Journal: Cell reports

Article Title: Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

doi: 10.1016/j.celrep.2023.113079

Figure Lengend Snippet: Figure 1. A combination of MEKi and CDK4/6i induces senescence in transformed and non-transformed cells (A) Representative images of MCF7 cells treated with DMSO, 10 Gy g-IR, or 100 nM MEKi and 500 nM CDK4/6i and stained for SA-b Gal activity every day for 7 days. Scale bar, 100 mm. (B) Percentage of SA-b Gal-positive cells treated with the indicated conditions as a function of time after treatment. Data are represented as mean ± SD from three independent experiments. p values were obtained from one-way ANOVA with multiple-comparison test. ns, not significant. (C) Scatterplot of CDK2 activity versus nuclear area (NA) of single cells. Color represents SA-b Gal activity in each cell. MCF7 cells were treated with 100 nM MEKi and 500 nM CDK4/6i for 2, 4, 6, and 7 days. Vertical line represents the 0.6 threshold, below which cells were considered to have low CDK2 activity. Horizontal line indicates the 200 mm2 threshold above which nuclei were considered large. (D) Quantification of cells (from C) that were either cycling (CDK2 > 0.6, NA < 200 mm2, log2[SA-b Gal] < 23.5), G0 senescent (CDK2 < 0.6, NA > 200 mm2, log2[SA-b Gal] > 23.5), G2 senescent (CDK2 > 0.6, NA > 200 mm2, log2[SA-b Gal] > 23.5), or G0/quiescent cells (CDK2 < 0.6, NA < 200 mm2, log2[SA-b Gal] < 23.5). N > 1,500 cells per condition. (E) Heatmap of SASP released by cells treated with DMSO, 10 Gy g-IR, or 100 nM MEKi and 500 nM CDK4/6i at day 7 of treatment. Conditioned media were collected at day 7 and analyzed by multiplex immunoassays. Data are fold change normalized to the DMSO-treated condition.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GeoMx MAPK Signaling Panel Human Protein Module for NGS NanoString Technologies GMX-PROMOD-NGS-HMAPK-12 Experimental models: Cell lines Human: MCF7 cells ATCC Cat#: HTB-22; RRID: CVCL_0031 Human: U2OS cells ATCC Cat#: HTB-96; RRID: CVCL_0042 Human: MCF10A cells ATCC Cat#: CRL-10317; RRID: CVCL_0598 Human: CAL33 cells DSMZ Cat#: ACC447 RRID: CVCL_1108 Human: Primary human lung fibroblasts ATCC Cat#: #PCS-201-013 Oligonucleotides On-Target plus control siRNA Dharmacon Cat#: D-001810-10-05 CDKN1B siRNA (On-Target plus pooled set of four) Dharmacon Cat#: L-003472-00-0005 CDKN1A siRNA (On-Target plus pooled set of four) Dharmacon Cat#: LQ-003471-00 TP53 (On-Target plus pooled set of four) Dharmacon Cat#: L-003329-00-0005 Primers for RT-PCR see Table S2 N/A Recombinant DNA CSII-pEF1a-H2B-mTurquoise Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mVenus Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mCherry Nathans et al.67 N/A CSII-pEF1a-mCherry-Geminin(aa1-110) Cappell et al.68 N/A pCMV(CAT)T7-SB100 Mates et al.69 Cat#: 34879 pSBtet-MYCT58A-emiRFP This work N/A pUltra-puro-RTTA2 Unpublished Gift from Yildirim Dogan and Kitai Kim Addgene #: 58750 FU-tet-o-hc-MYC Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19775 pLV-tetO-MYC T58A Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19763 TRIPZ hMYC lentiviral shRNA Dharmacon Cat#: RHS4696-200675280 TRIPZ non-silencing shRNA Dharmacon Cat#: RSH4743 Software and algorithms FIJI FIJI Version: 2.3.0/1.53f MATLAB MATHWORKS Version: 2019B MATLAB Tracking Scripts Cappell et al.68 N/A Prism Graphpad Version: 9.2.0 Illustrator Adobe Version: 26.3.1 Incucyte Cell-by-Cell Analysis Software Sartorius Version: 9600-0031 Halo imaging analysis software Indica Labs Version:3.3.2541.423 BD FACSDiVa Software BD Biosciences Version: 9.0 FlowJo FlowJo Version:10.8.01 Bio-Plex Manager software Bio-Rad Version: 6.2 Build175 Article ll OPEN ACCESS

Techniques: Transformation Assay, Staining, Activity Assay, Comparison, Multiplex Assay

Journal: Cell reports

Article Title: Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

doi: 10.1016/j.celrep.2023.113079

Figure Lengend Snippet: Figure 2. Cells maintain senescence after withdrawal of MEKi and CDK4/6i combination (A) Percentage confluence of cells treated with the indicated conditions as a function of time after treatment. Data are represented as mean ± SD from three independent experiments. p values were obtained from one-way ANOVA with multiple-comparison test. Vertical line denotes time at which drugs were washed off. 10 Gy g-IR-treated cells were used as a positive control. (B) Representative images of MCF7 cells treated with DMSO, 100 nM MEKi, 500 nM CDK4/6i, or a combination of MEKi and CDK4/6i for 7 days after which the treatments were washed off and the cells were grown in complete culture medium until day 17. Cells were stained for SA-b Gal activity at days 10 and 17. Cells exposed to 10 Gy g-IR at day 0 were used as a positive control. Scale bar, 100 mm. (C and D) Quantification of SA-b Gal-positive MCF7 (C) or MCF10A (D) cells treated with the indicated conditions as a function of time after treatment. Data are represented as mean ± SD from three independent experiments. p values were obtained from ANOVA with multiple-comparison test. (E) Heatmap of SASP factors released by cells treated with DMSO, 100 nM MEKi, 500 nM CDK4/6i, or a combination of MEKi and CDK4/6i. All treatments were washed off at day 7 and the cells were allowed to grow in complete medium until day 17. Conditioned media was collected at days 10 and 17 and analyzed by multiplex immunoassays. Data are SASP fold change normalized to DMSO-treated condition.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GeoMx MAPK Signaling Panel Human Protein Module for NGS NanoString Technologies GMX-PROMOD-NGS-HMAPK-12 Experimental models: Cell lines Human: MCF7 cells ATCC Cat#: HTB-22; RRID: CVCL_0031 Human: U2OS cells ATCC Cat#: HTB-96; RRID: CVCL_0042 Human: MCF10A cells ATCC Cat#: CRL-10317; RRID: CVCL_0598 Human: CAL33 cells DSMZ Cat#: ACC447 RRID: CVCL_1108 Human: Primary human lung fibroblasts ATCC Cat#: #PCS-201-013 Oligonucleotides On-Target plus control siRNA Dharmacon Cat#: D-001810-10-05 CDKN1B siRNA (On-Target plus pooled set of four) Dharmacon Cat#: L-003472-00-0005 CDKN1A siRNA (On-Target plus pooled set of four) Dharmacon Cat#: LQ-003471-00 TP53 (On-Target plus pooled set of four) Dharmacon Cat#: L-003329-00-0005 Primers for RT-PCR see Table S2 N/A Recombinant DNA CSII-pEF1a-H2B-mTurquoise Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mVenus Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mCherry Nathans et al.67 N/A CSII-pEF1a-mCherry-Geminin(aa1-110) Cappell et al.68 N/A pCMV(CAT)T7-SB100 Mates et al.69 Cat#: 34879 pSBtet-MYCT58A-emiRFP This work N/A pUltra-puro-RTTA2 Unpublished Gift from Yildirim Dogan and Kitai Kim Addgene #: 58750 FU-tet-o-hc-MYC Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19775 pLV-tetO-MYC T58A Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19763 TRIPZ hMYC lentiviral shRNA Dharmacon Cat#: RHS4696-200675280 TRIPZ non-silencing shRNA Dharmacon Cat#: RSH4743 Software and algorithms FIJI FIJI Version: 2.3.0/1.53f MATLAB MATHWORKS Version: 2019B MATLAB Tracking Scripts Cappell et al.68 N/A Prism Graphpad Version: 9.2.0 Illustrator Adobe Version: 26.3.1 Incucyte Cell-by-Cell Analysis Software Sartorius Version: 9600-0031 Halo imaging analysis software Indica Labs Version:3.3.2541.423 BD FACSDiVa Software BD Biosciences Version: 9.0 FlowJo FlowJo Version:10.8.01 Bio-Plex Manager software Bio-Rad Version: 6.2 Build175 Article ll OPEN ACCESS

Techniques: Comparison, Positive Control, Staining, Activity Assay, Multiplex Assay

Journal: Cell reports

Article Title: Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

doi: 10.1016/j.celrep.2023.113079

Figure Lengend Snippet: Figure 3. Probability of cells irreversibly committing to senescence increases with treatment duration (A) Experimental scheme showing that MCF7 cells were treated with DMSO or a combination of 100 nM MEKi and 500 nM CDK4/6i for increasing durations 1, 2, 3, 4, 5, 6, and 7 days before washing off the drugs. Cells were then grown in complete medium until day 17, when cells were fixed and stained for SA-b Gal activity. (B) Representative images of MCF7 cells treated as described in (A). Cells were fixed and stained for SA-b Gal activity using SPIDR- IF-SA-b Gal assay (green). Nuclei were stained with Hoechst (blue). Scale bar, 10 mm. (C) Quantification of SA-b Gal-positive cells from (B) plotted as a function of time exposed to treatment before washing off the drugs. Data are represented as mean ± SD from three independent experiments. (D) Single-cell CDK2 activity traces from MCF7 cells treated with DMSO or 100 nM MEKi and 500 nM CDK4/6i while in S/G2 phase (defined as CDK2 activity greater than 1 and APC/C activity less than 0.3 at the time of treatment). Vertical black lines indicate when drugs were added (solid) and washed off (dashed). Trace color denotes long-term fate of each cell: black, immediately built up CDK2 activity after mitosis (CDK2 rapid increase); cyan, variable time spent in a CDK2- low state following mitosis before eventually building up CDK2 activity (CDK2 delayed increase); pink, remained in CDK2-low state for the entire imaging period (CDK2 low). Black dot denotes when a cell went through mitosis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GeoMx MAPK Signaling Panel Human Protein Module for NGS NanoString Technologies GMX-PROMOD-NGS-HMAPK-12 Experimental models: Cell lines Human: MCF7 cells ATCC Cat#: HTB-22; RRID: CVCL_0031 Human: U2OS cells ATCC Cat#: HTB-96; RRID: CVCL_0042 Human: MCF10A cells ATCC Cat#: CRL-10317; RRID: CVCL_0598 Human: CAL33 cells DSMZ Cat#: ACC447 RRID: CVCL_1108 Human: Primary human lung fibroblasts ATCC Cat#: #PCS-201-013 Oligonucleotides On-Target plus control siRNA Dharmacon Cat#: D-001810-10-05 CDKN1B siRNA (On-Target plus pooled set of four) Dharmacon Cat#: L-003472-00-0005 CDKN1A siRNA (On-Target plus pooled set of four) Dharmacon Cat#: LQ-003471-00 TP53 (On-Target plus pooled set of four) Dharmacon Cat#: L-003329-00-0005 Primers for RT-PCR see Table S2 N/A Recombinant DNA CSII-pEF1a-H2B-mTurquoise Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mVenus Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mCherry Nathans et al.67 N/A CSII-pEF1a-mCherry-Geminin(aa1-110) Cappell et al.68 N/A pCMV(CAT)T7-SB100 Mates et al.69 Cat#: 34879 pSBtet-MYCT58A-emiRFP This work N/A pUltra-puro-RTTA2 Unpublished Gift from Yildirim Dogan and Kitai Kim Addgene #: 58750 FU-tet-o-hc-MYC Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19775 pLV-tetO-MYC T58A Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19763 TRIPZ hMYC lentiviral shRNA Dharmacon Cat#: RHS4696-200675280 TRIPZ non-silencing shRNA Dharmacon Cat#: RSH4743 Software and algorithms FIJI FIJI Version: 2.3.0/1.53f MATLAB MATHWORKS Version: 2019B MATLAB Tracking Scripts Cappell et al.68 N/A Prism Graphpad Version: 9.2.0 Illustrator Adobe Version: 26.3.1 Incucyte Cell-by-Cell Analysis Software Sartorius Version: 9600-0031 Halo imaging analysis software Indica Labs Version:3.3.2541.423 BD FACSDiVa Software BD Biosciences Version: 9.0 FlowJo FlowJo Version:10.8.01 Bio-Plex Manager software Bio-Rad Version: 6.2 Build175 Article ll OPEN ACCESS

Techniques: Staining, Activity Assay, Imaging

Journal: Cell reports

Article Title: Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

doi: 10.1016/j.celrep.2023.113079

Figure Lengend Snippet: Figure 4. Senescence entry and maintenance are associated with irreversible MYC loss (A) Signaling diagram showing major transcription factors as possible proteins mediating entry and commitment to cellular senescence. (B) Quantification of MYC, c-JUN, c-FOS, and c/EBPa mRNA levels normalized to control at different time points after adding MEKi + CDK4/6i. Data are rep- resented as mean ± SD from three independent experiments. (C) Immunoblotting of MYC, c-Jun, and c/EBPa at days 0, 2, 4, 6, and 7 post-treatment. Actin was used as the loading control. Representative blot from three independent experiments. (D) Representative images of MCF7 cells treated with MEKi + CDK4/6i for the indicated number of days. Cells were fixed and stained for MYC protein (red) and SA-b Gal activity (green). Nuclei were stained with Hoechst (blue). Scale bar, 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GeoMx MAPK Signaling Panel Human Protein Module for NGS NanoString Technologies GMX-PROMOD-NGS-HMAPK-12 Experimental models: Cell lines Human: MCF7 cells ATCC Cat#: HTB-22; RRID: CVCL_0031 Human: U2OS cells ATCC Cat#: HTB-96; RRID: CVCL_0042 Human: MCF10A cells ATCC Cat#: CRL-10317; RRID: CVCL_0598 Human: CAL33 cells DSMZ Cat#: ACC447 RRID: CVCL_1108 Human: Primary human lung fibroblasts ATCC Cat#: #PCS-201-013 Oligonucleotides On-Target plus control siRNA Dharmacon Cat#: D-001810-10-05 CDKN1B siRNA (On-Target plus pooled set of four) Dharmacon Cat#: L-003472-00-0005 CDKN1A siRNA (On-Target plus pooled set of four) Dharmacon Cat#: LQ-003471-00 TP53 (On-Target plus pooled set of four) Dharmacon Cat#: L-003329-00-0005 Primers for RT-PCR see Table S2 N/A Recombinant DNA CSII-pEF1a-H2B-mTurquoise Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mVenus Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mCherry Nathans et al.67 N/A CSII-pEF1a-mCherry-Geminin(aa1-110) Cappell et al.68 N/A pCMV(CAT)T7-SB100 Mates et al.69 Cat#: 34879 pSBtet-MYCT58A-emiRFP This work N/A pUltra-puro-RTTA2 Unpublished Gift from Yildirim Dogan and Kitai Kim Addgene #: 58750 FU-tet-o-hc-MYC Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19775 pLV-tetO-MYC T58A Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19763 TRIPZ hMYC lentiviral shRNA Dharmacon Cat#: RHS4696-200675280 TRIPZ non-silencing shRNA Dharmacon Cat#: RSH4743 Software and algorithms FIJI FIJI Version: 2.3.0/1.53f MATLAB MATHWORKS Version: 2019B MATLAB Tracking Scripts Cappell et al.68 N/A Prism Graphpad Version: 9.2.0 Illustrator Adobe Version: 26.3.1 Incucyte Cell-by-Cell Analysis Software Sartorius Version: 9600-0031 Halo imaging analysis software Indica Labs Version:3.3.2541.423 BD FACSDiVa Software BD Biosciences Version: 9.0 FlowJo FlowJo Version:10.8.01 Bio-Plex Manager software Bio-Rad Version: 6.2 Build175 Article ll OPEN ACCESS

Techniques: Control, Western Blot, Staining, Activity Assay

Journal: Cell reports

Article Title: Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

doi: 10.1016/j.celrep.2023.113079

Figure Lengend Snippet: Figure 5. Cells enter and maintain senescence by irreversible degradation of MYC (A and B) Quantification of SA-b Gal-positive and pRb-negative cells from Figures S5D and S5E treated with the indicated conditions. Data are represented as mean ± SD from three independent experiments. p values were obtained from one-way ANOVA with multiple-comparison test. ns, not significant. (C and D) Single-cell CDK2 activity traces from MCF7 cells transduced with teton-MYCT58A treated with 100 nM MEKi and 500 nM CDK4/6i for 7 days. At day 7, cells were washed to remove drugs and were treated with either DMSO (C) or 1 mM doxycycline (Dox) (D) when time-lapse imaging was started. Trace color denotes the long-term fate of each cell: cyan, started in the CDK2-low state before eventually building up CDK2 activity (CDK2 high); pink, remained in CDK2-low state for the entire imaging period (CDK2 low). Black dot denotes when a cell went through mitosis. (E and F) Single-cell CDK2 activity traces from MCF7 cells treated with 100 nM MEKi and 500 nM CDK4/6i for 7 days. At day 7, cells were washed to remove drugs and were treated with either DMSO (E) or 1 mM GSK3bi (F) when time-lapse imaging was started. (G) Bar graph depicting the percent of senescent cells that activated CDK2 activity and re-entered the cell cycle after treatment with a GSK3bi as described in (F). Data are represented as mean ± SD from two independent experiments. Example single-cell CDK2 traces can be found in Figure S5G. (H and I) Single-cell CDK2 activity traces from MCF7 cells exposed to 10 Gy g-IR. At day 7, cells were treated with either DMSO (H) or 1 mM GSK3bi (I) when time- lapse imaging was started. (J) Bar graph depicting the percent of senescent cells that activated CDK2 activity and re-entered the cell cycle after treatment with a GSK3bi as described in (I). Data are mean ± SD from two biological replicates. p value was obtained from Student’s t test. Example single-cell CDK2 traces can be found in (H) and (I). (K) Quantification of SA-b Gal-positive and pRb-negative cells from Figure S6A treated with the indicated conditions. Data are represented as mean ± SD from three independent experiments. p values were obtained from one-way ANOVA with multiple-comparison test. ns, not statistically significant. (L) Quantification of SA-b Gal-positive and pRb-negative cells from Figure S6B treated with the indicated conditions. Data are represented as mean ± SD from three independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GeoMx MAPK Signaling Panel Human Protein Module for NGS NanoString Technologies GMX-PROMOD-NGS-HMAPK-12 Experimental models: Cell lines Human: MCF7 cells ATCC Cat#: HTB-22; RRID: CVCL_0031 Human: U2OS cells ATCC Cat#: HTB-96; RRID: CVCL_0042 Human: MCF10A cells ATCC Cat#: CRL-10317; RRID: CVCL_0598 Human: CAL33 cells DSMZ Cat#: ACC447 RRID: CVCL_1108 Human: Primary human lung fibroblasts ATCC Cat#: #PCS-201-013 Oligonucleotides On-Target plus control siRNA Dharmacon Cat#: D-001810-10-05 CDKN1B siRNA (On-Target plus pooled set of four) Dharmacon Cat#: L-003472-00-0005 CDKN1A siRNA (On-Target plus pooled set of four) Dharmacon Cat#: LQ-003471-00 TP53 (On-Target plus pooled set of four) Dharmacon Cat#: L-003329-00-0005 Primers for RT-PCR see Table S2 N/A Recombinant DNA CSII-pEF1a-H2B-mTurquoise Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mVenus Spencer et al.28 N/A CSII-pEF1a-DHB(aa994-1087)-mCherry Nathans et al.67 N/A CSII-pEF1a-mCherry-Geminin(aa1-110) Cappell et al.68 N/A pCMV(CAT)T7-SB100 Mates et al.69 Cat#: 34879 pSBtet-MYCT58A-emiRFP This work N/A pUltra-puro-RTTA2 Unpublished Gift from Yildirim Dogan and Kitai Kim Addgene #: 58750 FU-tet-o-hc-MYC Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19775 pLV-tetO-MYC T58A Maherali et al.70 Gift from Konrad Hochedlinger Addgene #: 19763 TRIPZ hMYC lentiviral shRNA Dharmacon Cat#: RHS4696-200675280 TRIPZ non-silencing shRNA Dharmacon Cat#: RSH4743 Software and algorithms FIJI FIJI Version: 2.3.0/1.53f MATLAB MATHWORKS Version: 2019B MATLAB Tracking Scripts Cappell et al.68 N/A Prism Graphpad Version: 9.2.0 Illustrator Adobe Version: 26.3.1 Incucyte Cell-by-Cell Analysis Software Sartorius Version: 9600-0031 Halo imaging analysis software Indica Labs Version:3.3.2541.423 BD FACSDiVa Software BD Biosciences Version: 9.0 FlowJo FlowJo Version:10.8.01 Bio-Plex Manager software Bio-Rad Version: 6.2 Build175 Article ll OPEN ACCESS

Techniques: Comparison, Activity Assay, Transduction, Imaging