puromycin construct  (Gene Design Inc)

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Representative images of day 6 neurons after Lysotracker treatment. Lysotracker is shown in magenta, and DAPI in blue. Scale bar = 20 μm. (b) Quantification of Lysotracker signal in day 6 neurons. The relative Lysotracker intensity of CLCN4 -variant neurons compared to the WT was presented. Each dot represents one biological replicate. (n=3-4, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test * p =0.039 for variant A; *** p =0.0007 for variant B; ** p =0.0092 for variant C) The magenta dashed line indicates the value for WT. (c) Relative acidity in acidic subcellular organelles. Each dot represents one biological replicate. (n=3, mean ± SEM, unpaired Student’s t-test; p =0.2894) (d-g) Dynamics of endo-lysosomal vesicles. RAB5, RAB7, RAB11 and LAMP1 indicates early endosome, late endosome, recycling endosome and lysosome markers, respectively. (RAB5, n=6; RAB7, n=3; RAB11, n=4; and LAMP1, n=3 or 4; mean ± SEM, unpaired Student’s t-test) (d) Representative images of a neuron with GFP-labeled vesicular organelles. TUJ1 and DAPI are shown in red and blue, respectively. Scale bar = 10 μm. (e) Relative GFP intensity expressed in vesicular organelles (* p =0.0265 for RAB5, * p =0.0158 for RAB7, * p =0.0475 for RAB11, and ** p =0.0067 for LAMP1) (f) Number of GFP-expressing vesicles in neurons. (* p =0.0186 for RAB5, * *p =0.0066 for RAB7, and p =0.9031 for LAMP1) (g) Average distance of GFP-expressing vesicles from the center of nucleus, representing the mean linear distance of puncta from the center of the nucleus in each cell. (** p =0.0011 for RAB5, * p =0.0463 for RAB7, and * p =0.0103 for LAMP1) (h) Scheme of autophagic flux analysis using tandem fluorescent protein fused to LC3. (i) Representative images of day 6 neurons expressing tandem fluorescent-tagged LC3. Red: RFP, green: GFP, white: TUJ1. Scale bar = 5 μm. (j) Quantification of the GFP-/RFP+ puncta. (n=5, mean ± SEM, unpaired Student’s t-test; * p =0.0164)

Article Snippet: For specific subcellular vesicle visualization, lentiviral constructs encoding GFP-tagged RAB5 (Addgene #134858), RAB7 (Addgene #133027), RAB11 (Addgene #134860), and LAMP1 (Addgene #134868) were utilized.

Techniques: Variant Assay, Labeling, Expressing

Journal: Communications Biology

Article Title: The calcium pump PMCA4b promotes epithelial cell polarization and lumen formation

doi: 10.1038/s42003-025-07814-5

Figure Lengend Snippet: a Western blot analysis of parental, PMCA4 specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines; exp. denotes time of exposure. b E-cadherin immunostaining of parental, PMCA4-specific shRNA (sh-PMCA4) and GFP-PMCA4b-expressing MCF-7 cell lines. DAPI (4′, 6-diamidino-2-phenylindole, blue) labels nuclei. c Outline of the E-cadherin positive cell compartments on B generated by the ImageJ software. d Fluorescence intensity profiles of E-cadherin and GFP-PMCA4b corresponding to the white line across the cells shown in ( b ). e Statistical analysis of the number of E-cadherin positive vesicles in parental, PMCA4 specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cells. Graph displays the means of the number of intracellular vesicles per cell with 95% confidence intervals; data were collected from 2 independent experiments, n (MCF-7) = 13, n (sh-PMCA4) = 17, n (PMCA4b) = 13, n ( PMCA4b LA ) = 16 images. Data were analyzed with Kruskal–Wallis and Dunn’s multiple comparisons tests, adjusted p values: ** p < 0.01, *** p < 0.001. Non-significant differences are not indicated. f Model of the interaction between PMCA4b and the Scrib-Dlg1-Lgl polarity complex. PDZb denotes the PDZ-binding sequence motif of PMCA4b. g Dlg1 immunostaining of GFP-PMCA4b-expressing MCF-7 cells. Insets show magnification of the area framed by the dotted line. DAPI labels nuclei. h Dlg1 immunostaining of parental, PMCA4 specific shRNA (sh-PMCA4) and GFP-PMCA4b-expressing MCF-7 cells. DAPI labels nuclei. i Outline of the Dlg1 positive cell compartments on ( h ) generated by the ImageJ software. j Fluorescence intensity profiles of Dlg1 and GFP-PMCA4b corresponding to the white line across the cells shown on ( h ). k Statistical analysis of the number of Dlg1 positive particles in parental, PMCA4-specific shRNA (sh-PMCA4) and GFP-PMCA4b-expressing MCF-7 cells. Graph displays the means of the number of intracellular vesicles per cell with 95% confidence intervals; data were collected from 3 independent experiments, n (MCF-7) = 14, n (sh-PMCA4) = 15, n (PMCA4b) = 17, n ( PMCA4bLA ) = 14 images; data were analyzed with Kruskal–Wallis and Dunn’s multiple comparisons tests; adjusted p values: *** p < 0.001; non-significant difference is not labeled.

Article Snippet: In order to maintain the stable expression of PMCA4b transgenes and of the shRNA construct, 100 ng/ml puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology) was added to the culture media.

Techniques: Western Blot, shRNA, Expressing, Immunostaining, Generated, Software, Fluorescence, Binding Assay, Sequencing, Labeling

Journal: Communications Biology

Article Title: The calcium pump PMCA4b promotes epithelial cell polarization and lumen formation

doi: 10.1038/s42003-025-07814-5

Figure Lengend Snippet: a Western blot analysis for ezrin in parental, PMCA4-specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines. b Ezrin immunostaining of parental, PMCA4-specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines. DAPI labels nuclei. c Fluorescence intensity profiles of ezrin and GFP-PMCA4b corresponding to the white lines shown on ( b ). d Statistical analysis of cell polarization rate based on ezrin distribution in parental, PMCA4-specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell cultures. Data were collected from 16 fields of view from 2 independent experiments; n (MCF-7) = 1589, n (sh-PMCA4) = 1699, n (PMCA4b) = 1462, n ( PMCA4b LA ) = 1511 cells. Data were analyzed with chi-square test, p values: ** p < 0.01, *** p < 0.001; non-significant difference is not labeled.

Article Snippet: In order to maintain the stable expression of PMCA4b transgenes and of the shRNA construct, 100 ng/ml puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology) was added to the culture media.

Techniques: Western Blot, shRNA, Expressing, Immunostaining, Fluorescence, Labeling

Journal: Communications Biology

Article Title: The calcium pump PMCA4b promotes epithelial cell polarization and lumen formation

doi: 10.1038/s42003-025-07814-5

Figure Lengend Snippet: a Intracellular Ca 2+ was measured with the R-GECO fluorescence indicator in parental, PMCA4 specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cells. 10 μM ATP was added at the 120 s mark. Signals from individual cells are illustrated using different colored lines. Number of analyzed cells: n (MCF-7) = 12, n (sh-PMCA4) = 15, n (PMCA4b) = 18, n ( PMCA4b LA ) = 14. Data are derived from 2 independent experiments. b Area under curve (AUC) values were calculated from curves in panel ( a ) and analyzed with Kruskal–Wallis and Dunn’s multiple comparisons tests; adjusted p values: ** p < 0.01, *** p < 0.001; non-significant difference is not labeled. Graph displays means with 95% confidence intervals. c First peak F/F 0 maximum values were calculated from data in ( a ) and analyzed with Kruskal–Wallis and Dunn’s multiple comparisons tests; adjusted p values: ** p < 0.01, *** p < 0.001; non-significant difference is not labeled. Error bars show 95% confidence intervals.

Article Snippet: In order to maintain the stable expression of PMCA4b transgenes and of the shRNA construct, 100 ng/ml puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology) was added to the culture media.

Techniques: Fluorescence, shRNA, Expressing, Derivative Assay, Labeling

Journal: Communications Biology

Article Title: The calcium pump PMCA4b promotes epithelial cell polarization and lumen formation

doi: 10.1038/s42003-025-07814-5

Figure Lengend Snippet: a WGA uptake assay was performed on parental, PMCA4-specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines. After 10 min incubation with WGA, cells were washed and transferred to a 37 °C incubator for 20 min. Dashed white squares indicate areas for magnification on ( d ). DAPI labels nuclei. b Yellow lines connect the center of the cell nucleus with WGA positive endocytic vesicles accumulated in the cytosol. c Intracellular distribution analysis of WGA positive vesicles. Red line indicates the distribution fit curve. Data were collected from 2 independent experiments, n (MCF-7) = 1214, n (sh-PMCA4) = 922, n (PMCA4b) = 1021, n ( PMCA4b LA ) = 1493 vesicles from 15 to 28 cells, and analyzed as described in the Methods section. d Statistical analysis of the distribution of WGA positive vesicles is illustrated by a box plot diagram with medians, interquartile range boxes and whiskers. Interquartile rage boxes represent the middle 50% of the data. Whiskers represent the ranges for the bottom 25% and the top 25% of the data values. Data were the same as ( c ) and analyzed with Levene’s test. P values: ** P < 0.01, *** P < 0.001; non-significant difference is not labeled. e Magnification of th e areas indicated by the dashed white squares on ( a ). Yellow arrows indicate colocalized WGA and PMCA4b positive vesicles.

Article Snippet: In order to maintain the stable expression of PMCA4b transgenes and of the shRNA construct, 100 ng/ml puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology) was added to the culture media.

Techniques: shRNA, Expressing, Incubation, Labeling

Journal: Communications Biology

Article Title: The calcium pump PMCA4b promotes epithelial cell polarization and lumen formation

doi: 10.1038/s42003-025-07814-5

Figure Lengend Snippet: a Phalloidin staining of parental, PMCA4 specific-shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines. White arrows point to the pre-lumens. b Phalloidin staining of GFP-PMCA4b-expressing MCF-7 cells. The white line encircles a pre-lumen. White arrows point to phalloidin and PMCA4b double-positive vesicles. c WGA uptake assay on GFP-PMCA4b-expressing MCF-7 cells with 20 min incubation after WGA removal. Pre-lumen creating cells are encircled with white line. d A schematic figure of a typical pre-lumen structure in ( a ) 2D cell culture. e Statistical analysis of pre-lumen formation in control and NAV2729 treated parental, PMCA4 specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines. Graph displays means with 95% confidence intervals corrected with the total area of the images; data were collected from 2 independent experiments, n (sh-PMCA4) = 23, n (sh-PMCA4+NAV2729) = 21, n (MCF-7) = 26, n (MCF-7 + NAV2729) = 22, n (PMCA4b) = 25, n (PMCA4b+NAV2729) = 23, n ( PMCA4b LA ) = 23, n ( PMCA4b LA + NAV2729 ) = 17 images. Data were analyzed with Kruskal–Wallis and Dunn’s multiple comparisons tests, adjusted p values: ns p ≥ 0.05, * p < 0.05, ** p < 0.01 *** p < 0.001; non-significant differences are not labeled. f Phalloidin staining of control and NAV2729 treated PMCA4 specific shRNA (sh-PMCA4) and GFP-PMCA4b-expressing MCF-7 cell lines. Scaled-down insets show the GFP-PMCA4b signal. The white arrowhead points to a pre-lumen.

Article Snippet: In order to maintain the stable expression of PMCA4b transgenes and of the shRNA construct, 100 ng/ml puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology) was added to the culture media.

Techniques: Staining, shRNA, Expressing, Incubation, Cell Culture, Control, Labeling

Journal: Communications Biology

Article Title: The calcium pump PMCA4b promotes epithelial cell polarization and lumen formation

doi: 10.1038/s42003-025-07814-5

Figure Lengend Snippet: a Phalloidin staining of mammospheres formed by parental, PMCA4-specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines cultured in Matrigel for 10 days. b Schematic figures demonstrating structures of mammospheres based on results on ( a ). c Statistical comparisons of the ratios of mammospheres with central lumen in 3D cultures between parental, PMCA4-specific shRNA (sh-PMCA4), GFP-PMCA4b- and GFP-PMCA4b LA -expressing MCF-7 cell lines cultured in Matrigel. Graph displays means with 95% confidence intervals, n = 8 images with 10–15 mammospheres per image for each cell type. Data were analyzed with ordinary one-way ANOVA and Tukey’s multiple comparisons tests, adjusted p values: ns p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, non-significant differences are not labeled. d Transmission electron microscope images of 14 days old mammospheres formed by parental, PMCA4-specific shRNA (sh-PMCA4), GFP-PMCA4b and GFP-PMCA4b LA -expressing MCF-7 cell lines cultured in Matrigel. Insets show magnified areas indicated by the dashed black squares.

Article Snippet: In order to maintain the stable expression of PMCA4b transgenes and of the shRNA construct, 100 ng/ml puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology) was added to the culture media.

Techniques: Staining, shRNA, Expressing, Cell Culture, Labeling, Transmission Assay, Microscopy