Journal: Scientific Reports

Article Title: Inhibition of connexin 43 prevents trauma-induced heterotopic ossification

doi: 10.1038/srep37184

Figure Lengend Snippet: ( A ) Small hairpin RNAs (shRNA) were used to knock down Cx43 expression in MC-3T3 cells, and a scrambled sequence was used as a control. The expression of Cx43 was detected by Western blot. ( B ) MC-3T3 cells transfected with Cx43 shRNA were induced in osteogenic medium. Alizarin Red S (week 2) and ALP (week 1) staining was performed. ( C ) Calcium deposits and ALP activity were quantified. ( D ) mRNA expression of osteogenic marker genes was detected by real-time PCR at week 1 and 2. ( E ) ERK pathway activation and Cx43 expression were examined weekly by Western blot. ( F ) A densitometric analysis of p-ERK expression was performed (t-test, n = 3, *p < 0.05; NS, not significant).

Article Snippet: MC-3T3 cells were transfected with lentiviral particles loaded with either small hairpin RNA (shRNA) against Cx43 or ERK (Santa Cruz Biotechnology, Santa Cruz, CA; Cat No. sc-35091–SH for Cx43, Cat No. sc-44206-SH for ERK) or a scrambled control (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: shRNA, Knockdown, Expressing, Sequencing, Control, Western Blot, Transfection, Staining, Activity Assay, Marker, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Scientific Reports

Article Title: Inhibition of connexin 43 prevents trauma-induced heterotopic ossification

doi: 10.1038/srep37184

Figure Lengend Snippet: Tenotomy mice were injected with Cx43 shRNA or scrambled RNA weekly at the lesion site. ( A ) HO was visualized by a weekly radiograph. ( B ) The HO volumes were quantified. ( C ) At week 8, a three-dimensional reconstruction (Left), cross-section (Right upper) and sagittal section (Right below) of microCT scans of Cx43 shRNA- and control RNA-treated tenotomy mice were obtained. The red, green and blue lines represent the cross, sagittal and coronal plane in other sections, respectively. ( D ) The HO volumes were quantified. ( E ) The bone mineral density (BMD) of HO was qualified. ( F ) Expression of Cx43, p-ERK, ERK, Runx2, BSP and β-actin was examined by Western blot. ( G ) A densitometric analysis of Cx43, p-ERK, Runx2 and BSP protein expression was performed. (t-test, n = 3, *p < 0.05).

Article Snippet: MC-3T3 cells were transfected with lentiviral particles loaded with either small hairpin RNA (shRNA) against Cx43 or ERK (Santa Cruz Biotechnology, Santa Cruz, CA; Cat No. sc-35091–SH for Cx43, Cat No. sc-44206-SH for ERK) or a scrambled control (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Injection, shRNA, Control, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Inhibition of connexin 43 prevents trauma-induced heterotopic ossification

doi: 10.1038/srep37184

Figure Lengend Snippet: ( A ) Tenotomy mice were treated by local injection of Cx43 shRNA or intraperitoneal injection of U0126. HO tissues were decalcified, and expression of Cx43, p-ERK, ERK, Runx2 and BSP was detected by immunohistochemistry at week 8. ( B ) Cx43, p-ERK, Runx2 and BSP expression in the HO tissues was quantified.

Article Snippet: MC-3T3 cells were transfected with lentiviral particles loaded with either small hairpin RNA (shRNA) against Cx43 or ERK (Santa Cruz Biotechnology, Santa Cruz, CA; Cat No. sc-35091–SH for Cx43, Cat No. sc-44206-SH for ERK) or a scrambled control (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Injection, shRNA, Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: Inhibition of connexin 43 prevents trauma-induced heterotopic ossification

doi: 10.1038/srep37184

Figure Lengend Snippet: ( A ) Normal bones (n = 15) and HO tissues (n = 20) were decalcified. Expression of Cx43 and p-ERK was detected by immunohistochemistry. ( B ) Quantification of Cx43 and p-ERK expression. ( C ) A typical radiographic three-dimensional reconstruction CT scan is shown for elbow HO with weak and strong expression of Cx43. ( D ) The volumes of the HO with weak and strong Cx43 expression were quantified. (t-test, *p < 0.05). ( E ) The overall recurrence-free survival of patients with weak and strong expression of Cx43 is shown. ( F ) The overall recurrence-free survival of patients with weak and strong expression of p-ERK is shown. (log-rank test).

Article Snippet: MC-3T3 cells were transfected with lentiviral particles loaded with either small hairpin RNA (shRNA) against Cx43 or ERK (Santa Cruz Biotechnology, Santa Cruz, CA; Cat No. sc-35091–SH for Cx43, Cat No. sc-44206-SH for ERK) or a scrambled control (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Immunohistochemistry, Computed Tomography

Journal: Life Science Alliance

Article Title: Specific N-cadherin–dependent pathways drive human breast cancer dormancy in bone marrow

doi: 10.26508/lsa.202000969

Figure Lengend Snippet: (A) Representative images of tissue sections from biopsies of BC patients (A and S2), hematological malignancy (S3), and benign tumor (S3). The slides were colabeled with anti–CDH2-PE (red), anti–Cx43-AF488 (green), and anti–pan-cytokeratin-AF405 (blue). Images were acquired with EVOS FL Auto 2, 200× magnification. Black arrows show colocalized Cx43 and CDH2 in the white areas (red + green + blue). Inset, zoomed regions of colocalized proteins. (B) Table summarizes the total number of sections positive for colocalized CDH2, Cx43 in pan-cytokeratin + cells. ImageJ software was used to count the colocalized cells in 10 fields/slide. The last column shows the percentages of colocalized CDH2-Cx43. See also Table S1 and and . (C, D) Computational and functional prediction of CDH2–Cx43 interaction using ZDOCK (C) and STRING (D), respectively. CDH2, Cadherin-2 (N-cadherin); TJP1, tight junction protein 1; CTNNB1, β catenin; GJA1, gap junction alpha protein 1(Cx43); JUP, junction plakoglobin; NEDD4, neural precursor cell expressed, developmentally down-regulated 4. (E) Whole cell lysates from MDA-MB-231 and T47D were immunoprecipitated with anti-CDH2 or IgG and then electrophoresed on 12% SDS–PAGE. The membranes were blotted with anti-Cx43 (light band at 43 kD). (F) Whole-cell lysated from MDA-MB-231 cells were subjected to immunoprecipitation (IP) with anti-CDH2 or anti-Cx43. The membrane was blotted with anti-CDH2. (G) MDA-MB-231 was transfected with pCMV2-CDH2 Flag and pcDNA 3.2-Cx43-HA. Protein lysates were immunoprecipitated with anti-Flag or anti-IgG and blotted with anti-Flag or anti-HA. (H) Lysates from cancer stem cells, isolated from MDA-MB-231 were immunoprecipitated with anti-IgG, anti-Cx43, or anti-CDH2. The samples were electrophoresed and then blotted with anti-CDH2. (I) BM biopsy from BC patient (#3) was subjected Proximity Ligation Assay with anti-CDH2 and anti-Cx43. The proximity of the two antibodies was determined using EVOS FL Auto 2, 600× magnification. Control slide was labeled with isotype control. Source data are available for this figure.

Article Snippet: Human CDH2 and Cx43 siRNA and Risc free (control) were purchased from Dharmacon; human cyclin D1 promoter in pGL3-basic (research resource identifiers, RRID:Addgene_32726) and pcDNA 3.2 with Cx43-HA (RRID:Addgene_49851) were obtained from Addgene (which was a donation from Frank McCormick and Anne Brunet laboratory, respectively); pCMV2-CDH2-Flag from Sino Biologicals; and human shRNA pRFP-C-RS with scramble sequence, CDH2-shRNA, or Cx43 shRNA from OriGene Technologies.

Techniques: Software, Functional Assay, Immunoprecipitation, SDS Page, Membrane, Transfection, Isolation, Proximity Ligation Assay, Labeling

Journal: FEBS letters

Article Title: Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

doi: 10.1016/j.febslet.2013.12.023

Figure Lengend Snippet: A, An immunoblot for Cx43 showing a variable amount of endogenous Cx43 expression in HEK293 cells obtained from two different research laboratories. B, An immunoblot for Cx43 from the low Cx43 expressing HEK293-2 cells, a stable clone (80-1) of the same cell line after knockdown of Cx43 with an anti-Cx43 shRNA, and a scrambled control shRNA. Tubulin was used as a loading control. C, Quantification of the relative Cx43 expression levels from the HEK-2 cells, the Cx43 shRNA HEK-2 cell clone, and the scrambled shRNA control from three immunoblots like the one displayed in panel B. D, Anti-Cx43 antibody (Millipore #AB1728) was immunolocalized in HEK-2 cells with an Alexa Fluor555 conjugated secondary antibody. Gap junctions appeared as small puncta (arrows) located between adjacent HEK293 cells.

Article Snippet: We attempted to knockdown the endogenous Cx43 expression by stable transfection with a shRNA construct directed against human Cx43 (Origene HuSH-29 #SKU-TR312771; human GJA1 29mer shRNA).

Techniques: Western Blot, Expressing, Stable Transfection, Knockdown, shRNA, Control

Journal: FEBS letters

Article Title: Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

doi: 10.1016/j.febslet.2013.12.023

Figure Lengend Snippet: A, A whole cell patch clamp recordings from an HEK293-2 cell during repeated runs of a −40 to +30 mV voltage step under low (nominally zero) [Ca2+]o conditions. Activation of a membrane channel that remained open upon return to −40 mV was frequently evident. B, A whole cell recording from the same HEK-2 cell after the addition of 2 mM CaCl2 to the bath saline illustrates the reduction in observed endogenous channel activity. C, Ensemble averages of the five current traces displayed in panels A and B illustrating the difference in whole cell currents between 0 and 2 mM [Ca2+]o. D, The junctional current – voltage relationship acquired from this HEK-2 cell coupled to a partner HEK-2 cell via Cx43 gap junctions. The ratio of [Ca2+]o-sensitive nonjunctional membrane current to gap junction current was 0.04 in this HEK-2 cell pair.

Article Snippet: We attempted to knockdown the endogenous Cx43 expression by stable transfection with a shRNA construct directed against human Cx43 (Origene HuSH-29 #SKU-TR312771; human GJA1 29mer shRNA).

Techniques: Patch Clamp, Activation Assay, Membrane, Saline, Activity Assay

Journal: Journal of Cell Science

Article Title: Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium

doi: 10.1242/jcs.223313

Figure Lengend Snippet: Cx43 is located apically in the breast luminal epithelium. S1 non-neoplastic mammary epithelial cells were cultured in 2D (A,B) or in 3D (B­,D,E), as indicated, for 10 days. A thin section from breast tissue biopsy was used in C. (A) Western blot shows that Cx43, but not Cx26, is expressed in S1 cells; lamin B is used as loading control. (B) Immunostaining for Cx43 (red), with apical localization indicated by the arrow. (C) Immunohistochemistry for Cx43 (reddish-brown) in normal-appearing breast glandular tissue, with display of basal localization in myoepithelial cells (arrowheads) and apical localization in luminal cells (asterisks). Nuclei are counterstained with hematoxylin (blue). (D) Left: dual fluorescence staining for Cx43 (green) and a myoepithelial cell marker (α-smooth muscle actin protein, α-SMA; red) in normal-appearing breast glandular tissue. Cx43 staining overlap with α-SMA staining in myoepithelial cells appears in yellow (arrows). Right: dual immunostaining for Cx43 (red) and a lysosomal marker (lysosomal-associated membrane protein 2, LAMP-2) (green) in an acinus formed by S1 cells; the arrow points to a rare spot with colocalization (yellow). (E) Dual staining for Cx43 (red) and ZO-1 (green) or β-catenin (green). Colocalization of Cx43 and ZO-1 staining appears yellow (short arrows); cell–cell contacts with Cx43 aligned with β-catenin are indicated (long arrows). Nuclei are counterstained with DAPI (blue). Scale bars: 10 µm. Single immunofluorescence staining was done on multiple (>5) biological replicates (cell cultures and tissue samples); dual immunostaining was done on 2–3 biological replicates.

Article Snippet: For western blot analysis equal amounts of proteins were separated and immunoblotted with rabbit polyclonal antibodies against Cx43 (0.16 μg/ml; C6219 and 5 μg/ml; SAB4501175, Sigma-Aldrich; Ni et al., 2017 ), p-Akt (1:100; 9271, Cell Signaling Technology; Zhang et al., 2018 ), Akt (9272, 1:1000, Cell Signaling Technology; Geyer et al., 2018 ), β-catenin (0.04 μg/ml; sc7199, Santa Cruz Biotechnology; Nedvetsky et al., 2012 ); goat polyclonal antibody against ZO-2 (2 μg/ml, sc-8148, Santa Cruz Biotechnology; Talhouk et al., 2008 ); and monoclonal antibodies against Cx26 (0.5 μg/ml; 13-8100, Zymed Laboratories, San Francisco, CA), Cx43 (2 μg/ml; sc-271837, Santa Cruz Biotechnology; Lai et al., 2018 ) and ZO-1 (1 μg/ml; 33-9100, Life Technologies).

Techniques: Cell Culture, Western Blot, Immunostaining, Immunohistochemistry, Fluorescence, Staining, Marker, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium

doi: 10.1242/jcs.223313

Figure Lengend Snippet: 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.

Article Snippet: For western blot analysis equal amounts of proteins were separated and immunoblotted with rabbit polyclonal antibodies against Cx43 (0.16 μg/ml; C6219 and 5 μg/ml; SAB4501175, Sigma-Aldrich; Ni et al., 2017 ), p-Akt (1:100; 9271, Cell Signaling Technology; Zhang et al., 2018 ), Akt (9272, 1:1000, Cell Signaling Technology; Geyer et al., 2018 ), β-catenin (0.04 μg/ml; sc7199, Santa Cruz Biotechnology; Nedvetsky et al., 2012 ); goat polyclonal antibody against ZO-2 (2 μg/ml, sc-8148, Santa Cruz Biotechnology; Talhouk et al., 2008 ); and monoclonal antibodies against Cx26 (0.5 μg/ml; 13-8100, Zymed Laboratories, San Francisco, CA), Cx43 (2 μg/ml; sc-271837, Santa Cruz Biotechnology; Lai et al., 2018 ) and ZO-1 (1 μg/ml; 33-9100, Life Technologies).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation, Sequencing, shRNA, Fluorescence, Immunostaining, Cell Culture, Infection, Staining, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium

doi: 10.1242/jcs.223313

Figure Lengend Snippet: 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.

Article Snippet: For western blot analysis equal amounts of proteins were separated and immunoblotted with rabbit polyclonal antibodies against Cx43 (0.16 μg/ml; C6219 and 5 μg/ml; SAB4501175, Sigma-Aldrich; Ni et al., 2017 ), p-Akt (1:100; 9271, Cell Signaling Technology; Zhang et al., 2018 ), Akt (9272, 1:1000, Cell Signaling Technology; Geyer et al., 2018 ), β-catenin (0.04 μg/ml; sc7199, Santa Cruz Biotechnology; Nedvetsky et al., 2012 ); goat polyclonal antibody against ZO-2 (2 μg/ml, sc-8148, Santa Cruz Biotechnology; Talhouk et al., 2008 ); and monoclonal antibodies against Cx26 (0.5 μg/ml; 13-8100, Zymed Laboratories, San Francisco, CA), Cx43 (2 μg/ml; sc-271837, Santa Cruz Biotechnology; Lai et al., 2018 ) and ZO-1 (1 μg/ml; 33-9100, Life Technologies).

Techniques: Expressing, Western Blot, Infection, Plasmid Preparation, Stable Transfection, Cell Culture, Immunostaining

Journal: Journal of Cell Science

Article Title: Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium

doi: 10.1242/jcs.223313

Figure Lengend Snippet: 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.

Article Snippet: For western blot analysis equal amounts of proteins were separated and immunoblotted with rabbit polyclonal antibodies against Cx43 (0.16 μg/ml; C6219 and 5 μg/ml; SAB4501175, Sigma-Aldrich; Ni et al., 2017 ), p-Akt (1:100; 9271, Cell Signaling Technology; Zhang et al., 2018 ), Akt (9272, 1:1000, Cell Signaling Technology; Geyer et al., 2018 ), β-catenin (0.04 μg/ml; sc7199, Santa Cruz Biotechnology; Nedvetsky et al., 2012 ); goat polyclonal antibody against ZO-2 (2 μg/ml, sc-8148, Santa Cruz Biotechnology; Talhouk et al., 2008 ); and monoclonal antibodies against Cx26 (0.5 μg/ml; 13-8100, Zymed Laboratories, San Francisco, CA), Cx43 (2 μg/ml; sc-271837, Santa Cruz Biotechnology; Lai et al., 2018 ) and ZO-1 (1 μg/ml; 33-9100, Life Technologies).

Techniques: Expressing, Immunohistochemical staining, Staining, Stable Transfection, Cell Culture, Immunolabeling, Transduction, Sequencing, shRNA

Journal: Journal of Cell Science

Article Title: Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium

doi: 10.1242/jcs.223313

Figure Lengend Snippet: 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.

Article Snippet: For western blot analysis equal amounts of proteins were separated and immunoblotted with rabbit polyclonal antibodies against Cx43 (0.16 μg/ml; C6219 and 5 μg/ml; SAB4501175, Sigma-Aldrich; Ni et al., 2017 ), p-Akt (1:100; 9271, Cell Signaling Technology; Zhang et al., 2018 ), Akt (9272, 1:1000, Cell Signaling Technology; Geyer et al., 2018 ), β-catenin (0.04 μg/ml; sc7199, Santa Cruz Biotechnology; Nedvetsky et al., 2012 ); goat polyclonal antibody against ZO-2 (2 μg/ml, sc-8148, Santa Cruz Biotechnology; Talhouk et al., 2008 ); and monoclonal antibodies against Cx26 (0.5 μg/ml; 13-8100, Zymed Laboratories, San Francisco, CA), Cx43 (2 μg/ml; sc-271837, Santa Cruz Biotechnology; Lai et al., 2018 ) and ZO-1 (1 μg/ml; 33-9100, Life Technologies).

Techniques: Cell Culture, Transduction, Sequencing, shRNA

Journal: Journal of Cell Science

Article Title: Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium

doi: 10.1242/jcs.223313

Figure Lengend Snippet: 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.

Article Snippet: For western blot analysis equal amounts of proteins were separated and immunoblotted with rabbit polyclonal antibodies against Cx43 (0.16 μg/ml; C6219 and 5 μg/ml; SAB4501175, Sigma-Aldrich; Ni et al., 2017 ), p-Akt (1:100; 9271, Cell Signaling Technology; Zhang et al., 2018 ), Akt (9272, 1:1000, Cell Signaling Technology; Geyer et al., 2018 ), β-catenin (0.04 μg/ml; sc7199, Santa Cruz Biotechnology; Nedvetsky et al., 2012 ); goat polyclonal antibody against ZO-2 (2 μg/ml, sc-8148, Santa Cruz Biotechnology; Talhouk et al., 2008 ); and monoclonal antibodies against Cx26 (0.5 μg/ml; 13-8100, Zymed Laboratories, San Francisco, CA), Cx43 (2 μg/ml; sc-271837, Santa Cruz Biotechnology; Lai et al., 2018 ) and ZO-1 (1 μg/ml; 33-9100, Life Technologies).

Techniques: Immunostaining, Expressing, Western Blot, Transduction, shRNA, Sequencing, Plasmid Preparation

Journal: Journal of Cell Science

Article Title: Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium

doi: 10.1242/jcs.223313

Figure Lengend Snippet: Cx43 localization is altered in models of overweight condition. (A) Representative images of Cx43 localization in S1 acini, treated for 72 h on day 10 of culture (post-lumen assembly) with mammary adipose tissue explants (adipose coculture), with conditioned medium (CM) from adipose tissue, or with recombinant leptin (100 ng/ml). Nuclei were stained with DAPI. Arrows and arrowhead indicate non-apical distribution of Cx43. Scale bar: 10 µm. (B–D) Mean±s.e.m. quantification of apical Cx43 localization in acini cultured in the presence of adipose tissue explants or CM (B), and in acini treated post-lumen assembly with leptin, a leptin receptor antagonist (LA) (C), or leptin and PI3K inhibitor LY294002 (D). *P<0.05, one-way ANOVA with Tukey test. (E) Immunohistochemical analysis of Cx43 distribution in whole-mount mammary glands from mice fed control or obesity-inducing diets (OID). Cx43 signals were extracted from immunohistochemistry images using spectral imaging (bottom images). The arrow points to apical Cx43 signals. Cx43 localization was evaluated with a three-grade scale (0=no apical signal; 1=partial, discontinuous apical signal lining the lumen; 3=continuous apical signal) by two investigators blind to the treatment conditions (≥25 acini or mammary ducts analyzed for each animal). Cx43 localization is quantified in the graph. Data represented as mean±s.e.m.; *P<0.01; unpaired t-test. Scale bar: 25 µm.

Article Snippet: For western blot analysis equal amounts of proteins were separated and immunoblotted with rabbit polyclonal antibodies against Cx43 (0.16 μg/ml; C6219 and 5 μg/ml; SAB4501175, Sigma-Aldrich; Ni et al., 2017 ), p-Akt (1:100; 9271, Cell Signaling Technology; Zhang et al., 2018 ), Akt (9272, 1:1000, Cell Signaling Technology; Geyer et al., 2018 ), β-catenin (0.04 μg/ml; sc7199, Santa Cruz Biotechnology; Nedvetsky et al., 2012 ); goat polyclonal antibody against ZO-2 (2 μg/ml, sc-8148, Santa Cruz Biotechnology; Talhouk et al., 2008 ); and monoclonal antibodies against Cx26 (0.5 μg/ml; 13-8100, Zymed Laboratories, San Francisco, CA), Cx43 (2 μg/ml; sc-271837, Santa Cruz Biotechnology; Lai et al., 2018 ) and ZO-1 (1 μg/ml; 33-9100, Life Technologies).

Techniques: Recombinant, Staining, Cell Culture, Immunohistochemical staining, Immunohistochemistry, Imaging

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a , Upper Left: Contrast-enhanced MRI of representative patient with brain metastasis. Tumor (white) is surrounded by parenchymal reaction (dark grey). Upper Right: Hematoxallin-Eosin staining (H&E) of resected brain metastasis (T) and parenchyma (P). Lower Panels: Immunohistochemistry of adjacent sections for GFAP (Lower Left) and Cx43 (Lower Right). Scale bar, 10 μm. (n = 6 patient samples) b, Cx43 expression is increased in brain metastases compared with primary and normal tissue. Representative images of Cx43 staining in clinical samples from triple-negative breast cancer (TNBC) and non-small cell lung carcinoma (NSCLC). Proportion of CX43-positive samples was quantified in primary (1ry) tumours (TNBC n = 98, NSCLC n = 138), brain metastases (Mets) (TNBC n= 117; NSCLC n = 91) and normal lung tissues (n = 75) Scale bar, 100 μm. c , Upper: GFP+ H2030-BrM3 cells (green) are surrounded by GFAP+ activated astrocytes (red) in the brain parenchyma at early (day 7) and later (day 21) time points following intracardiac inoculation in mice. Blue, collagen IV (ColIV) staining in vessels. Scale bar, 10 μm. Lower: Cx43 staining (arrowhead) at the interface of GFP+ H2030-BrM3 (green) and GFAP+ astrocytes (blue). Scale bar, 10 μm. d-e, Gap junction communication between astrocytes and BrM cells. d, Time-lapse images of dye transfer from MDA231-BrM2 cells to astrocytes. See also . Scale bars, 100 μm. e, Quantification of dye transfer from astrocytes to cancer cells. Histograms show red fluorescent signal in parental (Par) and BrM cells. Values are mean ± S.E.M. (Data are from n=3 biological replicates over 3 independent experiments). f-i, Cx43 and PCDH7 western immunoblotting in the indicated parental and brain metastatic derivatives ( f , n=3 independent experiments), in brain metastatic cells compared to brain cell types ( g , n=2 independent experiments), and in MDA231 derivatives metastatic to brain, lung (LM) or bone (BoM) ( h , n=2 independent experiments). Full blots are shown in . i-j, Kaplan-Meier plots of brain metastasis-free survival in 189 cases of triple-negative breast cancer ( i ) and 129 cases (MSKCC set2) and 58 cases (GSE3141) of lung adenocarcinoma ( j ), based on Cx43/PCDH7 expression in the primary tumour.

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Staining, Immunohistochemistry, Expressing, Western Blot

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Representative images and quantification of Cx43 immunostaining in matched primary and brain metastatic samples from non-small lung carcinoma patients. Scale bar, 100 μm (n = 8 patients). b, Cancer cells used in this study. c, Astrocyte co-culture protects cancer cells. As illustrated in schema (left), cleaved caspase 3+/GFP+ apoptotic BrM cells were quantified by flow cytometery after sFasL- or chemo-treatments. (3 independent experiments). d, Flow cytometric quantification of dye transfer from astrocytes to MDA231-BrM2 cells over time. (3 independent experiments).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Immunostaining, Co-Culture Assay

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Cx43 and PCDH7 mRNA in parental (Par) and BrM cells. Values are mean ± S.E.M. (3 independent experiments in triplicate). b, Cx43 and PCDH7 western blotting in ErbB2 parental and brain cells, as well as Kras/p53 cell lines. (n = 3 independent experiments). c, Cx26 and Cx30 mRNA in MDA231 parental (Par) and the metastatic derivatives of brain (BrM2), lung (LM) and bone (BoM). d, Cx43 and PCDH7 mRNA in BrM cells compared to brain cells. (3 independent experiments). e, Kaplan-Meier plot illustrates the probability of cumulative metastasis free survival in 63 cases (GSE8893) of lung adenocarcinoma based on Cx43/PCDH7 expression in the primary tumour. f,g, Knockdown of Cx43 and PCDH7 with short hairpin RNAs (shRNA) as assessed by RT-PCR ( f ) and western blotting ( g ). Ctrl, control. Values are mean ± S.E.M. (3 independent experiments in triplicate).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Histograms (top) and quantification (bottom) of dye transfer from astrocytes to control and Cx43-depleted or PCDH7-depleted brain metastatic cells. Values are mean ± S.E.M. (Data are from n=3 biological replicates over 3 independent experiments). b-d, BLI ( c ) and quantification ( b, d ) of brain metastatic lesions formed by control, Cx43-depleted, or PCDH7-depleted brain metastatic cells in the MDA231 xenograft model or 393N1 syngeneic models of brain metastasis. Data are from n=3 independent experiments, n=8-10 mice per group. All source data from mouse experiments are in . e,f, Wild type (WT) or T154A mutant (Mut) Cx43 was re-expressed in Cx43-depleted MDA231-BrM2 cells (Cx43 sh2). The cells were subjected to astrocyte dye transfer analysis by flow cytometry ( e , 3 independent experiments), or to brain metastasis assays and BLI quantification ( f , n=2 independent experiments, 9 mice per group). g, Schematic summary of Cx43- and PCDH7-mediated interactions between cancer cells and astrocytes in brain metastasis.

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Mutagenesis, Flow Cytometry

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a,b, Histograms and quantification of dye transfer from astrocytes to control and Cx43-depleted or PCDH7-depleted Kras/p53-393N1 cells ( a ), and from astrocytes to control or Cx43-depleted MDA231-BrM2 cells, in comparison to Carbenoxolone (50 uM) treatment ( b ). c,d, PCDH7 in astrocytes facilitate gap junctions. PCDH7 immunoblotting of control or PCDH7-depleted astrocytes ( c ). Quantification of dye transfer from MDA231-BrM2 cells to PCDH7-depleted astrocytes ( d ). e, Quantification of dye transfer from human brain microvascular endothelial cells (HBMEC) to control, Cx43- or PCDH7-depleted MDA231-BrM2 cells. f, Dye transfer from MDA231-BrM2 cells to a mixed population of astrocytes and HBMEC. g, Quantification of dye transfer from control or Cx43-depleted MDA231-BrM2 cells to human microglia. For dye transfer assays, values are mean ± S.E.M. (≥ 2 independent experiments in triplicate).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Western Blot

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Schema illustrating split luciferase assay. Fusion constructs of PCDH7 and Cx43 were created with either the N or C terminal halves of luciferase, NLuc and CLuc, respectively. When these proteins are brought into proximity, luciferase is functionally reconstituted, producing photons of light. b, Cx43 and PCDH7 constructs fused with N-terminal and C-terminal firefly luciferase halves (N-luc) and (C-luc) were expressed in parental cell lines. The table (top) numerically identifies the cell line combinations used in the assays (bottom), and bioluminescence imaging (BLI) of a representative plate. c , Cx43 and PCDH7 western immunoblotting in cancer cells overexpressing fusion proteins. d, Quantification of BLI after co-culture of Cx43-CLuc/PCDH7-NLuc(+) cancer cells and astrocytes for 15 min. (3 independent experiments) e-g, Luciferase split assay to detect Cx43-E cadherin or Cx43-N cadherin interactions. Cell line combinations used in the assays are numerically identified in the table ( e ), and confirmed by western immunoblotting ( f ). Bioluminescence imaging (BLI) of a representative assay plate; cell line combinations are indicated numerically ( g ). (n ≥ 2 independent experiments).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Luciferase, Construct, Imaging, Western Blot, Co-Culture Assay

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Bioluminescent imaging (BLI) quantification of brain metastatic lesions formed by control (Ctrl), Cx43- or PCDH7-depleted H2030-BrM3 cells. (Data are from 2 independent experiments with 9 mice total per group). b, Representative images of GFP+ brain metastatic lesions formed by control, Cx43- or PCDH7-depleted MDA231-BrM2 cells. Brain sections or brain metastatic lesions are delineated by dotted white line or dotted red line, respectively. Scale bar, 1000 μm. c, BLI (images) and quantification (bar graph) of lung metastatic lesions formed by MDA231-BrM2 cells. Values are mean ± S.E.M. (Data are from 2 independent experiments with 5 mice total in each group). d,e, Gap junction-mediated brain metastasis requires channel function of Cx43. Wild type (WT) or T154A mutant (Mut) Cx43 was re-expressed in Cx43 depleted MDA231-BrM2 cells ( CX43 sh2). Cx43 expression was detected by western blotting ( d ) and brain metastasis formed by these cells was quantified by BLI ( e ). (Data are from 2 independent experiments with 10 mice total per group)

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Imaging, Mutagenesis, Expressing, Western Blot

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Cx43 and PCDH7 do not mediate trans-BBB Migration. Quantification of control (Ctrl), Cx43-or PCDH7-depleted MDA231-BrM2 cells in 7-day brain lesions was carried out as follows: at the indicated timepoint, mice were euthanized, brains were sectioned, 10% of the sections were immunostained stained, and all GFP(+) cells in these sections were counted. Values are mean ± S.E.M. (n=5 brains in each group). b, Cx43 and PCDH7 mediate cancer cell colonization in 14-day brain lesions. Sectioning and staining were carried out as described in (a). Representative images are GFP (green) and Ki67 (red) staining. DAPI, nuclear staining. Scale bar, 20 μm. Bar graph is the proportion of Ki67+ cancer cells. Values are mean ± S.E.M. (n=5 brains in each group). c, Cx43 and PCDH7 mediate cancer cell survival. MDA231 BrM2 cells expressing CX43sh, PCDH7sh or Control sh were deposited onto living brain sections, 5 brain slices were seeded with cancer cells of each type. After 48 h, slices were fixed and stained for GFP (green) and cleaved caspase 3 (Casp3)(red) staining. Representative images are shown, Scale bar, 30 μm. After staining, all GFP(+) cells were counted on each slice. GFP (+) cells with 3+ caspase staining were scored as “apoptotic”. Histogram is the proportion of caspase 3+ apoptotic cancer cells. Values are mean ± S.E.M. (n=5 brain slices in each group). d, Cx43 and PCDH7 do not affect vascular cooption of cancer cells in 14-day brain lesions. Representative images are GFP (green) staining and vascular structure filled with TRITC dextran (red). Scale bar, 20 μm. (2 independent experiments).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Migration, Staining, Expressing

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Schematic illustration of TRAP experimental set up to isolate translating mRNA from MDA231-BrM2 cells under 3 conditions (#1, #2, #3). b, Principle component (PC) analysis of TRAP mRNA sequencing. c. Scatter plot of log2 fold-changes regulated by astrocytes and gap junction communications between BrM cells and astrocytes. d, STAT1 and NF-κB p65 phosphorylation in H2030-BrM3 cells after a 2 h incubation with conditioned media (CM) from astrocyte co-cultures. CM were collected after 24 h co-culture of astrocytes with control or Cx43-depleted H2030-BrM3 cells. n=3 independent experiments.

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Sequencing, Incubation, Co-Culture Assay

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Signaling pathway analysis of TRAP-Seq data from MDA231-BrM2 cells after co-culture with astrocytes. Control (Ctrl) or Cx43-depleted MDA231-BrM2 cells expressing an L10a-GFP ribosomal protein fusion were co-cultured with astrocytes for 24 h prior to polysome immunoprecipitation and mRNA sequencing. Heatmap depicts blue (down-regulated) and red (up-regulated) pathways. n=2 biological replicates. b,c, STAT1 and NF-κB p65 phosphorylation in MDA231-BrM2 cells after a 2 h incubation with conditioned media (CM) from astrocyte co-culture. CM were collected after 24 h co-culture of astrocytes with control or Cx43-depleted MDA231-BrM2 cells ( b ), or from Cx43-depleted MDA231-BrM2 cells that were transduced with wild type Cx43 (WT) or Cx43(T154A) mutant (Mut) ( c ), n≥3 independent experiments both (b) and (c). d, Relative mRNA levels of IFNA and TNFA in human astrocytes re-isolated after co-culture with MDA231-BrM2 cancer cells. All values are mean ± S.E.M. (Data are n=3 biological replicates in 2 independent experiments). e-f, Quantification of BLI signal from brain metastases formed by control or STAT1-knockdown Kras/p53-393N1 cells (e), and SR-IκBα MDA231-BrM2 cells (f) (Data are from n=2 independent experiments, with 12 mice total per group).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Co-Culture Assay, Expressing, Cell Culture, Immunoprecipitation, Sequencing, Incubation, Transduction, Mutagenesis, Isolation

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Cytokine array analysis of conditioned media collected after 24 h co-culture of human astrocytes with control or Cx43-depleted MDA231-BrM2 cells. Log2 fold-changes were plotted. b, Schematic of co-culture conditioned media collection and human astrocyte re-isolation (left) ELISA of IFNα and TNFα in CM from astrocyte co-cultures with the indicated MDA231-BrM2 cells (right) All values are mean ± S.E.M. (≥2 independent experiments with 4 total replicates). c, Relative levels of cleaved caspase 3 in MDA231-BrM2 cells treated with various concentrations of carboplatin (Carbo) in the presence or absence of 10 units/ml (39 units/ng) IFNαA or 10 pg/ml TNFα. All values are mean ± S.E.M. (5 technical replicates over 3 independent experiments). d, STAT1 levels in control and STAT1-knockdown LLC BrM and 393N1 cells e. Quantification of BLI signal from brain metastases formed by syngeneic LLC-BrM control, or STAT1-knockdown cells. (Data are from 2 independent experiments with 12-15 mice total per group). f, NF-κB renilla luciferase reporter assay in MDA231-BrM cells expressing control pBABE or SR-IκBα vector. Vlues are mean ± S.E.M. (3 technical replicates).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Co-Culture Assay, Isolation, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Expressing, Plasmid Preparation

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, MDA231-BrM2 cells expressing control shRNA (Ctrl sh) or shRNA targeting Cx43 , were cultured for 18 h with or without astrocytes, and subjected to immunobloting analysis of phosphorylated TBK1 and IRF3 (n=3 independent experiments). b, Representative images of dual immunofluorescent staining of IRF3 and GFP. DAPI, nuclear staining. In co-cultures: white arrows, nuclear accumulation of IRF3 in astrocytes; green arrows, even distribution of IRF3 in GFP+ MDA231-BrM2 cells. Scale bar, 20 μm. (n=2 independent experiments). c, Mouse astrocytes (Astro) expressing control shRNA (Ctrl sh) or shRNA targeting STING , were cultured for 18 h with or without LLC BrM cells (BrM), and subjected to immunobloting analysis of phosphorylated IRF3 (c); d, LLC BrM growth in syngeneic C57Bl6 mice hosts wild-type (+/+) or knock-out (−/−) for STING. Quantification of BLI signal from brain metastases formed in +/+ and −/− host mice (e). (n=11 mice in each group; 2 independent experiments). e, MDA231-BrM2 alone, astrocytes alone, or 18 h co-cultures, were harvested for sample preparation and cGAMP analysis by LC-MS/MS. Representative chromatograms (e) and quantitation (f) are presented (n= 5 biological replicates in 3 independent experiments). Refer also to . g, Human astrocytes (Astro), were cultured for 18 h with or without H2030 BrM cells (BrM) expressing control shRNA (Ctrl sh) or shRNA targeting cGAS , and subjected to immunobloting analysis of phosphorylated IRF3, (2 independent experiments). h, Schematic summary of gap junction mediated anti-dsDNA response, production of IFNα and TNFα in astrocytes, and consequent activation of STAT1 and NF-κB pathways in cancer cells to support brain metastasis.

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Expressing, shRNA, Cell Culture, Western Blot, Staining, Knock-Out, Sample Prep, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Activation Assay

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Control or Cx43-deplated H2030-BrM3 cells were co-cultured for 18 h with or without astrocytes, and subjected to immunobloting analysis of phosphorylated TBK1 and IRF3 (n=3 independent experiments). b, Immunoblot of mouse astrocytes depleted on STING with short hairpins or control (non-silencing) sh. c, Mouse IFNa and TNFa were quantitated in the conditioned medium after co-culture (b) by ELISA. (2 independent experiments with 3 replicates each). d, LLC BrM growth in syngeneic C57Bl6 mice hosts wild-type (+/+) or knock-out (−/−) for STING. Diameter of brain metastases (d) Scale bar, 50 μm. Brains from all mice (n = 22) were sectioned, immunostained, and measured. All GFP(+) brain metastases were quantitated (2.8 +/− 0.67 metastases per +/+ mouse; 1.6 +/− 0.55 in STING −/− mice). e, Quantification of dsDNA in the indicated cellular fractions from 2×10 7 H2030-BrM3, MDA231-BrM2 or Human Astrocyte cells. Values are mean ± S.E.M. (n=3 biological replicates; 2 independent experiments). f, Ratio of cytosolic dsDNA and nuclear dsDNA in indicated cancer cells and non-neoplastic cells. g, Representative image of immunofluorescent staining of dsDNA, GFP, Cox IV (mitochondria marker) in MDA-BrM2 cells. h, cGAMP identification. The peak at 4.47 min contains all 3 SRM transitions specific for cGAMP. RT: retention time, AA: automatically integrated peak area. i, j, EdU labeled MDA231-BrM2 cells were co-cultured with astrocytes for 6 h. Transfer of EdU-labeled DNA from cancer cells to astrocytes was visualized using confocal microscopy ( i ), or quantified by flow cytometry ( j ). k, Immunoblot of H2030-BrM3 cells depleted of cGAS with short hairpins or control (non-silencing) sh. l, Human astrocytes (Astro), were cultured for 18 h with or without H2030 BrM cells (BrM) expressing control shRNA (Ctrl sh) or shRNA targeting cGAS ; Human IFNa and TNFa were quantitated in the conditioned medium by ELISA ( l ). (2 independent experiments in triplicate). m, Quantification of BLI signal from brain metastases formed by H2030 BrM3 cells depleted of cGAS with two independent sh. (Data are from 2 independent experiments with 6 mice total per group).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Cell Culture, Western Blot, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Knock-Out, Staining, Marker, Labeling, Confocal Microscopy, Flow Cytometry, Expressing, shRNA

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a, Dye transfer from astrocytes to MDA231-BrM2 cells in the presence of the indicated concentrations of Tonabersat (Tona) or meclofenamate (Meclo). (n ≥ 3 independent experiments). b, ELISA of IFNα and TNFα in conditioned media from co-cultured MDA231-BrM2 cell and astrocytes in the presence of Tonabersat (Tona) or meclofenamate (Meclo) ranging from 0.5 – 4 × 10 −4 M Tona or 0.25 – 2 × 10 −4 M Meclo. All graphs shown are mean ± S.E.M. (Data shown from 2 independent experiments with 4 replicates each). c, Tonabersat or meclofenamate was administered daily starting one day after cancer cell inoculation in mice. Brain metastatic lesions were quantified based on BLI. (n=2 independent experiments; 8 mice total per group). d, GFP staining of 14-day brain metastatic lesions. Representative images show large, progressive lesions. DAPI, nuclear staining. Scale Bar, 40μm. (n=10 mice). e,f,g, 14 days after inoculation with MDA231-BrM2 cells transduced with inducible control, CX43 or PCDH7 shRNAs, mice were treated with doxycycline and carboplatin. Representative images of matched ex vivo brain BLI and red fluorescence imaging (e). Brain metastatic lesions were quantified based on BLI (f). (2 independent experiments with n= 10 total mice per group). g, 14 days after inoculation with MDA231-BrM2 cells, mice were treated with Tonabersat, meclofenamate, and carboplatin. Following the indicated regimens, brain metastatic lesions were quantified based on BLI (i). (2 independent experiments with n=9 mice total per group).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Transduction, Ex Vivo, Fluorescence, Imaging

Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: a-d, Following treatment with Tonabersat (Tona) or meclofenamate (Meclo) ( a ), brain metastasis ( b ), primary tumour growth in mammary fat pads ( c ), or lung metastasis ( d ) was quantified by BLI. Values are mean ± S.E.M. (2 independent experiments with 10 mice total in each group). e, Human astrocytes were treated with Tonabersat or Meclofenamate at 200 uM and 100 uM, respectively for 12 h prior to transfection with cGAMP (4 μg/ml) with Lipofectamine 2000, or Lipofectamine alone.Conditioned media was collected 18 h later and assayed for human TNFαand IFNα by ELISA. (2 biological replicates). f, Knockdown of Cx43 and PCDH7 in MDA231-BrM2 cells with tet-on inducible short hairpin RNAs (shRNA), as assessed by RT-PCR ( f ) and western immunoblotting ( g ), after doxycycline treatment in vitro . 2 independent experiments. h, Brain ex vivo Bioluminescent imaging (BLI) 14 days after inoculation of MDA231-BrM2 cells (n = 10 mice).

Article Snippet: For Cx43 immunohistochemistry, normal lung tissue array (75 cases), primary triple negative breast cancer tissue array (98 cases) and primary non-small cell lung carcinoma tissue array (138 cases) were purchased from US Biomax.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, In Vitro, Ex Vivo, Imaging

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Expression of Cx43 in breast cancer cells during collective cancer cell invasion. (a) Original colored fluorescence overview of breast cancer lesions with zoomed insets on normal ducts and tumor invasive margins. (b) Trainable cell phenotyping function of the Inform software to identify different cell types within a multicellular tissue based on spectral fluorescence fingerprinting. Images display the results of the per-cell phenotyping analysis in normal ducts (luminal epithelial cells [LE]) and collective invasion (CI) patterns in fibrous and in adipose tissue. In each region, different cell types are marked by colored dots, as indicated. For all regions devoid of bilayered ducts, cells of epithelial origin, based on pan-cytokeratin, were considered as invasive breast cancer cells. (c) Unmixed composite images of Cx43 intensity in epithelial cancer cells within the tumor margins of representative samples with negative (upper panel) and mid-level (lower panel) Cx43 expression. (d) Heterogeneous expression of Cx43 within cancer cells of the collective invasion patterns in different tumor regions and samples. Values represent the percentage of Cx43 positive cancer cells. (e) Summary of Kaplan–Meier survival analysis correlating RFS with Cx43 expression in breast cancer patients categorized based on grade or molecular subtype. Bold and underlined values indicate positive (black) or inverse (green) association with Cx43 expression. P values, log-rank test. (f) Quantitative PCR for nine connexin subtypes relevant for the mammary gland (Cx26, Cx30, Cx30.1, Cx32, Cx36, Cx37, Cx40, Cx43, and Cx45). Connexin mRNA expression in 4T1 (black) and MMT (gray) cells from 3D collagen invasion cultures. Values represent mean normalized mRNA levels and SEM from three independent experiments. (g) Cx45 localization in collectively invading strands in 3D collagen. Confocal images represent maximum intensity projections from 3D confocal stacks. Arrowheads, cytoplasmic protrusions. Scale bars: 100 µm (c), 50 µm (c, inset), 20 µm (g).

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Expressing, Fluorescence, Software, Real-time Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Increased expression of Cx43 in breast cancer cells during collective cancer cell invasion in vivo and in vitro. (a) Multispectral microscopy of breast cancer tissue sections stained for Cx43 (green), pan-cytokeratin (red; epithelial cells), vimentin (white; stromal cells, cancer cells after epithelial-to-mesenchymal transition). The three panels represent example slices for normal ducts and the invasion zones in the collagen-rich fibrous or adipose tissue. Detection of Cx43 in myoepithelial cells surrounding the luminal epithelium and epithelial cancer cells along cell–cell junctions. Arrows and arrowheads depict Cx43 at cell–cell contacts with puncta or linear patterns, respectively. (b) Colocalization analysis of Cx43 versus pan-cytokeratin expression and classified myoepithelial cells, luminal epithelial cells, and invading cancer cells in fibrous or adipose tissue. Cx43 and pan-cytokeratin levels were acquired using supervised automated tissue segmentation software (Inform). (c) Cx43 levels in luminal epithelium cells (LEC) and cancer cells within the fibrous or adipose tissue from 13 clinical samples (see for sample details). Dotted line represents the threshold level for Cx43 negativity, determined by receiving operator characteristic (ROC) analysis with intensity values obtained from the luminal epithelial cells as control. (d) Kaplan–Meier survival plot predicting RFS for high versus low Cx43 expression in basal-type breast cancer patients . P values, log-rank test. (e and f) Distribution of Cx43 along cell–cell junctions (white arrowheads) and at the polar extensions (red arrowheads) of leader cells during collective invasion of 4T1 (e) and MMT (f) spheroids in 3D collagen. (g) Intensity distribution of Cx43 and F-actin along the circumference of a leader cell (LC; e, white dashed border). Values show the pixel intensity with background subtraction. Confocal images are displayed as maximum-intensity projections from a 3D confocal stack. (h) Cx43 expression and distribution during collective invasion of 4T1 cells in the stroma of the mouse mammary gland, 4 d after implantation and monitored by confocal microscopy. Scale bars: 100 µm (a, e, and f, overview), 50 µm (a, inset), 20 µm (e and f, spheroid 2; h), 10 µm (e, f, and h, inset). Cyt. Ext., cytoplasmic extension. Norm, normal.

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Expressing, In Vivo, In Vitro, Microscopy, Staining, Software, Control, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Collectively invading cancer cells maintain Cx43-mediated GJIC and extracellular nucleotide release . (a) β-Catenin localization along cell–cell junctions between both leader cells and follower cells in collective strands of 4T1 and MMT cells invading 3D fibrillar collagen. Arrowheads, cell–cell junctions. (b) Intensity distribution of β-catenin and F-actin along the circumference of a leader cell (white dashed border). Values show the pixel intensity with background subtraction. (c) Multicellular 3D gap FRAP. Single confocal slices of calcein-labeled invasive 4T1 strands before and after photobleaching. Contours indicate regions of interest for bleaching, including follower regions (red: R1; blue: R2), an invasive front (green: R3), and a single detached cell (SC; purple), in addition to the unbleached neighboring regions D1 and D2. (d) Normalized mean calcein fluorescence intensity of the selected regions. (e) Western blot for Cx43 and β-tubulin as loading control. Whole-cell lysates extracted from 4T1 and MMT cells with stable expression of shNT or shCx43; ±SD. (f and g) Fluorescence recovery of calcein-labeled MMT leader cells expressing shNT or shCx43 during invasion in 3D collagen (f) and physically connected cells in 2D culture (g). (h) Values represent the means and SEM of at least 19 bleached cells from 2D culture per condition from three independent experiments. P values, two-tailed unpaired Mann–Whitney test. (i) Relative dye transfer in parachute assay of 4T1 with nontargeting shRNA (NT) and Cx43 shRNA (Cx43) after 6 or 12 h of cell–cell (donor–receiver) encounter. Values represent the means and SEM from two independent experiments. (j) Summary of the effect of Cx43 down-regulation and/or CBX treatment on the reduction of individual nucleotide and nucleosides concentration detected in the media of 4T1 and MMT collagen cultures by HPLC (related to ). Concentrations from CBX-treated and Cx43 shRNA cultures were compared with control cells (Ctrl, untreated cells with nontargeting shRNA). P values, ANOVA with Dunnett’s multiple comparison test (ns, not significant). Scale bars: 20 µm (a, c, f, and g), 10 µm (a, inset).

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Labeling, Fluorescence, Western Blot, Control, Expressing, Two Tailed Test, MANN-WHITNEY, shRNA, Concentration Assay, Comparison

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Cx43 mediates GJIC and extracellular release of nucleotides/nucleosides during collective invasion. (a) Workflow of time-resolved 3D gap FRAP, including calcein labeling of multicellular spheroids in 3D collagen cultures and 3D gap-FRAP procedure of leader and follower cells within a 3D invasion strand by asymmetric region of interest selection and photobleaching, followed by recording the change in fluorescence intensity over time. (b) Single confocal slices of calcein-labeled invasive 4T1 strands before and after photobleaching. Dashed contours represent the bleached leader cell (LC; red), FC (blue), and SC (purple) in control media and LC in the presence of CBX (white). (c) Normalized calcein fluorescence intensity in bleached LCs, FCs, and SCs in the presence or absence of CBX. Values represent normalized mean fluorescence intensities with SEM; 11–16 cells per treatment condition from four independent experiments. (d) Effect of CBX on percentage fluorescence recovery after photobleaching of LCs and FCs. Values are represented as the means and SEM of three independent experiments. P values, two-tailed unpaired Mann–Whitney test. (e) Average fluorescence recovery in cells stably expressing control vector (shNT) or Cx43 shRNA (shCx43) during invasion in 3D collagen. Values represent the normalized mean intensities and SEM of four leader cells for shNT and pooled two leader cells and two follower cells for shCx43 condition. (f) Inhibition of fluorescence recovery of MMT cells after Cx43 down-regulation (cell groups in Petri dish culture). Normalized mean intensity and SEM of 19–22 cells from three independent experiments. P value, two-tailed unpaired Mann–Whitney test. (g and h) Parachute assay to measure de novo junction formation (g) followed by dye transfer into nonlabeled cells (h) and example histograms obtained by flow cytometry showing the time-dependent alterations of calcein label in donor and recipient cells. (i) Intensity of calcein in 4T1 receiver cells after 12 h of incubation with 4T1 donor cells in control conditions and during inhibition with CBX or after Cx43 down-regulation. Median (red line) intensities from four independent experiments. P values, Kruskal-Wallis test with Dunn’s multiple comparison test. (j and k) HPLC analysis of purines released from 4T1 and MMT spheroids into the supernatant after 24 h of invasion in 3D collagen. Values represent average concentrations of each purine (j) and the relative change of the total purine concentrations after treatment with CBX and/or Cx43 down-regulation in 4T1 and MMT cells (k). Mean values and SEM from four (j) or SD from three (k) independent experiments. P values, ANOVA Dunnett’s multiple comparison test. Scale bar: 20 µm (b). Neg ctrl, negative control; Norm., normalized; FC, follower cell; LC, leader cell; SC, single detached cell.

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Labeling, Selection, Fluorescence, Control, Two Tailed Test, MANN-WHITNEY, Stable Transfection, Expressing, Plasmid Preparation, shRNA, Inhibition, Flow Cytometry, Incubation, Comparison, Negative Control

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Cx43 dependence of leader cell function. (a and b) Resazurin cytotoxicity assay for 4T1 and MMT cells in 3D collagen for 24 h in the presence of vehicle control and escalating doses of CBX (a) and 18aGA (b). Values represent the mean relative fluorescence unit (RFU) with SEM of two or three independent experiments. P values, one-way ANOVA with Bonferroni multiple comparison test. (c) Bright-field images after 24 h of MMT cell invasion in 3D collagen in the presence of CBX or an inactive structurally homologous control compound (GLZ). Data represent the medians (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) of 8–11 MMT spheroids per condition pooled from three independent experiments. P value, two-tailed unpaired Mann–Whitney test. (d) Efficacy of collective invasion in 4T1 spheroids in the presence of escalating doses of 18aGA. Bright-field images after 24 h of culture. Arrowheads (c and d), tips of invasion strands. (e) 4T1 leader cell (LC) initiation in the presence of escalating doses of CBX. Data represent mean values and SEM from 23 to 40 spheroids per condition pooled from at least three independent experiments. P value, ANOVA with Bonferroni multiple comparison test. (f) Speed of MMT leader cells during collective invasion in 3D collagen. Values represent median (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) of seven or eight leader cells from one experiment. P value, two-tailed unpaired Mann–Whitney test. (g) Cumulative collective invasion of MMT cells. Data represent the medians (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) of 8–11 MMT spheroids per condition pooled from three independent experiments. P value, two-tailed unpaired Mann–Whitney test. (h) Cumulative collective invasion of 4T1 and MMT spheroids in the presence of escalating doses of 18aGA. Data represent mean values and SEM from 12 spheroids per condition pooled from three independent experiments. P value, ANOVA with Bonferroni multiple comparison test. Scale bars: 100 µm (c and d).

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Cell Function Assay, Cytotoxicity Assay, Control, Fluorescence, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Cx43 dependence of leader cell function. (a) Bright-field images of 4T1 spheroids after 24 h of invasion in 3D collagen in the presence of increasing concentration of CBX. Arrowheads, tips of invasion strands. (b) Mean cumulative length of collective invasion strands in response to CBX. Mean values and SEM from three independent experiments each comprising seven spheroids/condition. P values, Kruskal–Wallis test with Dunn’s multiple comparison test. (c) Maximum intensity projection from a confocal 3D stack, showing F-actin and DAPI in control and CBX-treated spheroids (100 µM). (d–g) Cx43-dependent collective invasion. Bright-field images (d), cumulative collective invasion (e and f), and leader cell initiation (g) of spheroids composed of 4T1 or MMT cells stably expressing Cx43 or nontargeting shRNA (NT). Values represent the medians (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) from three or four independent experiments with three to eight spheroids each. P values, two-tailed unpaired Mann–Whitney test. (h) Bright-field images of 4T1 spheroids in response to transient Cx43 down-regulation using two different Cx43 RNAi probes. (i) Number of leader cells (LCs) per spheroid. P values, Kruskal–Wallis test with Dunn’s multiple comparison test. (j) Maximum intensity projection from a confocal 3D stack showing mosaic spheroids composed of fluorescence-coded MMT cells stably expressing NT or Cx43 shRNA as indicated (1:1 ratio); arrowheads depict red or green leader cells. (k) Frequency of green or red leader cells. Bars represent the mean values and SEM pooled from one (j1) and three (j2) independent experiments. (l) Effect of CBX on the kinetics of established invasion strands. Mean length per invasion strand after addition of CBX at 0 h (intervention) and washout at 24 h (rescue). Data show the mean values and SEM from six to eight invasion strands from one spheroid per condition. Scale bars: 100 µm (a), 50 µm (c, d, and h), 25 µm (c, inset). LC, leader cell.

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Cell Function Assay, Concentration Assay, Comparison, Control, Stable Transfection, Expressing, shRNA, Two Tailed Test, MANN-WHITNEY, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Cx43 hemichannel function in leader cell induction by purine nucleotide release. (a–c) Rescue of invasion in 4T1 and MMT spheroids in 3D collagen in the presence of CBX and escalating doses of ADO; for comparison with untreated baseline levels refer to and . Representative bright-field images (a), cumulative collective invasion (b), and number of leader cells (LC) per spheroid (c). Mean values and SEM from four independent experiments, each comprising 5–11 spheroids/condition. P values, Kruskal–Wallis test with Dunn’s multiple comparison test. (d and e) Rescue of leader cell deficiency after Cx43 down-regulation by exogenous ADO. (d) Maximum intensity projection from a 3D confocal stack showing mosaic spheroids with MMT cells stably expressing NT or Cx43 shRNA (1:1 ratio) in the absence or presence of ADO and (e) frequency of LC with green color expressing Cx43 shRNA. Bars represent the means and SEM from three independent experiments, each comprising three to six spheroids. P values, two-tailed unpaired Mann–Whitney test. Scale bars: 50 µm (a), 100 µm (d).

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Comparison, Stable Transfection, Expressing, shRNA, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Cx43-mediated nucleotide release in LC initiation. (a) Leader cell (LC) initiation in MMT spheroids expressing shCx43 after 24 h of invasion in collagen in the presence and absence of exogenous ADO. Values display median (black line), 25th/75th percentiles (boxes) and maximum/minimum values (whiskers) of 30–35 spheroids per condition pooled from three independent experiments. P value, two-tailed unpaired Mann–Whitney test. (b) Purinergic receptor mRNA expression in 4T1 (black) and MMT (gray) cells during collective invasion in 3D collagen assessed by quantitative PCR. Values represent the mean normalized mRNA levels and SEM from three independent experiments. (c) MMT leader cell initiation in the presence of vehicle and escalating doses of PSB36. Values represent mean values and SEM of at least 20 spheroids per condition pooled from three independent experiments. P values, Kruskal–Wallis test with Dunn’s multiple comparison test. (d) Leader cell initiation in response to DCPX treatment 12–15 h of invasion of 4T1 cells in collagen. Values display medians (black lines) 25th/75th percentiles (boxes) and maximum/minimum values (whiskers), 23–27 spheroids from three independent experiments. P values, two-tailed unpaired Mann–Whitney test. (e) Mitotic figures in 4T1 spheroid after 24 h of invasion in collagen. Values represent average number and SEM of mitotic figures per spheroids, three spheroids (≥1,000 cell/spheroid) per condition. P value, two-tailed unpaired Mann–Whitney test. (f) Leader cell initiation of 4T1 and MMT spheroids after 12–15 h of invasion in collagen in the presence of ADORA2b inhibitor PSB1115. Values display medians (black lines) 25th/75th percentiles (boxes) and maximum/minimum values (whiskers), 4–12 spheroids from one experiment. P values, Kruskal–Wallis test with Dunn’s multiple comparison test. (g) Expression and distribution of ADORA1 in 4T1 spheroids treated with vehicle control or CBX for 24 h. Images represent single confocal slices. (h) ADORA1 expression of the outer cellular layer. Data represent the background-corrected mean fluorescence and SD of four (control) or six (CBX) spheroids from one experiment. P value, two-tailed unpaired Mann–Whitney test. Scale bars: 50 µm (g), 25 µm (g, inset).

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Comparison, Control, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Hemichannel-ADORA1 loop in other collective but not single-cell invasion models and downstream AKT activation. (a) Distribution of Cx43, β-catenin, and F-actin in NMuMG spheroids during collective invasion in 3D collagen after 68 h. Images represent maximum intensity projections from a 3D confocal stacks. Arrowheads, focal Cx43 hemichannel localization in leader cells. (b) Bright-field images of NMuMG spheroids embedded in 3D collagen for 68 h in the presence or absence of CBX. Arrowheads, protrusive leader cells. (c) Cumulative median length of invasion per spheroid (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) from 42 to 75 stands from six (control) and eight (CBX) spheroids (one experiment). P values, two-tailed unpaired Mann–Whitney test. (d) Bright-field images of NMuMG spheroids in 3D collagen after 48 h culture in the presence of PSB36 (5 or 20 µM). (e) Mean number of leader cells per spheroid and SEM from seven to nine spheroids per condition (one experiment). P values, Kruskal–Wallis test with Dunn’s multiple comparison test. (f) HPLC analysis of purines released from individualized 4T1 cells into the supernatant after 24 h of invasion in 3D collagen. Values represent concentrations of each purine from one experiment. (g) Speed of 4T1 leader vs individual cells in 3D collagen. Values represent the medians (black line), 25th/75th percentiles (box) and maximum/minimum (whiskers) of six leader cells (one experiment) and 19 individual cells tracked over 24 h (two independent experiments). P value, two-tailed unpaired Mann–Whitney test. (h and i) Percentage of individualized 4T1 cells with elongated morphology after 12, 24, and 40 h in collagen and treatment with CBX (h) and PSB36 (i). Data show the means and SEM from 17–33 cells per condition from two (h) or one (i) experiments. DPCPX, 8-cyclopentyl-1,3-dipropylxanthine. (j) Distribution of AKT, phospho-AKT473, and F-actin in MMT spheroid invasion culture. 3D confocal maximum intensity projections after 24 h of culture. Dashed line indicates the region of analysis in k. (k) Distribution of AKT and phospho-AKT473 and F-actin along extending protrusions of leader cells. Data represent the mean intensities and SEM of F-actin, AKT, and phospho-AKT473 from at least 12 leader cells from one experiment. (l) Phospho-AKT473 levels in response to 24 h of PSB36 treatment during 4T1 spheroid culture in 3D collagen. (m) Phospho-AKT473 levels in of 4T1 and MMT 2D cultures after ADO treatment for 0.5, 1, 3, 6, and 12 h. Numbers in (l, m) represent the ratio of phospho-AKT473 over total AKT mean intensities from one experiment. Scale bars: 50 µm (a, b, d, and j), 25 µm (a and j, insert).

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Activation Assay, Control, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Role of ADORA1 in preclinical spontaneous metastasis and prognostic relevance in clinical samples. (a) Detection of ADORA1 in normal ducts within clinical samples using multispectral microscopy. (b) Frequency of ADORA1-positive cancer cells with collective invasion patterns in breast lesions. Data represent the fibrous and adipose tissue from individual patients. (c) Overviews of lung sections isolated from DMSO and PSB36 (30 mg/kg) treated mice, stained for cytokeratin-8. Insets and arrowheads depict macro- and micrometastases. (d) Average number of micro- and macrometastases normalized per lung section. Data represent a total of 135–145 lung sections from three independent mice per group. (e) Relative frequency of micro- or macrometastases per treatment group: DMSO: 39 micrometastases, 59 macrometastases, 98 total; PSB36: 39 micrometastases, 91 macrometastases, 130 in total. Data show the mean percentages and SD from three mice. P value, two-tailed unpaired Mann–Whitney test. (f and g) Kaplan–Meier survival analysis predicting RFS in (f) basal-type breast cancer patients. (g) Correlation of ADORA1 or combined ADORA1 and Cx43 expression in clinical samples. Classification was based on tumor grade and molecular subtype . Bold and underlined values show the positive (black) or inverse (green) association with ADORA1/Cx43 expression. (f and g) P values, log-rank test. Scale bars: 100 µm (a), 50 µm (a and c, inset), 1,000 µm (c, overview).

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: Microscopy, Isolation, Staining, Two Tailed Test, MANN-WHITNEY, Expressing

Journal: The Journal of Cell Biology

Article Title: Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

doi: 10.1083/jcb.201911120

Figure Lengend Snippet: Histological and clinical classification of patient samples analyzed for Cx43 and/or ADORA1 expression

Article Snippet: The following antibodies were used: rabbit anti-human Cx43 (3512; CST), chicken anti-human vimentin (ab24525; Abcam), mouse anti-mouse β-catenin (clone 14/β-Catenin; BD Biosciences), rabbit anti-mouse AKT (9272; CST), mouse anti-human AKT (2920; CST); rabbit anti-human phospho-AKT Ser473 (4060; CST); mouse anti-human Pan Keratin (4545; CST); rabbit anti-rat ADORA1 (ab82477; Abcam); rabbit anti-human ADORA1 (ab124780; Abcam); and secondary polyclonal Alexa Fluor 488/647–conjugated goat anti-mouse, anti-rabbit, or anti-chicken antibody.

Techniques: