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human cdkn1a p21  (Addgene inc)


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    Addgene inc human cdkn1a p21
    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 <t>p21</t> (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.
    Human Cdkn1a P21, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human cdkn1a p21/product/Addgene inc
    Average 93 stars, based on 19 article reviews
    human cdkn1a p21 - by Bioz Stars, 2026-02
    93/100 stars

    Images

    1) Product Images from "Programming the elongation of mammalian cell aggregates with synthetic gene circuits"

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

    Journal: bioRxiv

    doi: 10.1101/2024.12.11.627621

    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.
    Figure Legend 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.

    Techniques Used: Growth Assay, Imaging, Microscopy, Activation Assay

    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.
    Figure Legend 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.

    Techniques Used: Marker, Activation Assay, Imaging

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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



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    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 <t>p21</t> (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.
    Mdm2, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cdkn1a mouse anti-human p21 (cdkn1a)  (Becton Dickinson)
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    Becton Dickinson cdkn1a mouse anti-human p21 (cdkn1a)
    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 <t>p21</t> (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.
    Cdkn1a Mouse Anti Human P21 (Cdkn1a), supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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.

    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

    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.

    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

    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.

    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

    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.

    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