Atenin (E-5), anti-Snail-1, anti-Twist, and anti-p21 (H-164)), Cell Signaling (anti-Slug), BD Transduction Laboratories (anti-E-cadherin), Calbiochem (p73 (ab-3)), ProSci incorporated (anti-PUMA), Sigma (anti-actin), and BioRad (Life Science Research, Hercules, CA; secondary antibodies against rabbit or mouse IgG conjugated with HRP).ReagentsGrowth factor-reduced Matrigel was purchased from BD Transduction Laboratories (Franklin Lakes, NJ). DMEM/F12 medium, donor horse serum, To-Pro-3 nucleus dye, and antimouse antibody conjugated to fluorophore 488 were purchased from Invitrogen (Carlsbad, CA). Hydrocortisone, insulin and cholera toxin were purchased from Sigma (St. Louis, MI). Recombinant human epidermal growth factor (EGF) was purchased from Title Loaded From File Peprotech (Rocky Hill, NJ). Normal goat serum was purchased from Jackson ImmunoResearch (West Grove, PA).Confocal Microscopy3-D structures in Matrigel were fixed in 4 paraformaldehyde at room temperature for 20 min and permeabilized with 0.5 Triton-X100 in PBS for 30 min at 4uC. After quenching with 100 mM glycine in PBS, the structures were pre-blocked in a primary blocking buffer A (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 0.1 BSA, 0.2 Triton X-100 and 0.05 Tween-20) containing 10 normal goat serum for 2 h and further blocked in a secondary blocking buffer B (buffer A plus 10 normal goat serum and 20 mg/mL of goat anti-mouse F[ab9]2 fragments) for 1 h. The structures were incubated overnight with primary antibodies at 4uC, washed thoroughly with buffer A with gentle shaking, and stained with FITC-conjugated secondary antibody (diluted 1:200 in buffer A) for 1 h. The structures were nuclear stained with 5 mg/mL of To-Pro-3 in PBS for 15 min 1315463 at room temperature. The To-Pro-3 stain was removed by washing the chamber slide with PBS for 5 min and then the slide was mounted under a glass cover slip with 0.1 para-phenylenediamine D (PPD) and 90 glycerol in PBS. Microscopic analysis was performed using a confocal microscopy system (Axiovert 100 m, Zeiss) and images were acquired using the software for Carl Zeiss Laser Scanning Microscope (LSM-510). The images of acinus structures were captured by the Z-stacking function for serial confocal sectioning at 2 mm intervals. Experiments were conducted in triplicate.Plasmid Constructions and Cell Line GenerationsTo produce p21 or PUMA shRNA under the control of the U6 promoter, two 62-base oligos were annealed and then cloned into pBabe-U6 shRNA expression vector [6], and the resulting plasmids were designed as pBabe-U6-shp21, or pBabe-U6shPUMA. To generate MCF10A cell lines with stable knockdown of DNp73 in combination with p21 or PUMA, the following plasmids, pBabe-U6-shDNp73, pBabe-U6-shp21 or pBabe-U6shPUMA, were co-transfected into MCF10A cells. The resulting knockdown cell lines were selected with puromycin and confirmed by RT-PCR and/or Western blot analysis. The shRNA oligos used are Title Loaded From File listed with the siRNA targeting region shown in uppercase (Table. 1).RT-PCR AnalysisTotal RNA was extracted from cells using TRIzol (Invitrogen Life Technoloogies, Inc.) according to manufacturer’s instructions. cDNA was synthesized using 23977191 M-MLV Reverse Transcriptase Kit (Promega Corporation) according to manufacturer’s protocol. The primers to detect DNp73 are sense 59-gatccatgccctcgtcccac-39 and antisense 59-ctgctcatctggtccatgg-39. The Actin gene was chosen as a loading control and detected with primers 59-ctgaagtaccccatcgagcacggca-39 (sense) and 59-ggatagcaca.Atenin (E-5), anti-Snail-1, anti-Twist, and anti-p21 (H-164)), Cell Signaling (anti-Slug), BD Transduction Laboratories (anti-E-cadherin), Calbiochem (p73 (ab-3)), ProSci incorporated (anti-PUMA), Sigma (anti-actin), and BioRad (Life Science Research, Hercules, CA; secondary antibodies against rabbit or mouse IgG conjugated with HRP).ReagentsGrowth factor-reduced Matrigel was purchased from BD Transduction Laboratories (Franklin Lakes, NJ). DMEM/F12 medium, donor horse serum, To-Pro-3 nucleus dye, and antimouse antibody conjugated to fluorophore 488 were purchased from Invitrogen (Carlsbad, CA). Hydrocortisone, insulin and cholera toxin were purchased from Sigma (St. Louis, MI). Recombinant human epidermal growth factor (EGF) was purchased from Peprotech (Rocky Hill, NJ). Normal goat serum was purchased from Jackson ImmunoResearch (West Grove, PA).Confocal Microscopy3-D structures in Matrigel were fixed in 4 paraformaldehyde at room temperature for 20 min and permeabilized with 0.5 Triton-X100 in PBS for 30 min at 4uC. After quenching with 100 mM glycine in PBS, the structures were pre-blocked in a primary blocking buffer A (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 0.1 BSA, 0.2 Triton X-100 and 0.05 Tween-20) containing 10 normal goat serum for 2 h and further blocked in a secondary blocking buffer B (buffer A plus 10 normal goat serum and 20 mg/mL of goat anti-mouse F[ab9]2 fragments) for 1 h. The structures were incubated overnight with primary antibodies at 4uC, washed thoroughly with buffer A with gentle shaking, and stained with FITC-conjugated secondary antibody (diluted 1:200 in buffer A) for 1 h. The structures were nuclear stained with 5 mg/mL of To-Pro-3 in PBS for 15 min 1315463 at room temperature. The To-Pro-3 stain was removed by washing the chamber slide with PBS for 5 min and then the slide was mounted under a glass cover slip with 0.1 para-phenylenediamine D (PPD) and 90 glycerol in PBS. Microscopic analysis was performed using a confocal microscopy system (Axiovert 100 m, Zeiss) and images were acquired using the software for Carl Zeiss Laser Scanning Microscope (LSM-510). The images of acinus structures were captured by the Z-stacking function for serial confocal sectioning at 2 mm intervals. Experiments were conducted in triplicate.Plasmid Constructions and Cell Line GenerationsTo produce p21 or PUMA shRNA under the control of the U6 promoter, two 62-base oligos were annealed and then cloned into pBabe-U6 shRNA expression vector [6], and the resulting plasmids were designed as pBabe-U6-shp21, or pBabe-U6shPUMA. To generate MCF10A cell lines with stable knockdown of DNp73 in combination with p21 or PUMA, the following plasmids, pBabe-U6-shDNp73, pBabe-U6-shp21 or pBabe-U6shPUMA, were co-transfected into MCF10A cells. The resulting knockdown cell lines were selected with puromycin and confirmed by RT-PCR and/or Western blot analysis. The shRNA oligos used are listed with the siRNA targeting region shown in uppercase (Table. 1).RT-PCR AnalysisTotal RNA was extracted from cells using TRIzol (Invitrogen Life Technoloogies, Inc.) according to manufacturer’s instructions. cDNA was synthesized using 23977191 M-MLV Reverse Transcriptase Kit (Promega Corporation) according to manufacturer’s protocol. The primers to detect DNp73 are sense 59-gatccatgccctcgtcccac-39 and antisense 59-ctgctcatctggtccatgg-39. The Actin gene was chosen as a loading control and detected with primers 59-ctgaagtaccccatcgagcacggca-39 (sense) and 59-ggatagcaca.
