Journal: Journal of Biological Chemistry

Article Title: Identification of the Drosophila Ortholog of HSPB8

doi: 10.1074/jbc.m110.127498

Figure Lengend Snippet: FIGURE 1. D. melanogaster HSP67Bc is a functional ortholog of human HSPB8. A and B, human HSPB8 and Dm-HSP67Bc co-immunoprecipitate with Dm-Starvin. HEK-293T cells were transfected with vectors encoding for D. melanogaster Myc-Starvin alone or together with either V5-HSPB8, V5-HSP67Bc, V5-L(2)efl, or V5-CG14207. 24 h post-transfection, the cell lysates were subjected to immunoprecipitation (IP) with an antibody against the V5 tag, and the immunoprecipitated complexes were analyzed by Western blotting (WB) using V5- and Myc-specific antibodies. Among the D. melanogaster sHSPs analyzed, HSP67Bc interacts with Dm-Starvin (B), similarly to human HSPB8 (A). C, like HSPB8, Dm-HSP67Bc also binds to BAG3, the human functional ortholog of Dm-Starvin. HEK293 cells were transfected with vectors encoding for human Myc-BAG3 alone or together with either V5-HSP67Bc, V5-L(2)efl, V5-CG14207, or V5-HSPB8 and V5-HSP70, both used as positive controls and subjected, 24 h post-transfection, to immu- noprecipitation with a V5-specific antibody. D, endogenous Hsp67Bc interacts with Starvin in vivo in fly head extracts. V5-starvin was expressed in flies under the control of the grm-GAL4 driver. Immunopre- cipitation with a specific V5 antibody was carried out using fly head protein extracts from control flies (grm/) and flies expressing V5-Starvin (gmr/V5-Stv). Interaction of endogenous Hsp67Bc with V5-Starvin was investigated by Western blotting using a specific rabbit polyclonal Hsp67Bc antibody. E, total levels of HSP67Bc are increased when it is co-expressed with Starvin. Drosophila Schneider S2 cells were trans- fected with vectors encoding for V5-HSP67Bc, V5-L(2)efl, and V5-CG14207 alone or in combination with V5-Starvin. The protein expression levels were analyzed by Western blotting 48 h post-transfection (aver- age values S.E. (error bars) of n 3–4 independent samples). F, human muscle tissue section showing that endogenous HSPB8 colocalizes with -actinin at the Z band. G, endogenous Dm-HSP67Bc colocalizes with -actinin at the Z band in third instar larvae muscles.

Article Snippet: Mouse monoclonal anti- -actinin and mouse monoclonal anti- -tubulin were from Sigma-Aldrich, whereas mouse monoclonal anti-Myc (9E10)was from theAmericanTypeCulture Collection.Mousemonoclonal anti-total-eIF2 and rabbit polyclonal anti-phospho-eIF2 were from Cell Signaling and Sigma-Aldrich, respectively.

Techniques: Functional Assay, Transfection, Immunoprecipitation, Western Blot, In Vivo, Control, Expressing, Muscles

Journal: Stem Cell Research & Therapy

Article Title: Hypoxia accelerates vascular repair of endothelial colony-forming cells on ischemic injury via STAT3-BCL3 axis

doi: 10.1186/s13287-015-0128-8

Figure Lengend Snippet: Hypoxic preconditioning of endothelial colony-forming cells (ECFCs) enhances functional recovery after limb ischemia. a Improvements in blood flow recovery were evaluated using LDPI analysis in the ischemic limbs of 8-week-old Balb/C nude mice injected with phosphate-buffered saline (PBS), nor-ECFCs, and hypo-ECFCs at 0, 4, 9, 18, and 28 days post-surgery. b The ratio of blood perfusion (blood flow of the left ischemic limb/blood flow of the right non-ischemic limb) was measured in the three groups. Values represent means ± SD. ** P < 0.01 vs. PBS group; ## P < 0.01 vs. nor-ECFCs. c , e Representative images of mouse-specific CD31 + and human-specific CD31 + tissue at 28 days after transplantation of nor-ECFC or hypo-ECFC into hindlimb ischemia (scale bar: 50 μm; n = 10). g Representative images of arteriole structures (α-SMA staining for arterioles, red fluorescence)-positive tissue at 28 days after transplantation of nor-ECFC or hypo-ECFC into hindlimb ischemia (scale bar: 50 μm; n = 10). Quantification ( d , f , h ) represents cell numbers analyzed per high-power field (HPF). Values represent means ± SD. ** P < 0.01 vs. nor-ECFCs; ## P < 0.01 vs. hypo-ECFCs. i H&E staining was used to produce histological images of tissue 28 days after transplantation of nor-ECFCs or hypo-ECFCs into hindlimb ischemia

Article Snippet: Immunofluorescence staining was performed using primary antibodies against mouse-specific (to confirm mouse vasculature) or human-specific (to confirm human ECFC-derived vessel formation) CD31, α-smooth muscle actin (α-SMA), human VEGF, phospho-STAT3, caspase-3, PCNA, Ki67 (Santa Cruz), human-specific CD31 (Novus Biologicals, Colorado, USA), and human nuclear antigen (HNA; Millipore) and secondary antibodies Alexa-488 and Alexa-594 (Life Technologies, Carlsbad, CA, USA).

Techniques: Functional Assay, Injection, Saline, Transplantation Assay, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity

doi: 10.1016/j.jbc.2023.104786

Figure Lengend Snippet: FADD is required for efficient ce ll cycle entry in lung cancer cells having oncogenic EGFR. A , the PIP-FUCCI dual reporter expression construct. The PIP-Cdt1 polypeptide consists of the Cdt1 1–17 PIP (PCNA-interacting protein) degron and an NLS fused to mVenus. The Gem 1–110 polypeptide consists of the D-box and the KEN-box degron motifs of Geminin fused to mCherry. The P2 self-cleaving peptide separates the two fluorescent polypeptides ( top panel ). The PIP-FUCCI cell cycle reporter is designed such that during G1, the Cdt1-mVenus polypeptide is stable, while the Geminin-mCherry polypeptide is degraded (APC/C-Cdh1 is activated) and hence cells demonstrate green fluorescence. Upon entry into S phase, inactivation of APC/C-Cdh1 results in stabilization of geminin-mCherry but degradation of Cdt1-mVenus resulting in red fluorescence. In the G2 phase, both polypeptides are stable and hence cell fluoresce yellow ( green + red ) ( lower panel ). A schematic of cell cycle transitions emphasizing APC/C-Cdh1 inactivation prior to entry into S phase and that APC/C-Cdh1 activity is characteristic of cells in G1. B , images of a single nucleus from confocal time-lapse imaging of stable PIP-FUCCI reporter expressing NCI-H1975 cells. Cell cycle transition from G1 to S ( green to red ) required 8 to 10 h, S to G2 ( red to yellow ) required 8 to 10 h and G2/M required 4 to 6 h. Scale bar is 10 μm. C , schematic of EGFR-mediated activation of the KRAS-MEK-ERK mitogenic signaling pathway in NCI-H1975 and HCC827 cells. D , live cell imaging of control siRNA (CTRL) and FADD KD NCI-H1975 cells was used to obtain fluorescence emission traces of individual cells. Traces were aligned in silico at the G1 to S transition (sharp decrease in Cdt1-mVenus signal and a steady increase in Geminin-mCherry signal). The first panel shows the mean of the median traces ( bold p lot ) of three independent experiments with variance shown in a lighter shade . Individual fluorescence traces of single cells (Cdt1-mVenus and Geminin-mCherry) are also shown. CTRL cells demonstrated an 8 to 10 h G1 phase, while a majority of FADD KD cells were in G1 during the entire imaging time or were in G1 for 24 h. n = 188 for CTRL and 152 for FADD KD cells. E , quantification of average time spent in S, G2/M, and G1 phase by CTRL cells and FADD KD cells reveal that in contrast to a 8 to 10 h G1, FADD KD cells had a G1 time of >20 h whereas there is no difference in previous S phase and G2/M time in CTRL versus FADD KD cells. n = 58, n = 35 and n = 123 for S, G2/M and G1 respectively. Quantifications are from three independent experiments. Error bars represents mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001, n.s. = non-significant. F , bar graph showing percentage of cells arrested in G1 in CTRL and FADD KD NCI-H1975 cells. 60% of FADD knockdown cells demonstrated an arrested G1, while none of the CTRL cells were arrested in G1, n = 165 for CTRL and n = 160 for FADD KD from three independent experiments. Error bars represents mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. G , quantification of number of cells that underwent at least one mitotic event during the 48 h live cells imaging experiment revealed that 100% of CTRL cells underwent mitosis, in contrast to only 5% of FADD KD cells. Error bars show the mean of three independent experiments ± SEM. Each dot represents average number of mitotic events per experiment with at least 100 cells analyzed per experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. H , Western blot analysis of CTRL ( left panel ) and FADD KD ( right panel ) cells synchronized in G2/M using the CDK1 inhibitor, CDK1i, and samples were collected at indicated timepoints after release from the block. Immunoblots were probed using S-phase and G2/M phase markers cyclin B1, cyclin A2, Securin, pRB, Phospho Histone-3, Aurora A, p27 as well as FADD and α-actinin as controls. CTRL cells show mitotic exit at approximately 8 h (degradation of cyclin A2, cyclin B1, Aurora A and Securin) and a transition from G1 to S was observed at 24 h post G2/M release (as observed by accumulation of cyclin A2, cyclin B1 and Securin). In contrast, FADD KD cells fail to accumulate each of these S phase markers at the expected time. I , quantification of cyclin B1 and cyclin A2 levels in CTRL and FADD knockdown NCI-H441 cells synchronized at G2/M using the CDK1 inhibitor, CDK1i. CTRL cells show an accumulation of cyclin B1 ( top panel ) and cyclin A2 ( bottom panel ) upon transition from G1 to S ( green line ), while FADD KD cells ( red line ) demonstrate a decreased efficiency of accumulation of each S phase cyclin. Error bars show mean values from three independent experiments with ± SEM. cyclin B1 and cyclin A2 were normalized to their respective mitotic levels. CTRL, Control; FADD, Fas-associated protein with death domain; NLS, nuclear localization signal.

Article Snippet: The following antibodies were used in this study: myc (9E10, SC-131, Mouse monoclonal) 1:1000 WB; cyclin B1 (sc-245, Mouse monoclonal) 1:1000 WB; were from Santa Cruz Biotechnology; FADD (2782, rabbit polyclonal) 1:1000; GST (26H1, mouse monoclonal) 1:1000 WB; cyclin A2 (4656, mouse monoclonal) 1:1000 WB; cyclin E1 (4129, mouse monoclonal) 1:1000 WB; cyclin E1 (13445, rabbit monoclonal) 1:1000 WB; Phospho-Histone H3 (3377, rabbit monoclonal) 1:1000 WB; Aurora A (14475, rabbit monoclonal) 1:1000 WB; α-Actinin (6487, rabbit monoclonal) 1:1000 WB; Rb (9309, mouse monoclonal) 1:100 IF; Phospho-Rb (8516, rabbit monoclonal) 1:1000 WB, 1:350 IF; Phospho-FADD (Ser194) Antibody (Human Specific) (2781 1:1000 WB, 1:100 IF) were from Cell Signaling Technology; Cdh1 (FZR1) (MABT1323 mouse monoclonal) was from Millipore Sigma; Alexa Fluor 405 and Alexa Fluor 647 -conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies (A48258, A32787) were from Thermo Fisher Scientific; (HRP)-conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies were from Jackson Immunoresearch: horseradish peroxidase secondary antibodies (715-035-151, 711-035-152).

Techniques: Expressing, Construct, Fluorescence, Activity Assay, Imaging, Activation Assay, Live Cell Imaging, Control, In Silico, Knockdown, Western Blot, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity

doi: 10.1016/j.jbc.2023.104786

Figure Lengend Snippet: FADD is required for efficient cell cycle entry in KRAS mutant lung cancer cells. A , schematic of mutant KRAS-mediated activation of the KRAS-MEK-ERK mitogenic signaling pathway in NCI-H44 cells. B , single cell traces of control siRNA (CTRL) or FADD siRNA (FADD KD)–treated NCI-H441 cells stably expressing the PIP-FUCCI reporter. Fluorescence traces were aligned in silico at the G1 to S transition (sharp decrease in Cdt1-mVenus signal and a steady increase in Geminin-mCherry signal). The first panel shows the mean of the median traces ( bold p lot ) of three independent experiments with variance shown in a lighter shade . Individual fluorescence traces of single cells (Cdt1-mVenus and Geminin-mCherry) are also shown. CTRL cells demonstrated a 10 h G1 phase, while FADD KD cells were in G1 for 20 h. n = 85 for CTRL and 65 for FADD KD cells. C , quantification of average time spent in S, G2/M, and G1 phase by CTRL and FADD KD NCI-H441 cells reveal that in contrast to 10 h G1, FADD KD cells had a G1 time of >20 h whereas there was no significant difference in previous S and G2/M times in CTRL versus FADD KD cells. n = 45, n = 42 and n = 105 for S, G2/M and G1 phases, respectively. Quantifications are from three independent experiments. Error bars represents mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001, n.s. = non-significant. D , bar graph showing percentage of cells arrested in G1 in CTRL or FADD KD NCI-H441 cells. 30% of FADD KD cells demonstrated an arrest in G1 compared to 0% of CTRL cells ( left panel ). n = 100 for CTRL and n = 100 for FADD KD. Error bars represents mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001, n.s. = non-significant. E , percentage of CTRL and FADD KD NCI-H441 cells that underwent mitosis during 48 h of imaging. Each data point represents a total of at least 100 cells from a single experiment. 100% of CTRL cells underwent mitosis whereas only 10% of FADD KD cells underwent mitosis during the entire imaging period. Error bars shows the mean of three independent experiments ± SEM. n = 300 for CTRL and n = 200 for FADD KD cells from three independent experiments. Error bars represents mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. F , Western blot analysis of CTRL ( left panel ) and FADD KD ( right panel ) cells synchronized in G2/M using CDK1i, and samples were collected at indicated timepoints after release from the block. Immunoblots were probed using S-phase and G2/M phase markers cyclin B1, cyclinA2, Securin, pRB, Phospho Histone-3, Aurora A, as well as FADD and α-actinin as controls. CTRL cells show mitotic exit at approximately 4 h (degradation of cyclin A2, cyclin B1, Aurora A and Securin) and a transition from G1 to S was observed at 20 h post G2/M release (as observed by accumulation of cyclin A2, cyclin B1 and Securin). In contrast, FADD KD cells fail to accumulate each of these S phase markers at the expected time. G , quantification of cyclin B1 and cyclin A2 levels in CTRL and FADD knockdown NCI-H441 cells synchronized at G2/M using CDK1i. Control (CT) cells show an accumulation of cyclin B1 ( left panel ) and cyclin A2 ( right panel ) upon transition from G1 to S ( green line ), while FADD KD cells ( red line ) demonstrate a decreased efficiency of accumulation of each S phase cyclin. Error bars show mean values from three independent experiments with ± SEM. cyclin B1 and cyclin A2 were normalized to their respective mitotic levels. CTRL, Control; FADD, Fas-associated protein with death domain.

Article Snippet: The following antibodies were used in this study: myc (9E10, SC-131, Mouse monoclonal) 1:1000 WB; cyclin B1 (sc-245, Mouse monoclonal) 1:1000 WB; were from Santa Cruz Biotechnology; FADD (2782, rabbit polyclonal) 1:1000; GST (26H1, mouse monoclonal) 1:1000 WB; cyclin A2 (4656, mouse monoclonal) 1:1000 WB; cyclin E1 (4129, mouse monoclonal) 1:1000 WB; cyclin E1 (13445, rabbit monoclonal) 1:1000 WB; Phospho-Histone H3 (3377, rabbit monoclonal) 1:1000 WB; Aurora A (14475, rabbit monoclonal) 1:1000 WB; α-Actinin (6487, rabbit monoclonal) 1:1000 WB; Rb (9309, mouse monoclonal) 1:100 IF; Phospho-Rb (8516, rabbit monoclonal) 1:1000 WB, 1:350 IF; Phospho-FADD (Ser194) Antibody (Human Specific) (2781 1:1000 WB, 1:100 IF) were from Cell Signaling Technology; Cdh1 (FZR1) (MABT1323 mouse monoclonal) was from Millipore Sigma; Alexa Fluor 405 and Alexa Fluor 647 -conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies (A48258, A32787) were from Thermo Fisher Scientific; (HRP)-conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies were from Jackson Immunoresearch: horseradish peroxidase secondary antibodies (715-035-151, 711-035-152).

Techniques: Mutagenesis, Activation Assay, Control, Stable Transfection, Expressing, Fluorescence, In Silico, Imaging, Western Blot, Blocking Assay, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity

doi: 10.1016/j.jbc.2023.104786

Figure Lengend Snippet: APC/C-Cdh1 ubiquitin ligase hyperactivity in FADD depleted cells requires Cdh1 expression. A , a schematic of the experimental design to investigate if failure to accumulate cyclin B1 during the G1 to S transition in the absence of FADD can be ascribed to hyperactive APC/C-Cdh1. Cells arrested at G2/M (using CDK1i) were released into G1 and treated with inhibitors of APC/C or the proteosome at the expected time for G1 to S transition (20 h). Cell extracts were prepared and analyzed for cyclin B1 levels 10 h post-treatment. B , immunoblot analysis for cyclin B1 levels in control siRNA-transfected (CTRL) as well as FADD siRNA transfected (FADD KD) cells treated with DMSO, MG132, ProTAME, or APCin. In contrast to DMSO-treated CTRL cells, FADD KD cells has decreased levels of cyclin B1. Treatment with MG132 (proteasomal inhibitor) and ProTAME (APC/C inhibitor) but not APCin (APC/C-Cdc20 inhibitor) resulted in an increase in cyclin B1 levels in FADD KD cells but not CTRL cells ( bottom panel ). C , live cell imaging of control siRNA (CTRL), FADD siRNA (FADD KD), Cdh1 siRNA (Cdh1 KD), and FADD+Cdh1 siRNA (FADD+Cdh1 KD) treated cells was used to acquire fluorescence emission traces over a >40 h time period. Graphs shown are the mean of the median traces from the two independent experiments with variance shown in a lighter shade . CTRL cells demonstrated a 8.5 h time in G1, while a majority of FADD KD cells were in G1 during the entire imaging period of 48 h. Cdh1 KD cells demonstrated a reduced G1 time of 6.5 h compared to CTRL cells (8.5 h) whereas simultaneous knockdown of FADD and Cdh1 also resulted in reduced G1 times of 6.5 h. D , quantification of average time spent in G1 phase by CTRL, FADD KD, Cdh1 KD, and FADD+Cdh1 KD NCI-H1975 cells. CTRL cells exhibited a 8.5 h time in G1 while FADD KD cells had a G1 time of 24 h. Cdh1 KD and FADD+Cdh1 KD cells had reduced mean time in G1 of 6.5 h. n = 50 cells for all conditions from two independent experiments. Error bars represents mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001, n.s. = non-significant. E , bar graph showing percentage of cells arrested in G1 in CTRL, FADD KD, Cdh1 KD, and FADD+Cdh1 KD NCI-H1975 cells. 70% of FADD KD cells demonstrated an arrest in G1 compared to <2% in CTRL, Cdh1 KD, and FADD+Cdh1 KD cells. n = 50 from two independent experiments. Error bars represents mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001, n.s. = non-significant. F , Western blot analysis of CTRL, FADD KD, Cdh1 KD and FADD+Cdh1 KD NCI-H1975 cells collected 48 h after siRNA treatment. Immunoblots were probed using Cdh1, FADD or α-actinin antibodies. G , functional domains within human FADD showing the Death Effector Domain at the N-terminus and the Death Domain at the C-terminus, as well as putative D-box and KEN-box motifs ( top panel ). Expression constructs used to investigate the interaction of FADD with Cdh1. GST-tagged FADD WT , FADD Dbox (RESL to AAAA), and FADD KEN (KEN to AAA) mutants of FADD as well as with myc-tagged human Cdh1 were constructed. H , lysates from HEK293FT cells expressing the indicated constructs were immunoprecipitated using myc antibodies (myc-trap nanobodies) to immunoprecipitate myc-Cdh1. The precipitates were immunoblotted with myc antibody to detect the immunoprecipitated myc-CDH1 as well as a GST antibody to detect GST-FADD coimmunoprecipitation. FADD, Fas-associated protein with death domain.

Article Snippet: The following antibodies were used in this study: myc (9E10, SC-131, Mouse monoclonal) 1:1000 WB; cyclin B1 (sc-245, Mouse monoclonal) 1:1000 WB; were from Santa Cruz Biotechnology; FADD (2782, rabbit polyclonal) 1:1000; GST (26H1, mouse monoclonal) 1:1000 WB; cyclin A2 (4656, mouse monoclonal) 1:1000 WB; cyclin E1 (4129, mouse monoclonal) 1:1000 WB; cyclin E1 (13445, rabbit monoclonal) 1:1000 WB; Phospho-Histone H3 (3377, rabbit monoclonal) 1:1000 WB; Aurora A (14475, rabbit monoclonal) 1:1000 WB; α-Actinin (6487, rabbit monoclonal) 1:1000 WB; Rb (9309, mouse monoclonal) 1:100 IF; Phospho-Rb (8516, rabbit monoclonal) 1:1000 WB, 1:350 IF; Phospho-FADD (Ser194) Antibody (Human Specific) (2781 1:1000 WB, 1:100 IF) were from Cell Signaling Technology; Cdh1 (FZR1) (MABT1323 mouse monoclonal) was from Millipore Sigma; Alexa Fluor 405 and Alexa Fluor 647 -conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies (A48258, A32787) were from Thermo Fisher Scientific; (HRP)-conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies were from Jackson Immunoresearch: horseradish peroxidase secondary antibodies (715-035-151, 711-035-152).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Control, Transfection, Live Cell Imaging, Fluorescence, Imaging, Knockdown, Functional Assay, Construct, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity

doi: 10.1016/j.jbc.2023.104786

Figure Lengend Snippet: FADD KEN but not FADD WT or FADD Dbox conditional overexpression leads to APC/C-Cdh1 hyperactivity during the G1/S transition. A , a schematic of the experimental approach. Doxycycline inducible halo FADD overexpressing NCI-H441 cells were synchronized at G2/M using CDK1i and released. Live cell imaging of cyclin accumulation using Western blot analysis (see below). B , single cell traces of NT CTRL, FADD WT , FADD Dbox , and FADD KEN overexpressing NCI-H441 cells stably expressing PIP-FUCCI reporter synchronized at G2/M using CDK1 inhibitor CDK1i. APC/C-Cdh1 activity was monitored using Geminin-mCherry fluorescence. Accumulation of mCherry fluorescence beginning 16 to 18 h post-release is indicative of APC/C-Cdh1 inactivation during the G1 to S phase transition in no-treatment control cells (NT CTRL). Analogous to NT CTRL cells, 100% of FADD WT and FADD Dbox cells demonstrated accumulation of Geminin-mCherry fluorescence (indicative of APC/C-Cdh1 inactivation) at the expected 16 to 18 h post G2/M release, whereas APC/C-Cdh1 inactivation was delayed (24–32 h) and observed in only 72% of FADD KEN cells. 38% of FADD KEN cells failed to inactivate APC/C-Cdh1 (shown in grey ) during the 48 h imaging time. n = 100 independent traces of each condition are shown, from at least three independent experiments. C , Western blot analysis of NT CTRL as well as inducible FADD WT , FADD Dbox , and FADD KEN overexpressing NCI-H441 cells were synchronized at G2/M using CDK1i, released and samples were collected at various timepoints for analysis of S-phase and G2/M phase markers cyclin B1, cyclin A2, Securin, Phospho Histone-3, Aurora A, Halo (FADD transgenes are Halo-tagged) and α-actinin. NT CTRL, FADD WT and FADD Dbox cells demonstrate elevated levels of G2/M proteins (cyclin B1, cyclin A2, Aurora A, Securin at 0 h) which are depleted upon mitotic exit (8 h), and reaccumulated upon entry into S as a result of APC/C-Cdh1 inactivation at 20 h post G2/M release. In contrast to NT CTRL as well as FADD WT , FADD Dbox overexpressing cells, FADD KEN overexpressing cells demonstrate decreased levels of each of these proteins in G2/M (0 h) as well as a failure to efficiently accumulate cyclin A2, cyclin B1, and Securin at the expected time. D , quantification of cyclin B1 in NT CTRL, FADD WT ,FADD Dbox and FADD KEN NCI-H441 cells synchronized at G2/M using CDK1i. Control (NT CTRL), FADD WT , and FADD Dbox cells show an accumulation of cyclin B1 upon transition from G1 to S while FADD KEN cells demonstrate a decreased efficiency of accumulation of cyclin B1. Error bars show mean values from two independent experiments with ± SEM. cyclin B1 was normalized to its 0 h timepoint level. E , percentage of NT CTRL, or doxycycline-inducible FADD WT , FADD Dbox , and FADD KEN overexpressing NCI-H441 cells undergoing mitosis during 48 h of imaging (Imaging was initiated 24 h after the knockdown). Each data point represents a total of at least 100 cells from a single experiment. Error bars show the mean of three independent experiments ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001, n.s., non-significant. F , lysates from G1 synchronized (as described in Fig. 5 A ) NCI-H441 cells expressing indicated doxycycline-inducible Halo-tagged FADD WT or Halo-tagged FADD KEN (KEN to AAA) were immunoprecipitated using Cdc27(APC3) specific antibodies to immunoprecipitate the entire APC/C complex. Lysates were immunoblotted using Cdc27 or FADD antibodies. 10% immunocomplexes were immunoblotted with Cdc27 antibodies whereas 90% immunocomplexes were immunoblotted with FADD antibodies to detect co-immunoprecipitated FADD WT or FADD KEN along with Cdc27 ( upper panel ). Reverse IP of the same samples to confirm APC/C-Cdh1 interaction are shown in Fig. S7 E . The adjacent cartoon presents our working model for FADD dependent inactivation of APC/C-Cdh1 through its KEN-box dependent interaction with Cdh1. FADD WT inhibits APC/C-Cdh1 during the G1 to S transition. Based on the above results, we propose that FADD KEN interaction with a different subunit of the APC/C but not Cdh1 leads to APC/C-Cdh1 hyperactivity in a dominant-negative fashion, wherein endogenous wild-type FADD is unable to inhibit APC/C-Cdh1 activity. FADD, Fas-associated protein with death domain.

Article Snippet: The following antibodies were used in this study: myc (9E10, SC-131, Mouse monoclonal) 1:1000 WB; cyclin B1 (sc-245, Mouse monoclonal) 1:1000 WB; were from Santa Cruz Biotechnology; FADD (2782, rabbit polyclonal) 1:1000; GST (26H1, mouse monoclonal) 1:1000 WB; cyclin A2 (4656, mouse monoclonal) 1:1000 WB; cyclin E1 (4129, mouse monoclonal) 1:1000 WB; cyclin E1 (13445, rabbit monoclonal) 1:1000 WB; Phospho-Histone H3 (3377, rabbit monoclonal) 1:1000 WB; Aurora A (14475, rabbit monoclonal) 1:1000 WB; α-Actinin (6487, rabbit monoclonal) 1:1000 WB; Rb (9309, mouse monoclonal) 1:100 IF; Phospho-Rb (8516, rabbit monoclonal) 1:1000 WB, 1:350 IF; Phospho-FADD (Ser194) Antibody (Human Specific) (2781 1:1000 WB, 1:100 IF) were from Cell Signaling Technology; Cdh1 (FZR1) (MABT1323 mouse monoclonal) was from Millipore Sigma; Alexa Fluor 405 and Alexa Fluor 647 -conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies (A48258, A32787) were from Thermo Fisher Scientific; (HRP)-conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies were from Jackson Immunoresearch: horseradish peroxidase secondary antibodies (715-035-151, 711-035-152).

Techniques: Over Expression, Live Cell Imaging, Western Blot, Stable Transfection, Expressing, Activity Assay, Fluorescence, Sublimation, Control, Imaging, Knockdown, Immunoprecipitation, Dominant Negative Mutation

Journal: Cell Reports

Article Title: Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation

doi: 10.1016/j.celrep.2019.05.005

Figure Lengend Snippet: Activation of YAP Mediates Activity of miRNAs that Induce Cardiomyocyte Proliferation (A) Experimental scheme to test activation of TEAD-dependent transcription. (B) TEAD-luciferase reporter analysis of CMs transfected with the indicated miRNA mimics. Transfection efficiency was standardized over a constitutively expressed Renilla luciferase reporter. Transfection of a constitutively active YAP plasmid (pYAP5SA) served as a positive control. Data are mean ± SEM (n = 5 independent experiments); ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (C) Induction of YAP nuclear translocation by treatment of CMs with pro-proliferative miRNAs. The western blots show the levels of nuclear and cytoplasmic YAP1 and phospho-YAP1 (P-YAP1) 72 h after transfection in a representative experiment. PARP1 and GAPDH were used for loading control of the nuclear and cytoplasmic fractions, respectively. (D) Quantification of YAP nuclear translocation in CMs after miRNA mimics transfection. Results are shown as a ratio of YAP to nuclear PARP1. Data are mean ± SEM (n = 5 independent experiments); ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (E) Efficacy of YAP1 downregulation using a specific siRNA. On the left side, representative western blotting. On the right side, quantification of 3 independent experiments. Data are mean ± SEM; ∗∗ p < 0.01; t test. (F) Induction of CM proliferation by miRNA mimics is blunted by YAP knockdown. CMs were transfected with pro-proliferative miRNAs alone or in combination with an anti-YAP siRNA or the same amount of a non-targeting siRNA. The graph shows the percentage of sarcomeric α-actinin-positive cells that have incorporated EdU in a 72 h period. Data are mean ± SEM (n = 4 independent experiments); ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (G) Representative images of CMs treated with the indicated miRNAs with or out without a siRNA against YAP. Scale bar, 100 μm.

Article Snippet: CMs were then stained overnight at 4 ° C with mouse monoclonal antibody against sarcomeric α-actinin (Abcam) diluted in blocking solution.

Techniques: Activation Assay, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Positive Control, Translocation Assay, Western Blot

Journal: Cell Reports

Article Title: Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation

doi: 10.1016/j.celrep.2019.05.005

Figure Lengend Snippet: Disruption of the Sarcomeric Architecture in Mitotic Cardiomyocytes Treated with miR-199a-3p and Cofilin2 siRNA (A and B) Representative pictures showing G2/M cells upon treatment with miR-199a-3p (A) or anti-Cofilin2 siRNA (B), showing nuclear positivity for phospho-histone H3 (pH3(S10), red) and diffused cytoplasmatic staining for α-actin (green). Nuclei are counterstained with Hoechst 33342 (blue). (C) Percentage of pH3(S10) + , α-actinin + cells (CMs) after treatment with the indicated miRNAs or Cofilin2 siRNA. Cel-miR-67 and a non-targeting (NT) siRNA served as negative controls for miRNA and siRNA experiments respectively. Data are from the analysis of over 400 CMs from four different experiments; shown are mean ± SEM; ∗∗ p < 0.01, ∗ p < 0.05; one-way ANOVA. (D) Representative pictures of G2/M CMs (defined as in C), showing nuclear positivity for phospho-histone H3 (pH3(S10), red) and disruption of the sarcomeric architecture, as concluded from diffused staining of α-actinin (red). Nuclei are counterstained with Hoechst 33342 (blue). (E) Mitotic CMs in subsequent phases of mitosis (from A to D). Staining as in (D).

Article Snippet: CMs were then stained overnight at 4 ° C with mouse monoclonal antibody against sarcomeric α-actinin (Abcam) diluted in blocking solution.

Techniques: Staining

Journal: Cell Reports

Article Title: Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation

doi: 10.1016/j.celrep.2019.05.005

Figure Lengend Snippet: Mechanism for YAP Activation by miR-199a-3p and Other miRNAs (A) Schematic representation of the Hippo pathway with the indication of the predicted target proteins according TargetScan software predictions. (B) Real-time RT-PCR quantification of TAOK1 and β-TrCP mRNAs in CMs treated with miR-199a-3p mimic. Data are mean ± SEM of 3 independent experiments; ∗ p < 0.05; t test. (C) Real-time RT-PCR quantification of STK38L mRNA levels in CMs treated with the indicated miRNA mimics. Data are mean ± SEM of 3 independent experiments; ∗ p < 0.05; one-way ANOVA. (D and E) Representative western blots (D) and quantification (E) showing downregulation of TAOK1, β-TrCP and STK38L proteins in cells treated with miR-199a-3p mimic. Data are mean ± SEM of 3 independent experiments; ∗ p < 0.05; t test. (F) Experimental flow chart of 3′ UTR luciferase assays. Fluc and Rluc: firefly and Renilla luciferase genes, respectively. (G and H) TAOK1 (G) and β-TrCP (H) are direct targets of miR-199a-3p. Results of 3′ UTR luciferase assays with miR-199a-3p. Renilla luciferase (Rluc) values were normalized over firefly (Fluc) values to standardize for transfection efficiency. Control refers to transfection of a Renilla luciferase gene with no 3′ UTR. Data are mean ± SEM (n = 3 independent experiments); ∗ p < 0.05; t test. (I) Results of 3′ UTR assays for STK38L in cells transfected with the indicated miRNA mimics. (J and K) Representative western blots (J) and quantification (K) showing downregulation of TAOK, β-TrCP, and STK38L in CMs transfected with the respective siRNAs. (L) Downregulation of TAOK1 and β-TrCP stimulates CM proliferation in a YAP-dependent manner. The graph shows the percentage of sarcomeric α-actinin-positive cells that have incorporated EdU in a 72 h period after treatment with specific siRNAs alone or in combination with an anti-YAP siRNA. Data are mean ± SEM (n = 4 independent experiments); ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (M) Downregulation of TAOK1 and β-TrCP stimulate transcriptional TEAD activity. CMs were transfected with siRNAs against TAOK1 or β-TrCP in combination with a TEAD-firefly luciferase reporter. The results were normalized to those of a constitutively expressed Renilla luciferase construct. Data are mean ± SEM over a non-targeting (NT) siRNA (n = 5 independent experiments); ∗ p < 0.05; one-way ANOVA.

Article Snippet: CMs were then stained overnight at 4 ° C with mouse monoclonal antibody against sarcomeric α-actinin (Abcam) diluted in blocking solution.

Techniques: Activation Assay, Software, Quantitative RT-PCR, Western Blot, Luciferase, Transfection, Activity Assay, Construct

Journal: Cell Reports

Article Title: Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation

doi: 10.1016/j.celrep.2019.05.005

Figure Lengend Snippet: Treatment of CMs with miRNAs Downregulates Cofilin2 and Induces Remodeling of the Actin Cytoskeleton (A) Immunofluorescence pictures showing remodeling of the actin cytoskeleton upon miRNA treatment. F-actin is visualized using fluorescent phalloidin (green), CMs with an anti-α -actinin antibody (red), nuclei with Hoechst (blue). F indicates fibroblasts (α-actinin-negative cells). (B) Percentage of CMs with a rounded shape (as in the representative images in A) after treatment of CMs with the indicated miRNAs. Data are from the analysis of over 400 CMs from four different experiments; shown are mean ± SEM; ∗∗ p < 0.01; one-way ANOVA. (C) Simplified view of proteins involved in actin cytoskeleton remodeling which are predicted to be targeted by the indicated pro-proliferative miRNAs. (D) Results of 3′ UTR assays to evaluate direct targeting of Cofilin2 (Cfl2) by the investigated miRNAs. Experiments were performed as in Figure 2 F. Data are mean ± SEM (n = 3 independent experiments); ∗ p < 0.05; ∗∗ p < 0.01; one-way ANOVA. (E) Results of real-time RT-PCR quantification of the Cofilin2 mRNA in CMs treated with the indicated miRNA mimics. An siRNA against Cofilin2 (siCfl2) was used as a positive control. Data are mean ± SEM (n = 3 independent experiments); ∗ p < 0.05; ∗∗ p < 0.01; one-way ANOVA. (F) Effect of miRNAs on Cofilin2 protein levels in CMs. Strip in the upper part: representative western blot showing downregulation of Cofilin2 in CMs transfected with the indicated miRNA mimics. Graph in the lower part: quantification of the levels of Cofilin2. Data are mean ± SEM (n = 3 independent experiments); ∗ p < 0.05; ∗∗ p < 0.01; one-way ANOVA. The dotted, red line shows the levels Cofilin2 in CMs treated with the control cel-miR-67 miRNA. (G) Same as in (A) in CMs treated with an anti-Cofilin2 siRNA or with a non-targeting (NT) siRNA control. (H) Percentage of CMs with a rounded shape (as in the representative images in G) after treatment of CMs with the Cofilin2 siRNA. Data are from the analysis of over 400 CMs from four different experiments; shown are mean ± SEM; ∗∗ p < 0.01; t test.

Article Snippet: CMs were then stained overnight at 4 ° C with mouse monoclonal antibody against sarcomeric α-actinin (Abcam) diluted in blocking solution.

Techniques: Immunofluorescence, Quantitative RT-PCR, Positive Control, Stripping Membranes, Western Blot, Transfection

Journal: Cell Reports

Article Title: Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation

doi: 10.1016/j.celrep.2019.05.005

Figure Lengend Snippet: Perturbation of the Actin Cytoskeleton Activates YAP Nuclear Translocation and Activity (A) Downregulation of Cofilin2 activates TEAD reporter activity. The graph reports the results of TEAD-firefly luciferase reporter analysis of CMs transfected with miR-199a-3p and Cofilin2 siRNA. Experiments were performed as in Figure 1 A. Transfection efficiency was standardized over a constitutively expressed Renilla luciferase reporter. Data are mean ± SEM (n = 4 independent experiments); ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (B) Downregulation of Cofilin2 activates CM replication in a YAP-dependent manner. The graph shows the percentage of α-actinin-positive cells that have incorporated EdU in a 72 h period after treatment with anti-Cofilin2 siRNA or miR-199a-3p mimic alone or in combination with an anti-YAP siRNA. Data are mean ± SEM (n = 4 independent experiments); ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (C) Representative pictures showing CMs incorporating EdU after treatment with an anti-Cofilin2 siRNA as in (B) in the absence or presence of an anti-YAP siRNA. (D) Real-time RT-PCR analysis of the levels of the two TEAD responsive genes CTFG and CyR61 in CMs transfected with anti-Cofilin2 siRNA. Data are mean ± SEM (n = 4 independent experiments); ∗∗ p < 0.01; t test. (E) Treatment of CMs with cytochalasin D blocks nuclear translocation of YAP. Representative blots showing the levels of nuclear and cytoplasmic YAP1 and phospho-YAP1 (P-YAP1) in CMs treated with cytochalasin D for the indicated time points. GAPDH and p84 were used for loading controls of cytoplasmic and nuclear fractions, respectively. (F) Quantification of YAP nuclear translocation in CMs treated with cytochalasin D. Data are mean ± SEM (n = 3 independent experiments); ∗∗ p < 0.01; one-way ANOVA. (G) Treatment with cytochalasin D blocks transcription of YAP-responsive genes. The graph shows the levels of the CTFG and CyR61 mRNAs, measured by real-time RT-PCR, in CMs treated with cytochalasin D for the indicated time points. Data are mean ± SEM (n = 3 independent experiments). All times showed statistical significance at p < 0.01; one-way ANOVA.

Article Snippet: CMs were then stained overnight at 4 ° C with mouse monoclonal antibody against sarcomeric α-actinin (Abcam) diluted in blocking solution.

Techniques: Translocation Assay, Activity Assay, Luciferase, Transfection, Quantitative RT-PCR

Journal: Cell Reports

Article Title: Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation

doi: 10.1016/j.celrep.2019.05.005

Figure Lengend Snippet:

Article Snippet: CMs were then stained overnight at 4 ° C with mouse monoclonal antibody against sarcomeric α-actinin (Abcam) diluted in blocking solution.

Techniques: Recombinant, Transfection, In Vivo, Luciferase, Imaging, Expressing, Sequencing, Negative Control, Plasmid Preparation, Mutagenesis, Software, Functional Assay

Journal: Cell Transplantation

Article Title: Construction of Pedicled Smooth Muscle Tissues by Combining the Capsule Tissue and Cell Sheet Engineering

doi: 10.1177/0963689718821682

Figure Lengend Snippet: Characterization of SMC sheets. (A) Phase-contrast micrographs of rabbit primary bladder SMCs. (B, C) Phase-contrast and macroscopic images of SMC sheets. (D) Cross-sectional views of SMC sheets colored by hematoxylin and eosin staining. (E–H) The SMC sheet was stained with α-SMA (red color) and anti-desmin (green color) antibodies, while the nuclei were stained with DAPI (blue color). (I–K) Viability assay of the SMCs after 5-day culture. Live cells (green color); dead cells (red color). (L) Viability assay of the SMCs killed by 70% methanol as control. (M–O) Viability assay of the harvested SMC sheet. Live cells (green color); dead cells (red color). (P) For the viability, there was no significant difference between the sheets before and after harvest. The data are expressed as the mean ± SD, and the error bars represent the SD. n.s. denotes not significant. (Q–T) Immunofluorescent staining of Caspase-3, Ki-67, collagen IV and laminin (red color), respectively; nuclei were counterstained with DAPI (blue color). Scale bar = 100 μm (A, B, I–O); Scale bar = 50 μm (D–H, Q–T).

Article Snippet: For immunohistochemistry, 4 µm-thick sections were deparaffinized and blocked with donkey serum for 30 min. To accurately measure the thickness of the SMC sheets transplanted in vivo , sections were incubated with mouse anti-alpha smooth muscle actin (α-SMA) monoclonal antibody (1:1000; Abcam, Cambridge, UK).

Techniques: Staining, Viability Assay

Journal: Cell Transplantation

Article Title: Construction of Pedicled Smooth Muscle Tissues by Combining the Capsule Tissue and Cell Sheet Engineering

doi: 10.1177/0963689718821682

Figure Lengend Snippet: Outcome of SMC sheets transplanted onto two vascular beds on day 2. The transplanted cell sheets were labeled with CM-DiI (red color; first column). The width of red-fluorescent dye increased with the number of cell sheets, while the fluorescence intensity in the expander capsule transplantation group was stronger than that in the subcutaneous transplantation group. Nuclei were counterstained with DAPI (blue color; second column). The first and second column images were merged (third column). Hematoxylin and eosin staining of transplanted SMC sheets onto two vascular beds on day 2 (fourth column) showed engineered smooth muscle tissues located on the surface of two vascular beds and erythrocytes were found in the vessels of cell sheet grafts. Immunostaining of α-SMA was conducted to measure the thickness of cell sheet grafts (fifth column). Bidirectional arrows indicate viable cell sheet grafts. Immunostaining of CD31 was conducted to evaluate vessel density of cell sheet grafts (sixth column). “1-layer C” denotes 1-layer SMC sheets transplanted onto the capsule tissue. “1-layer S” denotes 1-layer SMC sheets transplanted onto the subcutaneous tissue. “2-layer C” denotes 2-layer SMC sheets transplanted onto the capsule tissue. “2-layer S” denotes 2-layer SMC sheets transplanted onto the subcutaneous tissue. “3-layer C” denotes 3-layer SMC sheets transplanted onto the capsule tissue. “3-layer S” denotes 3-layer SMC sheets transplanted onto the subcutaneous tissue. Scale bar = 100 μm (first–fifth column); Scale bar = 50 μm (sixth column).

Article Snippet: For immunohistochemistry, 4 µm-thick sections were deparaffinized and blocked with donkey serum for 30 min. To accurately measure the thickness of the SMC sheets transplanted in vivo , sections were incubated with mouse anti-alpha smooth muscle actin (α-SMA) monoclonal antibody (1:1000; Abcam, Cambridge, UK).

Techniques: Labeling, Fluorescence, Transplantation Assay, Staining, Immunostaining

Journal: Cell Transplantation

Article Title: Construction of Pedicled Smooth Muscle Tissues by Combining the Capsule Tissue and Cell Sheet Engineering

doi: 10.1177/0963689718821682

Figure Lengend Snippet: Outcome of SMC sheets transplanted onto two vascular beds on day 7. The transplanted cell sheets were labeled with CM-DiI (red color; first column). The width of cell sheets traced with red fluorescence increased compared with those on day 2, indicating that the transplanted cells proliferated and formed thicker tissues. Nuclei were counterstained with DAPI (blue color; second column). The first and second column images were merged (third column). Hematoxylin and eosin staining of transplanted SMC sheets onto two vascular beds on day 7 showed engineered smooth muscle tissues on two vascular beds became thicker and contained plenty of small functional vessels (fourth column). Immunostaining of α-SMA was conducted to measure the thickness of cell sheet grafts (fifth column). Bidirectional arrows indicate viable cell sheet grafts. Immunostaining of CD31 was conducted to evaluate vessel density of cell sheet grafts (sixth column). The denotations are the same as those of . Scale bar = 100 μm (first–fifth column); Scale bar = 50 μm (sixth column).

Article Snippet: For immunohistochemistry, 4 µm-thick sections were deparaffinized and blocked with donkey serum for 30 min. To accurately measure the thickness of the SMC sheets transplanted in vivo , sections were incubated with mouse anti-alpha smooth muscle actin (α-SMA) monoclonal antibody (1:1000; Abcam, Cambridge, UK).

Techniques: Labeling, Fluorescence, Staining, Functional Assay, Immunostaining