Journal: Science Advances

Article Title: Targeting oncoproteins with a positive selection assay for protein degraders

doi: 10.1126/sciadv.abd6263

Figure Lengend Snippet: ( A ) Immunoblot and ( B ) RT-qPCR analysis of the NCI-H1876 SCLC cell line that endogenously expresses ASCL1 infected to express the indicated sgRNAs. n = 3 biological replicates. ( C and E ) Immunoblot and ( D and F ) RT-qPCR analysis of NCI-H1876 human SCLC cells (C and D) and 97-2 mouse SCLC cells (E and F) after treatment with the CDK2 PROTAC degraders (TMX-2138 and TMX-2172) or the indicated negative controls, all used at 500 nM for either 36 hours (C and D) or 8 hours (E and F). Neg Deg, negative control degrader ZXH-7035. n = 3 biological replicates. ( G ) Immunoblot analysis and ( H ) quantification of ASCL1 protein levels in 97-2 cells first treated with the CDK2 PROTAC degrader or negative control (500 nM) for 4 hours and then treated with cycloheximide (CHX) (150 μg/ml) for the indicated times. S.E., short exposure; L.E., long exposure. n = 4 biological replicates. In all experiments, error bars represent SD except in (H), where error bars represent SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following compounds were purchased: POM (Selleck, #S1567), LEN (Selleck, #S1029), MG132 ( N -carbobenzyloxy- l -leucyl- l -leucyl- l -leucinal; Thermo Fisher Scientific, #47479020MG), MLN4924 (Active Biochem, #A-1139), MLN7243 (Thermo Fisher Scientific, #NC1129906), Spautin-1 (BioTechne; #5197/10), cycloheximide (VWR, #97064-724), BVdU (Chem-Impex International Inc., catalog no. 27735), actinomycin D (Thermo Fisher Scientific, #11805017), and dinaciclib (Selleck, #S2768).

Techniques: Western Blot, Quantitative RT-PCR, Infection, Negative Control

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Diagram showing the strategy employed to shortlist AICAR-binding proteins. The three steps include in silico screening using FINDSITE comb2.0 , protein expression assay, and thermal stability assay. b Time-dependent western blotting and relative quantification of protein expression for AICAR-binding proteins. The treatment responses on H1975 cells treated with 1 mM AICAR for 1 and 2 h were grouped by strong inactivation (type 1) and weak response (type 2). GAPDH was used as a loading control. N = 2–3 replicates. c Dose-dependent western blotting and relative quantification of protein expression for MUC1-CT and TMEM70. H1975 cells were treated with increasing doses of AICAR (0, 0.4, 1.3, and 4.4 mM) for 22 h, followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. d Thermal stability assay for MUC1-CT and TMEM70. H1975 cells were treated with 1 mM AICAR for 15 min. The cell pellets were heated for 3 min at their respective temperature (37–55 °C), followed by a western blot assay. N = 2 replicates. e Immunofluorescence staining for MUC1-CT in H441 cells. The cells were treated with 0.3 mM AICAR for 4 h and then incubated with rabbit anti-MUC1-CT primary antibody followed by goat anti-rabbit IgG conjugated with Alexa Fluor 555. The nucleus was counterstained with DAPI. The images were taken using a Keyence fluorescent microscope. Scale bar, 50 μm. f qRT-PCR analysis for MUC1-CT targeting genes. Expression levels for CTGF , PGM2 , and ENO1 were analysed by qRT-PCR in H1975 cells treated with 0.3 mM AICAR for 4 h. GAPDH was used as an endogenous control. N = 3 replicates. g qRT-PCR analysis for MUC1 expression in H1975 cells with MUC1 overexpression (OE). The cells were transfected with a lentiviral vector containing MUC1 or scrambled control, followed by 0.5 µg/ml puromycin selection. The relative MUC1 expression level in scrambled control cells was calibrated as 1. GAPDH was used as an endogenous control. N = 3 replicates. h Organoid formation assay for AICAR treatment response in H1975 cells overexpressing MUC1 . The cells with MUC1 overexpression or a scrambled control vector were plated at 2000 cells per well and treated with vehicle or 0.3 mM AICAR continuously for nine days. Images were taken with an EVOS microscope, and the treatment responses were quantified using a 3D Celltiter-Glo assay. N = 4–5 replicates. Scale bar, 300 µm. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , f , h ); Welch’s t -test ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Binding Assay, In Silico, Expressing, Stability Assay, Western Blot, Control, Immunofluorescence, Staining, Incubation, Microscopy, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Selection, Tube Formation Assay, Glo Assay

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Top KEGG signalling pathways differentially expressed in H1975 cells treated with AICAR. The cells were treated with 1 mM AICAR for 4 h, followed by whole transcriptomic analysis. N = 3 replicates. b Enrichment plot by gene set enrichment analysis for the JAK-STAT signalling pathway in H1975 cells treated with AICAR. Profile of the running enrichment score (ES) (top) and positions of gene set members on the rank-ordered list (bottom) were shown. N = 3 replicates. c A heat map showing top enriched genes of the JAK-STAT signalling pathway in H1975 cells treated with AICAR compared with vehicle-treated cells. d Longitudinal analysis of p-JAK1, p-TYK2, and MUC1-CT expression. H1975 cells were treated with 1 mM AICAR for 0, 1, and 2 h followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. e Confocal images for co-localisation of MUC1-CT and p-JAK1 in lung cancer cells. H1975 cells were co-incubated with Armenian hamster anti-MUC1-CT and rabbit anti-p-JAK1 primary antibodies for 2 h. Then the cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 555. The nucleus was counterstained with DAPI. The images (top: lower magnification; bottom: higher magnification) were taken using a Zeiss confocal fluorescent microscope. Scale Bar, 50 µm (top) and 20 µm (bottom). f Duolink ligation assay and confocal imaging for physical MUC1-JAK1 interactions. H441 cells were treated with vehicle or 1 mM AICAR for 1 h. After treatment, the cells were incubated with mouse anti-ZO-1 primary antibody overnight, followed by anti-mouse IgG conjugated with Alexa Fluor 488. Then the cells were co-incubated with anti-MUC1 and anti-JAK1 primary antibodies, followed by incubation with proximity ligation assay probes conjugated with Cy3, ligation, and amplification steps. The nucleus was counterstained with DAPI. The images were taken using a Zeiss confocal fluorescent microscope, and the Duolink dots were quantified using Image J. Scale Bar, 20 µm. g Cell viability assay of H1975 cells treated with AICAR and VX-509. Cells were plated in a 96-well plate and treated with AICAR (1 mM) with or without VX-509 (10 μM). The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated control group. N = 3–4 replicates. Data are mean ± s.e.m. and were analysed with Welch’s t -test ( a , b , f ); Brown-Forsythe and Welch one-way ANOVA ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Expressing, Western Blot, Control, Incubation, Microscopy, Ligation, Imaging, Proximity Ligation Assay, Amplification, Viability Assay

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL ( T790M; L858R )-induced lung tissues from transgenic mice. The mice were fed doxycycline (Dox)-impregnated food pellets for 0, 1, and 2 weeks followed by whole lung-tissue extraction. N = 2 replicates. b qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues. Gapdh was used as an endogenous control. EG0, EG1, and EG2 represent tissues from the mice fed with dox-impregnated food pellets for 0, 1, and 2 weeks, respectively. N = 3 replicates. c Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL -induced lung tissues after EGFR inactivation. The mice fed with Dox-impregnated food pellets for 8 weeks were given either the same Dox diet for an additional 2 weeks (EG10) or a regular diet for 2 weeks (EG8off2). Then the whole lung tissues were extracted for protein expression assay. N = 2 replicates. d qRT-PCR analysis of gene expression for EGFR and MUC1 in mouse EGFR TL -induced lung tissues after EGFR inactivation. The lung tissues from EG14 and EG8OFF2 mice were extracted for RNA analysis. GAPDH was used as an endogenous control. N = 3 replicates. e Western blotting and quantitative analysis for p-EGFR (Y1068) and EGFR expression in H1975 cells with MUC1 overexpression (OE). β-actin was used as a loading control. N = 3 replicates. f Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT expression in H1975 cells treated with 1 mM AICAR for one and 2 h. β-actin was used as a loading control. N = 3 replicates. g qRT-PCR analysis for MUC1 gene expression in H1975 cells with MUC1 knockdown. The cells were transfected with a lentiviral vector containing shRNA against MUC1 (shMUC1) or a scrambled control vector (sh-Control), followed by a 0.5 µg/ml puromycin selection. GAPDH was used as an endogenous control. N = 3 replicates. h Cell viability assay of H1975 cells treated with osimertinib and VX-509. 3000 cells with MUC1 knockdown (sh-MUC1) and a negative control vector (sh-control) were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), or both. The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated sh-control group. N = 4 replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( c , d , e , g ); one-way ANOVA ( a , b ); Brown-Forsythe and Welch ANOVA ( f , h ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Expressing, Control, Over Expression, Knockdown, Transfection, Plasmid Preparation, shRNA, Selection, Viability Assay, Negative Control, Two Tailed Test

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues from transgenic mice. The far normal and tumour tissues from transgenic mice 14 weeks after Dox induction were extracted for RNA analysis. Gapdh was used as an endogenous control. N = 3 replicates. b Differential MUC1 gene expression in lung adenocarcinoma (LUAD) compared with tumour-adjacent tissues by analysing the TCGA_LUAD dataset. N = 59 (normal), and N = 517 (tumour). c Patients’ overall survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 135. d Patients’ disease-free survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 105. e Xenograft tumour growth in mice treated with AICAR. The xenograft tumour was pre-established by implanting 1 million H1975 cells subcutaneously. When the tumour reached 45 mm 3 , the mice were treated with 300 mg/kg/day AICAR in PBS or a vehicle subcutaneously for ten days. The tumour was measured with a digital caliper, and the tumour size was calculated. N = 7 replicates. f Xenograft tumour images and relative weight quantification from mice treated with AICAR or PBS. The average tumour weight from the PBS-treated group is normalised as 1. N = 7 replicates. g Mouse body weight after treatment with AICAR or PBS for ten days. N = 7 replicates. h , i H&E staining of subcutaneous tumours ( h ) and liver tissues ( I ) from H1975 cell line-derived xenograft (CDX) treated with PBS or AICAR. Scale bar, 125 μm. j – l Immunofluorescence staining for Ki-67 ( j ), γ-H 2 AX ( k ), and p21 Cip1 ( l ) in subcutaneous tumours from H1975 CDX treated with PBS or AICAR. Scale bar, 100 μm. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b ); log-rank test ( c , d ); Welch’s t -test ( e – g ). * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Quantitative RT-PCR, Expressing, Transgenic Assay, Control, Staining, Derivative Assay, Immunofluorescence, Two Tailed Test

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a A diagram showing mechanisms of AICAR’s anticancer roles. In MUC1-dependent tumours, AICAR treatment directly binds and degrades MUC1-CT, increasing DNA damage in tumour cells. The degraded MUC1-CT de-stabilises p-EGFR and p-JAK1, further inactivating tumour-supportive signals. Created with BioRender.com. b Treatment response to VX-509 and osimertinib and AICAR in H1975 cells. 3000 cells were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), AICAR (1 mM), or a combination. The cell viability was measured 3 days after treatment. Values were normalised to a vehicle-treated group. N = 4 replicates. c , d Growth of PDX ( c ) and transgenic mouse EGFR TL -induced lung tumour ( d )-derived organoids treated with AICAR, osimertinib, and VX-509. 2000 cells were plated in organoid-culture media followed by treatments with AICAR (1 mM), osimertinib (0.5 μM), VX-509 (10 μM), or combinations for 10 days. The media were replenished every three days. The 3D cultures’ size was measured on day ten by ImageJ. The organoid tumour area in the vehicle-treated group was normalised as 100%. Scale bar, 50 μm. N = 6–12 replicates. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , d ). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Transgenic Assay, Derivative Assay