Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 expression is associated with senescence. A Human fibroblasts were exposed to 20 Gy ionizing irradiation (IR). On day 1, 5 and 7, NLRP1 and senescence protein expression were analyzed by immunoblotting. B Representative images of Ki67 and NLRP1 immunofluorescence. Scale bar = 50 μm. C , D IL-1β, IL18, IL-6 and IL-8 were quantified by ELISA. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 differences between time points after irradiation and day 0. E , F Human fibroblasts were treated with Valboropro (Vbpro) to induce NLRP1 expression. After 24 h, NLRP1 and IL-6 protein expression were analyzed by immunoblotting and cytokines were analyzed by ELISA. Human fibroblasts were irradiated ( G ) or stimulated with palbociclib (Palbo) ( H ) to induce two different senescence models. Then, cells were transfected with a non-targeting control siRNA (Control) or with siRNAs against NLRP1 (siNLRP1). Expression of NLRP1 and senescence-associated proteins p16, p21, p53 and IL-6 were assessed by immunoblotting and IL6, IL-8 or IL-18 were quantified by ELISA. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 irradiated vs. control. aaa P < 0.001, IR + siRNA vs. IR cells (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: Expressing, Irradiation, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 contributes to cellular senescence in vivo. A Nlrp1 protein expression in liver from WT mice at 1 month after IR. B Serum levels of IL-18 after IR. C Effect of IR on the bodyweight of WT and Nlrp1 knockout (KO) mice. D Protein expression in liver from IR and non-IR WT and Nlrp1 KO mice of senescent markers (IL-6, p16 and p21). Densitometry in Supplementary Fig. 6. E Serum levels of IL-6 in serum from IR and non-IR WT and Nlrp1 KO mice. F Heat map depicting expression of 44 mouse cytokines in serum at 5 weeks after IR of WT and Nlrp1 KO mice. n = 6 mice per group. G IL-6 releases from healthy fibroblasts was assessed after 24 and 48 h of incubation with media containing serum from IR and non-IR WT and Nlrp1 KO mice. H Representative liver section stainings of hematoxylin and eosin (H&E). I Representative liver section immunostainings of NLRP1 and p16. All data are presented as means ± SEM, n = 6–8 mice per group; * P < 0.05, ** P < 0.005, *** P < 0.001 irradiated vs. control. aa P < 0.005 IR WT vs. IR KO mice; bb P < 0.005 IR KO vs. IR KO mice (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: In Vivo, Expressing, Knock-Out, Incubation, Irradiation, Control

Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 senses DNA damage dependent of cGAS activation. A Protein expression levels of NLRP1 and cGAS after 24 h exposition to gDNA from non-irradiated and irradiated cells. B IL-6 and IL-18 release to the medium after the same experimental condition. Levels were determined by ELISA assay. C , D Protein expression levels of NLRP1 and cGAS and IL-6 and IL-18 release after 24 h exposition to a non-irradiated or irradiated synthetic double-stranded DNA sequence, poly(dA-dT), Cytokine levels were determined by ELISA assay. Cytokine levels were determined by ELISA assay. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 gDNA vs. control cells. a P < 0.05, aa P < 0.005, aaa P < 0.001, IR gDNA vs. control cells. E Human fibroblasts were irradiated to induces senescence. Then, cells were transfected with a non-targeting control siRNA (Control) or with siRNAs against cGAS (sicGAS). Expression of NLRP1, NLRP3, IL-1β, cGAS and senescent protein p16, p21 was assessed by immunoblotting. F IL-18 release of non-irradiated, irradiated and irradiated and transfected with siRNAs against cGAS. Cytokine levels were determined by ELISA assay. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, IR vs. control cells. aa P < 0.005, IR vs. sicGAS (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: Activation Assay, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Transfection, Western Blot

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The total p21 protein expression (pg/mL) in normal cell line-HUVEC and in tumor cell line-PE/CA-PJ49 cells treated 24 h with CisPt and/or RSV, CRM. The experiments were performed in triplicates. Results are expressed as mean values of three determinations ± standard deviation (SD). (** p < 0.005, *** p < 0.0005; **** p < 0.00005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Standard Deviation

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effects of CisPt, RSV, CRM treatment applied alone or in combination for 24 h on the level of p21 protein expression (n-fold p21protein expression) in normal cell line-Huvec and in tumor cell line-PE/CA-PJ49. The n-fold p21expression was calculated using the formula: n-fold p21expression = p21 pg/mL treatment/p21 pg/mL control.

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Control

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effect of treatment with CisPt, RSV, CRM applied independently or in combination on P21 gene expression in tumor cells PE/CA-PJ49 compared to normal cells HUVEC. Each sample was performed in duplicate. The samples were analyzed using the formula 2-ΔΔCt = gene expression. (* p < 0.05, ** p < 0.005; *** p < 0.0005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Pathway Analysis Identifies Amphiregulin as a Key Factor for Cisplatin Resistance of Human Breast Cancer Cells

doi: 10.1074/jbc.m706287200

Figure Lengend Snippet: FIGURE 3. Analysis of AKT kinase, downstream signaling reveals inactiva- tion of the p53 pathway in MCF-7 CisR cells. A, quantification of AKT kinase activity. To measure AKT kinase activity, a solid phase ELISA, which utilizes a specific synthetic peptide as a substrate and a polyclonal antibody that rec- ognizes the phosphorylated form of the substrate, was used. (n 3, ***, p 0.001). B, detection of p53 protein by immunoblotting (IB) using a polyclonal affinity-purified goat Ab specific for p53. MCF-7 cells (lane 1) and MCF-7 CisR cells (lane 2). Lane M, molecular weight marker. p53 is indicated by an arrow. C, quantification of p53 by a sandwich ELISA that measures human total p53 in cell lysates (n 3, ***, p 0.001). D, p21 expression indicates p53 pathway activity. Detection of p21 in whole cell lysates of MCF-7 (lane 1) and MCF-7 CisR cells (lane 2) by immunoblotting using a polyclonal affinity-purified goat Ab specific for p21. Lane M, molecular weight marker. p21 indicated by an arrow. E, to quantify the levels of p21 protein a sandwich ELISA that measures p21 in cell lysates was used. (n 3, ***, p 0.001). F, to quantify BCL-2 expression an ELISA that detects human BCL-2 in cell lysates was used (n 3, ***, p 0.001).

Article Snippet: For immunoblotting we used a polyclonal affinity-purified goat Ab specific for p53 at a concentration of 1 g/ml (AF1355, R & D Systems, Wiesbaden, Germany) and a polyclonal affinity-purified goat Ab specific for p21 at a concentration of 1 g/ml (AF1047, R & D Systems, Wiesbaden, Germany).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Affinity Purification, Molecular Weight, Marker, Sandwich ELISA, Expressing

Journal: Oncotarget

Article Title: Dietary NiCl₂ causes G₂/M cell cycle arrest in the broiler's kidney.

doi: 10.18632/oncotarget.5934

Figure Lengend Snippet: Figure 3: Changes of p-ATM, p53, p21, p-Chk1, p-Chk2 and p-cdc25C protein expression levels in the kidney at 42 days of age. (Immunohistochemistry, ×400).

Article Snippet: Table 1: Antibodies used in immunohitochemistry Name Company Cat# Dilution p-ATM Bioss, China bs-2272R 1:100 p-Chk1 Bioss, China bs-5251R 1:100 p-Chk2 Bioss, China bs-3721R 1:100 p53 Boster, China BM0101 1:100 p21 Boster, China BA0272 1:100 p-cdc25C Bioss, China bs-3482R 1:100 p-cdc2 Boster, China BM0027 1:100 cyclinB1 Bioss, China bs-0572R 1:100 PCNA Boster, China BM0104 1:100 Oncotarget35969www.impactjournals.com/oncotarget or P < 0.01) in the 900 mg/kg groups at 14 days of age and in the three NiCl2-treated groups from 28 to 42 days of age in comparison with those in the control group.

Techniques: Expressing, Immunohistochemistry

Journal: Oncotarget

Article Title: Dietary NiCl₂ causes G₂/M cell cycle arrest in the broiler's kidney.

doi: 10.18632/oncotarget.5934

Figure Lengend Snippet: Figure 5: Changes of the mean density of p-ATM, p53, p21, p-Chk1, p-Chk2 and p-cdc25C protein expression in the kidney. Data are presented with the mean ± standard deviation (n=5×5) *P<0.05, compared with the control group **P<0.01, compared with the control group.

Article Snippet: Table 1: Antibodies used in immunohitochemistry Name Company Cat# Dilution p-ATM Bioss, China bs-2272R 1:100 p-Chk1 Bioss, China bs-5251R 1:100 p-Chk2 Bioss, China bs-3721R 1:100 p53 Boster, China BM0101 1:100 p21 Boster, China BA0272 1:100 p-cdc25C Bioss, China bs-3482R 1:100 p-cdc2 Boster, China BM0027 1:100 cyclinB1 Bioss, China bs-0572R 1:100 PCNA Boster, China BM0104 1:100 Oncotarget35969www.impactjournals.com/oncotarget or P < 0.01) in the 900 mg/kg groups at 14 days of age and in the three NiCl2-treated groups from 28 to 42 days of age in comparison with those in the control group.

Techniques: Expressing, Standard Deviation, Control

Journal: Oncotarget

Article Title: Dietary NiCl₂ causes G₂/M cell cycle arrest in the broiler's kidney.

doi: 10.18632/oncotarget.5934

Figure Lengend Snippet: Figure 7: Changes of ATM, p53, p21, Chk1, Chk2 and cdc25 mRNA expression levels in the kidney. Data are presented with the mean ± standard deviation (n=5) *P<0.05, compared with the control group **P<0.01, compared with the control group.

Article Snippet: Table 1: Antibodies used in immunohitochemistry Name Company Cat# Dilution p-ATM Bioss, China bs-2272R 1:100 p-Chk1 Bioss, China bs-5251R 1:100 p-Chk2 Bioss, China bs-3721R 1:100 p53 Boster, China BM0101 1:100 p21 Boster, China BA0272 1:100 p-cdc25C Bioss, China bs-3482R 1:100 p-cdc2 Boster, China BM0027 1:100 cyclinB1 Bioss, China bs-0572R 1:100 PCNA Boster, China BM0104 1:100 Oncotarget35969www.impactjournals.com/oncotarget or P < 0.01) in the 900 mg/kg groups at 14 days of age and in the three NiCl2-treated groups from 28 to 42 days of age in comparison with those in the control group.

Techniques: Expressing, Standard Deviation, Control

Journal: EMBO Reports

Article Title: Praja2 controls P-body assembly and translation in glioblastoma by non-proteolytic ubiquitylation of DDX6

doi: 10.1038/s44319-025-00425-5

Figure Lengend Snippet: ( A ) β-galactosidase staining at pH 6 on U87MG WT and praja2KO cells. Scale bar: 40 μm. ( B ) Quantitative analysis of three biological independent experiments as reported in ( A ), mean ± SD is indicated. t test **** P < 0.0001. ( C ) Lysates from U87MG (WT) and praja2KO cells were immunoblotted with anti-p21 and anti-α-tubulin. ( D ) Quantitative analysis of three biological independent experiments as reported in ( C ), mean ± SD is indicated. t test * P = 0.0459 ( E ). 5 × 10 6 U87MG (WT) and U87MG praja2KO cells were implanted into CD1 nude mice. Six weeks later, the mice were killed, and the tumors were excised and weighed. Tumor sections were fixed and doubly stained with hematoxylin/eosin or subjected to immunohistochemistry with anti-praja2 antibody. Scale bar (referred to ×40 magnification): 50 μm. ( F ) Quantitative analysis of the tumors size (at 6 weeks) shown in ( E ). Three WT and four praja2KO mice were analyzed. Data are expressed as mean value ± SEM. t test * P = 0.0441 ( G ). Representative imaging of immunostaining for ki67, p53, and p21 in mice subcutaneously injected with U87MG (WT) (upper panels) or U87MG praja2KO (lower panels) cells. Scale bar (referred to ×40 magnification): 50 μm. .

Article Snippet: Slides were fixed with 70% ethanol and immunostained using a Benchmark Ultra XT (Roche) for the proliferation marker ki67 (prediluted from Roche), praja2 (1:200, Novus Biological), p21 (1:100, Proteintech), and p53 (1:100, Proteintech).

Techniques: Staining, Immunohistochemistry, Imaging, Immunostaining, Injection

Journal: bioRxiv

Article Title: Programming the elongation of mammalian cell aggregates with synthetic gene circuits

doi: 10.1101/2024.12.11.627621

Figure Lengend Snippet: a-b. Spheroid growth assay: 1000 transceiver (B-type) cells are dispensed in a U-bottom well, and their growth is captured by imaging on days 2 and 7. On the right, representative microscope images of a spheroid of B-type cells with inducible p53, either preactivated (B’-type) by culture on GFP, or inhibited (B-type) by dox at the indicated time points. mCherry signal is rendered as red. Scale bar 500um. c. Growth index graph for the indicated conditions. Per each effector, a condition without synNotch activation (dox, gray dots), and one with synNotch activation (via plate-bound GFP, red dots) are reported. For details on methods of growth index quantification see Methods. Growth index of 0 means no growth at all; growth index of 1 means duplication of volume every day. d. Schematic of spheroid fluidity assay. For more details, see Fig. S3. Briefly, transceiver (B-type) cells with diverse inducible effectors were preactivated or preinhibited before 1000-cell spheroid seeding as explained above. The resulting spheroids were brought together in homotypic pairs and imaged every hour for 24 hours. e. Microscope images of spheroid fluidity assay with no effectors (top row), inducible p21 (middle row), and inducible p21+CA-RhoA (bottom row). The first three columns (0h, 12h, 24h) are brightfield images taken from activated (B’-type) cells. The fourth column (24h) represents brightfield images taken from inactivated B-type cells of the same genetic effectors. Scale bar 500um. f. Fluidity index graph for the indicated conditions. Per each effector, a condition without synNotch activation (dox, gray dots), and one with synNotch activation (via plate-bound GFP, red dots) are reported. For details on methods of fluidity index quantification see Methods and Fig. S3d. High fluidity index means the spheroids are fluid and tend to fuse fast; low fluidity index means the spheroids are more viscous and tend to fuse more slowly. For c. and f. Brown-Forsythe and Welch ANOVA test results: * is p<0.1; ** is p<0.01 all other differences between measurements in ON vs. OFF transceivers were non-significant.

Article Snippet: Mouse WNT5A was amplified from plasmid pRK5-mWnt5a [bought from Addgene, catalog number #42279]; human CA-RhoA (a constitutively active mutant of RHOA, RHOA Q63L ) was amplified from plasmid pRK5-myc-RhoA-Q63L [bought from Addgene, catalog number #12964] CA-MLCK (constitutively active mutant of human MLCK, MLCK ED785-786KK ) was amplified from plasmid pSLIK CA MLCK [bought from Addgene, catalog number #84647]; KD-MLCK (kinase-dead mutant of human MLCK, MLCK E1626K ) was amplified from plasmid Hela kinase-dead MLCK-GFP [bought from Addgene, catalog number #46317]; human CDKN1A (p21) was amplified from plasmid Flag p21 WT [bought from Addgene, catalog number #16240]; human CAV1 (Caveolin-1) was amplified from plasmid mCherry-Caveolin-C10 [bought from Addgene, catalog number #55008]; mouse SOX9 was amplified from plasmid tetO.Sox9.Puro [bought from Addgene, catalog number #117269]; human RAB5A DN (dominant negative form of RAB5A, RAB5A S34N ) was amplified from plasmid mCherry-Rab5DN(S34N) [bought from Addgene, catalog number #35139]; human CDKN2A (P16INK4a, p16) was amplified from plasmid pQCXIH-CDKN2A [bought from Addgene, catalog number #37104]; human CDKN1B (p27) was amplified from plasmid pcDNA3-myc3-p27 [bought from Addgene, catalog number #19937]; human TP53 (tumor protein 53, p53) was amplified from plasmid pLenti6 / V5-p53_wt p53 [bought from Addgene, catalog number #22945]; mCherry was amplified from plasmid pHR TRE3GS->mCherry_pGK->BFP [bought from Addgene, catalog number #162231]; human LOXL2 was amplified from plasmid pQXCINeo-LOXL2-BC [bought from Addgene, catalog number #134763]; human TG2 was amplified from plasmid W118-1_hTGM2-Flag [bought from Addgene, catalog number #180407]; human Cdh1 (ECAD) was amplified from plasmid E-cadherin-GFP [bought from Addgene, catalog number #28009]; mouse Cdh2 (NCAD) was amplified from a plasmid given by the Lim lab ; human Cdh3 (PCAD) was amplified from plasmid pcDNA3 P-cad [bought from Addgene, catalog number #47502]; H2B-miRFP703 was amplified from plasmid pH2B-miRFP703 [bought from Addgene, catalog number #80001].

Techniques: Growth Assay, Imaging, Microscopy, Activation Assay

Journal: bioRxiv

Article Title: Programming the elongation of mammalian cell aggregates with synthetic gene circuits

doi: 10.1101/2024.12.11.627621

Figure Lengend Snippet: a. Schematic of elongation assay: spheroids of 200 sender (A-type) cells (blue) and 4,000 transceiver (B-type) cells (gray) are seeded separately. All B-type cells in this figure have constitutive N-cadherin, anti-GFP-synNotch that activate GFP-lig for signal propagation, and one or more inducible effectors as indicated below. After 2 days, the two resulting spheroids are assembled in an individual well, and the assembloid is imaged daily henceforth. b. Elongation assay with B-type cells that activate only mCherry reporter and no effectors. Left, schematic of B cell circuit. Right, micrographs of Day0 and Day6 of two conditions: top row, condition where synNotch signaling in B-type cells is allowed to happen; bottom row, condition where synNotch signaling in B-type cells is impaired with the small molecule Dox. In the micrographs the far-red signal from the constitutive far-red marker in A-type cells is rendered in blue, and the GFP signal is rendered in green. Scale bar is 500um (shown at the bottom, valid for all panels). c. Graph of aspect ratio (AR) over time for n=1 experiment, n=3-6 technical replicates. The continuous black line is for samples with signaling; the dashed black line is for samples with DOX, i.e. without signaling. AR =1 for a circle, AR>1 proportionally to the elongation of the ellipsoid. For more details on AR calculation see methods. d. Elongation assay with B cells that activate p21 as proliferation effector. Left, schematic of B cell circuit. Right, micrographs of Day0 and Day6 of two conditions: top row, condition where synNotch signaling in B cells is allowed to happen; bottom row, condition where synNotch signaling in B cells is impaired with the small molecule Dox. In the micrographs the far-red signal from the constitutive far-red marker in A cells is rendered in blue, the mCherry signal in B’ cells is rendered in red, and the GFP signal is not shown. Scale bar is 500um (shown at the bottom, valid for all panels). e. Graph of aspect ratio (AR) over time for n=2 +signaling experiments and n=1 no signaling experiment, each averaged from 3-6 technical replicates. The continuous black line is for samples with signaling; the dashed black line is for samples with DOX, i.e. without signaling. AR =1 for a circle, AR>1 proportionally to the elongation of the ellipsoid. For more details on AR calculation see methods. f. Elongation assay with B cells that activate p21 and CA-MLCK upon synNotch signaling activation. Left, schematic of B cell circuit. Right, micrographs of Day0 and Day6 of two conditions: top row, condition where synNotch signaling in B cells is allowed to happen; bottom row, condition where synNotch signaling in B cells is impaired with the small molecule Dox. In the micrographs the far-red signal from the constitutive far-red marker in A cells is rendered in blue, the mCherry signal in B’ cells is rendered in red, and the GFP signal is not shown. Scale bar is 500um (shown at the bottom, valid for all panels). g. Graph of aspect ratio (AR) over time for n=2 +signaling experiments and n=1 no signaling experiment, each averaged from 3-6 technical replicates. AR =1 for a circle, AR>1 proportionally to the elongation of the ellipsoid. The continuous green line is for samples with signaling; the dashed green line is for samples with DOX, i.e. without signaling. For more details on AR calculation see methods. h. Elongation assay with B cells that activate p21 and CA-RhoA upon synNotch signaling activation. Left, schematic of B cell circuit. Right, micrographs of Day0 and Day6 of two conditions: top row, condition where synNotch signaling in B cells is allowed to happen; bottom row, condition where synNotch signaling in B cells is impaired with the small molecule Dox. In the micrographs the far-red signal from the constitutive far-red marker in A cells is rendered in blue, the mCherry signal in B’ cells is rendered in red, and the GFP signal is not shown. Scale bar is 500um. i. Graph of aspect ratio (AR) over time for n=2 +signaling experiments and n=1 no signaling experiment, each averaged from 3-6 technical replicates. AR =1 for a circle, AR>1 proportionally to the elongation of the ellipsoid. The continuous blue line is for samples with signaling; the dashed blue line is for samples with DOX, i.e. without signaling. For more details on AR calculation see methods. For more details on AR calculation see methods. Day 1 to 10 daily imaging time series and AR quantifications can be found in Fig. S8.

Article Snippet: Mouse WNT5A was amplified from plasmid pRK5-mWnt5a [bought from Addgene, catalog number #42279]; human CA-RhoA (a constitutively active mutant of RHOA, RHOA Q63L ) was amplified from plasmid pRK5-myc-RhoA-Q63L [bought from Addgene, catalog number #12964] CA-MLCK (constitutively active mutant of human MLCK, MLCK ED785-786KK ) was amplified from plasmid pSLIK CA MLCK [bought from Addgene, catalog number #84647]; KD-MLCK (kinase-dead mutant of human MLCK, MLCK E1626K ) was amplified from plasmid Hela kinase-dead MLCK-GFP [bought from Addgene, catalog number #46317]; human CDKN1A (p21) was amplified from plasmid Flag p21 WT [bought from Addgene, catalog number #16240]; human CAV1 (Caveolin-1) was amplified from plasmid mCherry-Caveolin-C10 [bought from Addgene, catalog number #55008]; mouse SOX9 was amplified from plasmid tetO.Sox9.Puro [bought from Addgene, catalog number #117269]; human RAB5A DN (dominant negative form of RAB5A, RAB5A S34N ) was amplified from plasmid mCherry-Rab5DN(S34N) [bought from Addgene, catalog number #35139]; human CDKN2A (P16INK4a, p16) was amplified from plasmid pQCXIH-CDKN2A [bought from Addgene, catalog number #37104]; human CDKN1B (p27) was amplified from plasmid pcDNA3-myc3-p27 [bought from Addgene, catalog number #19937]; human TP53 (tumor protein 53, p53) was amplified from plasmid pLenti6 / V5-p53_wt p53 [bought from Addgene, catalog number #22945]; mCherry was amplified from plasmid pHR TRE3GS->mCherry_pGK->BFP [bought from Addgene, catalog number #162231]; human LOXL2 was amplified from plasmid pQXCINeo-LOXL2-BC [bought from Addgene, catalog number #134763]; human TG2 was amplified from plasmid W118-1_hTGM2-Flag [bought from Addgene, catalog number #180407]; human Cdh1 (ECAD) was amplified from plasmid E-cadherin-GFP [bought from Addgene, catalog number #28009]; mouse Cdh2 (NCAD) was amplified from a plasmid given by the Lim lab ; human Cdh3 (PCAD) was amplified from plasmid pcDNA3 P-cad [bought from Addgene, catalog number #47502]; H2B-miRFP703 was amplified from plasmid pH2B-miRFP703 [bought from Addgene, catalog number #80001].

Techniques: Marker, Activation Assay, Imaging

Journal: bioRxiv

Article Title: Programming the elongation of mammalian cell aggregates with synthetic gene circuits

doi: 10.1101/2024.12.11.627621

Figure Lengend Snippet: a. Schematic of elongation assay: spheroids of 200 sender (A-type) cells (blue) and 4,000 transceiver (B-type) cells (gray) are seeded separately. B-type cells in this figure do not have constitutive N-cadherin. After 2 days, the two resulting spheroids are assembled in an individual well, and the assembloid is imaged daily henceforth. b, d, and f. For each row, on the left is a schematic of the gene circuits engineered in the transceiver cells. Sender and transceiver spheroids seeded as explained above were either cultured in doxycycline (dox, “no signaling” condition) or in control medium (“+ signaling” condition). Day 1 and 6 micrographs are shown for each condition, with miRFP703 represented in blue, GFP represented in green in b, and mCherry represented in red in d and f. The rightmost images are brightfield micrographs highlighting shape changes brought by activation of the synthetic gene circuit at day 6. b. Transceivers only induce GFPlig when activated. d. GFPlig, N-cad and mCherry are induced upon transceiver activation. f. GFPlig, N-cad, p21 and mCherry are induced upon transceiver activation. c, e, and g. Quantification of the aspect ratio (AR) over time of the conditions immediately on the left of each graph. See Methods and Figure S7 for more details on how the AR is measured. Briefly, the minimum AR that can be measured is 1 which corresponds to a perfect circle, any AR >1 corresponds to an elongated shape. Dashed lines represent AR measurements in the “no signaling” condition, and continuous lines measurements in the “+signaling” condition. The shaded regions represent the standard deviation from n=3-6 technical replicate in the n=1 experiment. Day 1 to 10 daily imaging time series and AR quantifications can be found in Fig. S10.

Article Snippet: Mouse WNT5A was amplified from plasmid pRK5-mWnt5a [bought from Addgene, catalog number #42279]; human CA-RhoA (a constitutively active mutant of RHOA, RHOA Q63L ) was amplified from plasmid pRK5-myc-RhoA-Q63L [bought from Addgene, catalog number #12964] CA-MLCK (constitutively active mutant of human MLCK, MLCK ED785-786KK ) was amplified from plasmid pSLIK CA MLCK [bought from Addgene, catalog number #84647]; KD-MLCK (kinase-dead mutant of human MLCK, MLCK E1626K ) was amplified from plasmid Hela kinase-dead MLCK-GFP [bought from Addgene, catalog number #46317]; human CDKN1A (p21) was amplified from plasmid Flag p21 WT [bought from Addgene, catalog number #16240]; human CAV1 (Caveolin-1) was amplified from plasmid mCherry-Caveolin-C10 [bought from Addgene, catalog number #55008]; mouse SOX9 was amplified from plasmid tetO.Sox9.Puro [bought from Addgene, catalog number #117269]; human RAB5A DN (dominant negative form of RAB5A, RAB5A S34N ) was amplified from plasmid mCherry-Rab5DN(S34N) [bought from Addgene, catalog number #35139]; human CDKN2A (P16INK4a, p16) was amplified from plasmid pQCXIH-CDKN2A [bought from Addgene, catalog number #37104]; human CDKN1B (p27) was amplified from plasmid pcDNA3-myc3-p27 [bought from Addgene, catalog number #19937]; human TP53 (tumor protein 53, p53) was amplified from plasmid pLenti6 / V5-p53_wt p53 [bought from Addgene, catalog number #22945]; mCherry was amplified from plasmid pHR TRE3GS->mCherry_pGK->BFP [bought from Addgene, catalog number #162231]; human LOXL2 was amplified from plasmid pQXCINeo-LOXL2-BC [bought from Addgene, catalog number #134763]; human TG2 was amplified from plasmid W118-1_hTGM2-Flag [bought from Addgene, catalog number #180407]; human Cdh1 (ECAD) was amplified from plasmid E-cadherin-GFP [bought from Addgene, catalog number #28009]; mouse Cdh2 (NCAD) was amplified from a plasmid given by the Lim lab ; human Cdh3 (PCAD) was amplified from plasmid pcDNA3 P-cad [bought from Addgene, catalog number #47502]; H2B-miRFP703 was amplified from plasmid pH2B-miRFP703 [bought from Addgene, catalog number #80001].

Techniques: Cell Culture, Control, Activation Assay, Standard Deviation, Imaging

Journal: bioRxiv

Article Title: Programming the elongation of mammalian cell aggregates with synthetic gene circuits

doi: 10.1101/2024.12.11.627621

Figure Lengend Snippet: a. 3D Morphospace graph. Throughout the graph, A-type cells are in blue, B-type cells are in gray, and B’-type cells are in red. The axes are as follows: x-axis, the ratio of growth index in B vs B’-type cells (which is proportional to B’-type cells growth arrest); y-axis, 1/tissue fluidity index of B’-type cells, which is a measure proportional to B’-type cell spheroid viscosity; and z-axis, a qualitative measure of the isolation of the B-type cells cluster from the B’-type cells cluster (correlating with low heterotypic/high homotypic adhesion of B and B’-type cells). The light gray box is bounded by the highest value which can realistically be achieved from each axis according to our in vitro data. At the 8 vertices of this white cube are placed the corresponding endpoint (7 days) elongation assay snapshots from in silico implementations. Corresponding circuits were numbered as in this figure, from 000 to 111, in Table 1.2. The green bounding lines define the volume in the morphospace that contains implementations that have been realized in vitro . On the bounding lines are shown selected examples of in vitro realizations, from day 6 micrographs where the resulting spheroid structures are isolated from the background via image processing. They go from a-f. For all of them, A-type cells have GFP ligand and P-cadherin expression, and spheroids were seeded from 200 cells before fusion. B-type cells were engineered from the “fast” clone chassis, and expressed the following constitutive of inducible effectors: a: constitutive N-cad, no inducible effectors (and thus no induced mCherry), also shown in ; b: inducible p21, also shown in Fig.S10a; c: inducible N-cad+p21, also shown in ; d: constitutive N-cad and inducible p21, also shown in ; e: constitutive N-cad, inducible p21+CA-MLCK, also shown in ; f: constitutive N-cad, inducible p21+CA-RhoA, also shown in . For each one of these are also shown the corresponding in silico implementation (a*-f*, circled by a dashed line), which in the graph are connected to the corresponding in vitro implementation with a dashed line. For the complete in silico genomes for a*-f* See Table 1.3. See text for further description. In silico – day 7 for the vertices; for the asterisks is variable, see Fig. S12a, last column.

Article Snippet: Mouse WNT5A was amplified from plasmid pRK5-mWnt5a [bought from Addgene, catalog number #42279]; human CA-RhoA (a constitutively active mutant of RHOA, RHOA Q63L ) was amplified from plasmid pRK5-myc-RhoA-Q63L [bought from Addgene, catalog number #12964] CA-MLCK (constitutively active mutant of human MLCK, MLCK ED785-786KK ) was amplified from plasmid pSLIK CA MLCK [bought from Addgene, catalog number #84647]; KD-MLCK (kinase-dead mutant of human MLCK, MLCK E1626K ) was amplified from plasmid Hela kinase-dead MLCK-GFP [bought from Addgene, catalog number #46317]; human CDKN1A (p21) was amplified from plasmid Flag p21 WT [bought from Addgene, catalog number #16240]; human CAV1 (Caveolin-1) was amplified from plasmid mCherry-Caveolin-C10 [bought from Addgene, catalog number #55008]; mouse SOX9 was amplified from plasmid tetO.Sox9.Puro [bought from Addgene, catalog number #117269]; human RAB5A DN (dominant negative form of RAB5A, RAB5A S34N ) was amplified from plasmid mCherry-Rab5DN(S34N) [bought from Addgene, catalog number #35139]; human CDKN2A (P16INK4a, p16) was amplified from plasmid pQCXIH-CDKN2A [bought from Addgene, catalog number #37104]; human CDKN1B (p27) was amplified from plasmid pcDNA3-myc3-p27 [bought from Addgene, catalog number #19937]; human TP53 (tumor protein 53, p53) was amplified from plasmid pLenti6 / V5-p53_wt p53 [bought from Addgene, catalog number #22945]; mCherry was amplified from plasmid pHR TRE3GS->mCherry_pGK->BFP [bought from Addgene, catalog number #162231]; human LOXL2 was amplified from plasmid pQXCINeo-LOXL2-BC [bought from Addgene, catalog number #134763]; human TG2 was amplified from plasmid W118-1_hTGM2-Flag [bought from Addgene, catalog number #180407]; human Cdh1 (ECAD) was amplified from plasmid E-cadherin-GFP [bought from Addgene, catalog number #28009]; mouse Cdh2 (NCAD) was amplified from a plasmid given by the Lim lab ; human Cdh3 (PCAD) was amplified from plasmid pcDNA3 P-cad [bought from Addgene, catalog number #47502]; H2B-miRFP703 was amplified from plasmid pH2B-miRFP703 [bought from Addgene, catalog number #80001].

Techniques: Viscosity, Isolation, In Vitro, In Silico, Expressing

Journal: Molecular Medicine Reports

Article Title: Prognostic and predictive roles of microRNA-411 and its target STK17A in evaluating radiotherapy efficacy and their effects on cell migration and invasion via the p53 signaling pathway in cervical cancer

doi: 10.3892/mmr.2019.10826

Figure Lengend Snippet: Reverse transcription-quantitative polymerase chain reaction primer sequences.

Article Snippet: The membranes were incubated with phosphorylated (p)-STK17A antibody (cat. no. 14433-1-AP; Proteintech, Wuhan, China; 1:1,000), p-p21 WAF1 antibody (cat. no. AP01654PU-N; Origene; 1:10,000), p-p53 antibody (cat. no. MABE518; Merck KGaA; 1:1,000), TAp63 antibody (cat. no. TA311397; Origene; 1:1,000), and GAPDH antibody (cat. no. 10494-1-AP; Proteintech; 1:2,000) at 4°C overnight.

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Molecular Medicine Reports

Article Title: Prognostic and predictive roles of microRNA-411 and its target STK17A in evaluating radiotherapy efficacy and their effects on cell migration and invasion via the p53 signaling pathway in cervical cancer

doi: 10.3892/mmr.2019.10826

Figure Lengend Snippet: miR-411 activates the p53 signaling pathway and negatively regulates STK17A in cervical cancer cells. (A) Determination by reverse transcription-quantitative polymerase chain reaction analysis demonstrated that ectopic expression of miR-411 and siRNA-mediated knockdown of STK17A decreased the mRNA expression of STK17A, but increased the mRNA expression of p53, p21 WAF1 and TAp63; miR-411 inhibitor increased the mRNA expression of STK17A, but decreased the mRNA expression of p53, p21 WAF1 and TAp63. (B) Determination by western blot analysis and (C) quantification demonstrated that ectopic expression of miR-411 and siRNA-mediated knockdown of STK17A decreased the protein expression of STK17A, but increased the protein expression of p53, p21 WAF1 and TAp63; miR-411 inhibitor increased the protein expression of STK17A, but decreased the protein expression of p53, p21 WAF1 and TAp63. *P<0.05, vs. NC group; # P<0.05, vs. miR-411 inhibitor + siRNA-STK17A group. The experiment was repeated three times and data were compared by one-way analysis of variance and analyzed by Tukey's post hoc test. NC, negative control; miR-411, microRNA-411; STK17A, serine/threonine kinase 17a; siRNA, small interfering RNA.

Article Snippet: The membranes were incubated with phosphorylated (p)-STK17A antibody (cat. no. 14433-1-AP; Proteintech, Wuhan, China; 1:1,000), p-p21 WAF1 antibody (cat. no. AP01654PU-N; Origene; 1:10,000), p-p53 antibody (cat. no. MABE518; Merck KGaA; 1:1,000), TAp63 antibody (cat. no. TA311397; Origene; 1:1,000), and GAPDH antibody (cat. no. 10494-1-AP; Proteintech; 1:2,000) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Negative Control, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: The embryonic transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies

doi: 10.1038/cddis.2013.208

Figure Lengend Snippet: Brachyury modulates expression of cell cycle regulatory proteins. Western blot analysis of Brachyury and the cell cycle regulatory proteins Rb, cyclin D1 and p21 was performed using ( a ) the A549 tumor cell pair at indicated times after release from cell cycle arrest by serum starvation or ( b ) asynchronous cultures of H460 control versus Brachyury shRNA-1 and -2 cells. ( c ) Two tumor cell lines derived from single-cell cloning of H460 cells were analyzed for expression of Brachyury in relation to Rb and p21. ( d ) H460 cells transfected with p21 expression vector or pCMV control were analyzed for p21 and Rb expression by western blot and ( e ) growth kinetics over a 5-day period. ( f ) Indicated cells were treated with cytotoxic therapies and assayed for survival in comparison with untreated cells. ( g ) The H460 cell pair transfected with a pool of nonspecific control siRNA or p21-specific siRNAs was treated with γ -radiation (1 Gy) and evaluated for cell death by using CellTiter-Glo (Promega). (** P <0.01; *** P <0.001)

Article Snippet: The full-length human Brachyury construct (pCMV-Neo-Brachyury, accession number NM_003181.2), a p21 expression vector (accession number NM_078467.1) and empty-vector control (pCMV-Neo) were purchased from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Expressing, Western Blot, shRNA, Derivative Assay, Clone Assay, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: The embryonic transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies

doi: 10.1038/cddis.2013.208

Figure Lengend Snippet: Brachyury drives repression of p21. ( a ) A ChIP assay was employed utilizing control IgG versus anti-Brachyury antibody with template DNA obtained from indicated cells. Shown at the top is the T-box palindromic consensus element juxtaposed with the half-binding site (shaded) located in the human p21 promoter (wild type), and a mutated p21 promoter containing point mutations in the T-box-binding site (denoted by arrowheads). Depicted below is the gel electrophoresis of PCR products amplified from the indicated immunoprecipitated DNA template sources using p21 promoter-specific primers. M corresponds to the DNA ladder. ( b ) Luciferase reporter assay was used to analyze H460 cells utilizing a wild-type p21 promoter driving the expression of the luciferase gene. ( c ) Luciferase reporter assay with H460 control.shRNA cells utilizing a wild-type versus mutated human p21 promoter driving the expression of the luciferase gene. Values shown represent the average of two technical replicates corrected for background±S.E.M. (* P <0.05). ( d ) Synchronized A549 pCMV and pBrachyury cells or asynchronous H460 control.shRNA and H460 Brachyury.shRNA2 were treated with indicated doses of radiation; cells collected after 24 h were analyzed for p21 induction by western blot. Intensity of p21 detection was quantitated and compared against β -actin or GAPDH (bottom panels)

Article Snippet: The full-length human Brachyury construct (pCMV-Neo-Brachyury, accession number NM_003181.2), a p21 expression vector (accession number NM_078467.1) and empty-vector control (pCMV-Neo) were purchased from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Binding Assay, Nucleic Acid Electrophoresis, Amplification, Immunoprecipitation, Luciferase, Reporter Assay, Expressing, shRNA, Western Blot