lentiviral muc1 shrna  (Santa Cruz Biotechnology)

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: Fig. 4 Activated EGFR upregulates MUC1-CT expression. 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: Expressing, Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, 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: Fig. 6 AICAR combined with osimertinib and VX-509 block 3D structure formation in patient and transgenic mouse-derived tumours. 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: Blocking Assay, Transgenic Assay, Derivative 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: Fig. 4 Activated EGFR upregulates MUC1-CT expression. 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: Expressing, Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, 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: Fig. 6 AICAR combined with osimertinib and VX-509 block 3D structure formation in patient and transgenic mouse-derived tumours. 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: Blocking Assay, Transgenic Assay, Derivative 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: Fig. 4 Activated EGFR upregulates MUC1-CT expression. 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: Expressing, Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, 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: Fig. 6 AICAR combined with osimertinib and VX-509 block 3D structure formation in patient and transgenic mouse-derived tumours. 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: Blocking Assay, Transgenic Assay, Derivative Assay