. (B) Representative immunostaining of endogenous active phospho-paxillin detected by confocal imaging. Scale bar, 25 µm. Green boxed regions correspond to zoom panels (right; scale bar, 5 µm), which highlight the localization of phospho-paxillin at the cell periphery. (C) Representative immunostaining of endogenous active phospho-FAK tyrosine kinase detected by confocal imaging. Scale bar, 25 µm. Green boxed regions correspond to zoom panels (right; scale bar, 5 µm), which highlight the localization of phospho-FAK at the cell periphery. (D) Densities of endogenous phospho-Src, phospho- paxillin, and phospho-FAK determined from cell images as in A–C. Data averaged from three independent experiments. n ≥56 cells for phospho-Src, n = 35 cells for phospho-paxillin, and n ≥103 cells for phospho-FAK per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (E) Representative SIM images of cells immunostained for phospho-Src (green) and paxillin (pink). Scale bar, 5 µm. White boxed regions correspond to zoom panels (right; scale bar, 2 µm), highlighting the localization of phospho-Src and paxillin at FAs. (F) Representative SIM images of cells immunostained for phospho-paxillin (green) and paxillin (pink). Scale bar, 5 µm. White boxed regions correspond to zoom panels (right; scale bar, 2 µm), highlighting the localization of phospho-paxillin and paxillin at FAs. (G) Representative SIM images of cells immunostained for phospho-FAK (green) and paxillin (pink). Scale bar, 5 µm. White boxed regions correspond to zoom panels (right; scale bar, 2 µm), highlighting the localization of phospho-paxillin and paxillin at FAs. (H) Density of phospho-Src, phospho-paxillin, and phospho-FAK staining at individual FAs from cell images as in E–G. Data averaged from two independent experiments. n = 50 FAs total from 15 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001; **, P < 0.01. (I) Densities of signals at FAs for different components: GFP-paxillin and mCherry-zyxin densities

Article Snippet: For immunoprecipitations (Fig. 6 I and Fig. S5 E), cells were transfected as described above with plasmids expressing fulllength APC (WT or m4), GFP-LC3 (11546; Addgene), GFPLAMP1 (34831; Addgene), and/or GFP empty vector (54522; Addgene).

Techniques: Expressing, Construct, Live Cell Imaging, Immunostaining, Imaging, MANN-WHITNEY, Two Tailed Test, Staining

Journal: The Journal of cell biology

Article Title: The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover.

doi: 10.1083/jcb.201904165

Figure Lengend Snippet: Figure 6. APC-m4 alters autophagosome dynamics at FAs. All data are from live cell TIRF imaging of migrating MDA-MB-231 cells expressing APC constructs (APC-WT or APC-m4), along with markers for autophagosomes (GFP-LC3) and FAs (mCherry-zyxin). For all panels, data are averaged from at least three experiments. (A) Representative time-lapse imaging showing autophagosomes (GFP-LC3, cyan) and FAs (mCherry-zyxin, pink). Time = 0 corresponds to maximum mCherry-zyxin fluorescence intensity (peak FA growth). Scale bar, 3 µm. (B) Percentage of mature FAs targeted by autophagosomes, analyzed from experiments as in A. n > 800 FAs per condition from n ≥20 cells per condition. Error bars, SEM. Statistical significance calculated by one-way ANOVA Dunn’s multiple comparisons test: n.s., not significant. (C) Histograms showing distributions of mature FAs targeted by autophagosomes in the 30-min observation window, from experiments as in A. n = 40 FAs from n > 5 cells per condition. (D) Scatter plot showing dwell times of autophagosomes at FAs, analyzed from experiments as in A. n ≥42 autophagosomes per condition from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (E) Scatter plot showing time after first appearance of an autophagosome at the FA to complete FA disassembly, analyzed from experiments as in A. n = 31 FAs (APC-WT) or n = 50 FAs (APC-m4) from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (F) Overlaid histograms showing time elapsed from peak FA maturity (time = 0) to arrival of autophagosome during the 40-min observation window. Negative numbers correspond to rare events in which auto- phagosomes arrive before FA peak maturation. Data are analyzed from experiments as in A. n = 20 FAs from n > 5 cells per condition. (G) Percentage of mature FAs targeted by GFP-NBR1 receptor, analyzed from live imaging experiments as in A, except using cells expressing mCherry-zyxin and GFP-NBR1. n = 100 FAs (from 15 cells) per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not sig- nificant. (H) Scatter plot showing dwell times of GFP-NBR1 interactions with FAs, analyzed from live imaging experiments as in G. n = 40 GFP-NBR1 visits to

Article Snippet: For immunoprecipitations (Fig. 6 I and Fig. S5 E), cells were transfected as described above with plasmids expressing fulllength APC (WT or m4), GFP-LC3 (11546; Addgene), GFPLAMP1 (34831; Addgene), and/or GFP empty vector (54522; Addgene).

Techniques: Imaging, Expressing, Construct, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Journal of Pharmaceutical and Biomedical Analysis

Article Title: Forced degradation studies of medroxyprogesterone acetate injectable suspensions (150 mg/ml) with implementation of HPLC, mass spectrometry, and QSAR techniques

doi: 10.1016/j.jpba.2020.113352

Figure Lengend Snippet: Chemical structures for medroxyprogesterone acetate (MPA) and its known impurities . A - 6-hydroxymedroxyprogesterone acetate (6β-hydroxy-6-methyl-3,20-dioxopregn-4-en-17-yl acetate); B – medroxyprogesterone (17-hydroxy-6α-methylpregn-4-ene-3,20-dione); C - 6α,17α-dimethyl-3,17-dioxo- d -homoandrost-4-en-17α-yl acetate; D - 6-epimedroxyprogesterone acetate (6β-methyl-3,20-dioxopregn-4-en-17-yl acetate); E - 6-methylenehydroxyprogesterone acetate (6-methylidene-3,20-dioxopregn-4-en-17-yl acetate); F - 4,5-dihydromedroxyprogesterone acetate (6α-methyl-3,20-dioxo-5β-pregn-17-yl acetate); G - megestrol acetate (6-methyl-3,20-dioxopregna-4,6-dien-17-yl acetate); H - hydroxyprogesterone acetate (3,20-dioxopregn-4-en-17-yl acetate); I - 17β-hydroxy-6α,17α-dimethyl- d -homoandrost-4-en-3,17-dione.

Article Snippet: Medroxyprogesterone acetate for system suitability CRS (contains impurities A, B, C, D, E, G, I) and medroxyprogesterone acetate for peak identification (containing impurity F) were obtained from the European Pharmacopoeia through the European Directorate for the Quality of Medicines and Healthcare (EDQM, Strausbourg, France).

Techniques:

Journal: Journal of Pharmaceutical and Biomedical Analysis

Article Title: Forced degradation studies of medroxyprogesterone acetate injectable suspensions (150 mg/ml) with implementation of HPLC, mass spectrometry, and QSAR techniques

doi: 10.1016/j.jpba.2020.113352

Figure Lengend Snippet: Example chromatogram for Reference Solution A with assignments for respective medroxyprogesterone impurities [ , , ], including relative retention times and +/- 2% RRT range observed for implemented chromatographic conditions.

Article Snippet: Medroxyprogesterone acetate for system suitability CRS (contains impurities A, B, C, D, E, G, I) and medroxyprogesterone acetate for peak identification (containing impurity F) were obtained from the European Pharmacopoeia through the European Directorate for the Quality of Medicines and Healthcare (EDQM, Strausbourg, France).

Techniques: