Loss of GJIC disrupts acinar differentiation and primes luminal epithelial cells for cell cycle entry. S1 cells were cultured in 3D for 10 days and treated with AGA (50 μM) or vehicle (A–F). (A) One cell per acinus was ionophoretically microinjected with a mixture of Lucifer Yellow (LY, 2.5% w/v) and rhodamine B isothiocyanate-dextran (RD, 2% w/v) in 0.1 mM LiCl. Cells were observed with epifluorescence microscopy 15 min following injection. Representative acini are shown (n=10 acini in different cell cultures). The gap junction impermeable dye, RD (red), marks the injected cells; LY diffuses throughout the acinus when gap junctions are functional. (B) Phase contrast images with accompanying drawings of a normal-looking acinus (left panel) and an acinus with cells positioned abnormally (right panel, arrows). (C) Immunostaining for β-catenin (green) and DAPI (blue) to better visualize the arrangement of cells. The correct acinar morphology is defined as one layer of cells; the arrow indicates an aberrantly localized (central) cell observed on the optical section through the middle of an acinus. Graph shows the mean±s.e.m. percentages of acini with correct morphology; at least 100 acini analyzed per condition; n=3 (biological replicates corresponding to distinct cell cultures usually of different passages). (D) Mean±s.e.m. percentages of BrdU-positive cells at day 4 of 3D culture; a minimum of 500 cells analyzed per condition; n=3. (E) Dual immunostaining for apical polarity marker ZO-1 (red) and basal polarity marker α6 integrin (green); arrowheads indicate aberrant basal localization of ZO-1. Graph shows mean±s.e.m. percentages of S1 acini with apical location of ZO-1 during (days 1–10) or following (days 9–12) acinar differentiation; at least 100 acini analyzed per condition; n=3. (F) Western blot for ZO-1 shows no effect of AGA treatment on expression levels; lamin B is used as loading control. (G) S1 cells were cultured for nine days in 3D with or without AGA (50 µM) followed by incubation with 100 ng/ml of insulin-like growth factor I (IGF-1) or vehicle for 36 h. Graph shows mean±s.e.m. percentages of cells positive for cell proliferation marker Ki67. In representative dual fluorescence immunostaining for ZO-1 (green) and Ki67 (red) under different treatment conditions, nuclei are counterstained with DAPI (blue). Diffuse ZO-1 distribution is indicated by arrowheads; positive intranuclear Ki67 localization is shown by arrows in some of the cells. At least 100 cells analyzed for each condition, n=3. **P<0.01, ***P<0.001; unpaired t-test (C,D,E) and one-way ANOVA with Dunn's comparison (G). Scale bar: 10 µm.

Downregulation of Cx43 expression disrupts GJIC and apical polarity in S1 cells. (A) Western blot (upper image) and quantitative real-time PCR (bottom graph) for Cx43 expression in S1 cells following retroviral delivery of empty vector (EV) or non-specific sequence shRNA (NSS) used as negative controls, and Cx43-specific shRNA (shRNA). Lamin B serves as loading control; n=3. Cx43 mRNA expression normalized to EV control; data represented as mean±s.e.m. (B) Fluorescence immunostaining for Cx43 (red) in S1 cells treated as indicated and cultured for 10 days in 2D or 3D conditions. Arrows indicate Cx43 foci. Graph shows the mean±s.e.m. percentages of acini that express Cx43 in each treatment condition; at least 200 acini were analyzed per condition; n=3. (C) S1 cells infected with NSS (upper panel) or Cx43 shRNA (lower panel) were cultured in 3D for 10 days and microinjected with 3% NB in 0.15 M LiCl, followed by dual fluorescence staining with streptavidin–FITC and an antibody against Cx43 (red). Merged images show the extent of NB spread within the acini; n=10 acini. (D) Dual immunostaining of acini for Cx43 (red) and β-catenin (green; indicating cell–cell limits) in S1 cells treated as indicated. The peripheral organization of cells around a hollow center (left lower panel) is considered morphologically correct. Graph shows mean±s.e.m. percentages of acini with correct morphology; at least 100 acini analyzed per condition; n=3. (E) Dual immunostaining for Cx43 (red) and ZO-1 (green) in acini formed by S1 cells treated as indicated. The arrow points to the peripheral location of ZO-1 and the asterisk indicates the central location of a cell (optical section through the middle of the acinus), illustrating abnormal morphogenesis. Graph shows mean±s.e.m. percentages of acini with apical ZO-1 staining; at least 100 acini analyzed per condition; n=3. (F) Western blots show unchanged levels of total ZO-1 expression in 10-day-old S1 acini treated as indicated. Lamin B was used as loading control. (G,H) Western blots for Cx43 and co-immunoprecipitated β-catenin (G) or ZO-2 (H) following immunoprecipitation with Cx43 antibody in S1 acini. *P<0.05, **P<0.01, ***P<0.001; one way ANOVA, with Dunn's comparison (A,B), nonpaired t-test (D,E). Nuclei are counterstained with DAPI (blue). Scale bar: 10 µm.

Cx43 expression induces apical polarity in MCF10A acini. (A) Western blots for Cx43 and ZO-1 expression from 10-day 2D and 3D cultures of MCF10A cells that were uninfected (Un) or infected with empty vector (EV) for controls, or infected with HuCx43 vector (HuCx43). Lamin B is used as loading control. (B) MCF10A cells stably infected with EV (MCF10A/EV) or with HuCx43 (MCF10A/HuCx43) were cultured in 3D for 10 days to induce acinar differentiation. Graphs show mean±s.e.m. percentages of acinar structures expressing Cx43 (left) and of acinar structures with Cx43 apically localized among those expressing Cx43 (right). (C) Representative images of dual immunostaining for Cx43 (green) and ZO-1 (red) in acini formed by MCF10A/HuCx43 and by MCF10A/EV with (Cx43+) and without (Cx43−) Cx43 expression. Red arrows on merged image indicate the apical location of ZO-1. Nuclei are counterstained with DAPI (blue). Graph shows mean±s.e.m. percentages of acini with apically localized ZO-1 among those with apical Cx43 and non-apical Cx43. A minimum of 100 acini were analyzed in each condition although the number of acini analyzed was less in EV population due to the paucity of structures expressing ZO-1 in C; n=3; unpaired-test (B) and one-way ANOVA with Dunn's comparison (C), **P<0.01, ***P<0.001 (significance only shown for comparison of interest in C). Scale bar: 10 µm.

Absence of luminal expression of Cx43 is associated with cell multilayering. (A–C) Immunohistochemical staining for Cx43 was performed on archival breast tissue biopsy sections from 22 women with no history of breast cancer. Graphs show mean±s.e.m. percentages of acini with apical localization of Cx43 in the luminal epithelium (in addition to basal myoepithelial localization; see drawing in red) and basal localization of Cx43 only (A); percentages of acini displaying typical (one layer of luminal cells at the inner side of the layer of myoepithelial cells) or atypical (piling up of cells) organization depending on the location of Cx43 (B), n=22 acini; paired t-test. (C) Representative images of acini with basal (red arrows) and apical (orange arrowheads) Cx43 localization in a typical (normal-appearing) epithelial structure and only basal Cx43 (red arrows) in an atypical structure. Nuclei are stained with hematoxylin (blue). (D) S1 cells stably silenced for Cx43 expression were cultured in 3D for 10 days and immunolabeled for Cx43. Graph shows mean±s.e.m. percentages of acini with a monolayer of cells in acini of cells transduced with non-specific sequence (NSS) control and Cx43 shRNA. Shown are representative images of a monolayered acinus with Cx43 (red) apically localized and a multilayered (arrows) acinus lacking Cx43 staining. Nuclei are stained with DAPI. At least 100 acini analyzed; n=3, unpaired t-test. **P<0.01, ***P<0.001. Scale bars: 10 µm.

Cx43-mediated GJs regulate mitotic spindle orientation (MSO). (A–D) S1 cells were cultured in 3D and treated, pre-lumen assembly, with vehicle or with AGA until day 4. Resulting multicellular structures were dual-immunostained for Ki67 (red) and α-tubulin (green). MSO was analyzed based on the directionality of the α-tubulin poles, either parallel to the basement membrane (or tangential to the circumference of the growing acini), which is the proper MSO to maintain a monolayered epithelium, or non-tangential to the circumference (conducive to cell multilayering) as drawn in A, and shown on representative structures in B. Red arrows in α-tubulin panels point to mitotic spindles and double-headed arrows in merge panels indicate MSO; the asterisk indicates the center of the acinar structure; nuclei are counterstained with DAPI (blue). (C) Graph shows mean±s.e.m. percentages of S1 cells that demonstrate proper MSO; at least 60 mitotic cells analyzed per group; n=3; non-paired t-test. (D) Mean±s.e.m. percentages of acinar structures with mitotic cells that show ‘correct’ MSO depending on the location of Cx43 (usually only one mitotic cell seen per acinus). At least 60 cells in each treatment group analyzed; n=3. (E) Representative acinar structures of S1 cells transduced with non-specific sequence (NSS) shRNA or with Cx43 shRNA (shRNA-Cx43) and immunostained for α-tubulin (red); double-headed arrows indicate MSO; the asterisk indicates the center of the acinar structure; nuclei are counterstained with DAPI (blue). Graph shows mean±s.e.m. percentages of S1 cells that show proper MSO; at least 60 mitotic cells analyzed per group; n=3; unpaired t-test. (F,G) MCF10A/HuCx43 and MCF10A/EV cells were cultured in 3D for four days and dual immunostained for Cx43 and α-tubulin. Graphs show mean±s.e.m. percentages of cells that display ‘correct’ MSO; n=3 (F) and percentages of MCF10A/HuCx43 acini with mitotic cells that that show ‘correct’ MSO depending on the location of Cx43 (G). At least 60 mitotic cells analyzed per group; n=3. *P<0.05, **P<0.01, ***P<0.001; unpaired t-test (C,F,G), one-way ANOVA with Dunn's comparison (D). Scale bars: 10 µm.

Regulation of MSO by Cx43 involves PI3K–pAKT. (A,B) S1 cells were treated with vehicle (control) or with AGA, or with 1 μM of PI3K inhibitor LY294002 (LY), or with AGA+LY294002 pre-lumen assembly and until day 4 in 3D culture. Graphs show mean±s.e.m. percentages of cells with proper MSO (i.e. tangential to the acinus circumference) based on immunostaining for α-tubulin and analysis of at least 60 mitotic cells per group (A), and mean±s.e.m. ratios of expression levels of downstream effector of PI3K, p-AKT, over AKT, normalized to the control group based on quantification of western blot bands (B); n=3, one-way ANOVA with Dunn's comparison. A representative image of western blots for one of the replicates is also shown in B; lamin B is used as loading control. (C) Representative images of western blots for PI3K, AKT and p-AKT expression from 3D cultures of S1 cells transduced with Cx43 shRNA (shRNA), non-specific sequence shRNA (NSS) or empty vector (EV). Graph shows mean±s.e.m. relative levels of p-AKT expression compared to total AKT upon Cx43 silencing, normalized to expression in NSS control cells. (D) S1 cells were treated with 5 μM aPKC pseudosubstrate inhibitor until day 4 of 3D culture. Graph shows mean±s.e.m. percentages of cells with MSO tangential to the acini circumference based on α-tubulin immunostaining and analysis of a minimum of 60 cells; n=3; *P<0.05, ***P<0.001; unpaired t-test.