myc t58a  (ATCC)

Journal: Communications Biology

Article Title: Activation of Kv11.1 potassium channel suppresses non-small cell lung cancer growth by promoting c-Myc degradation

doi: 10.1038/s42003-025-08917-9

Figure Lengend Snippet: A Western blot showing the effect of NS1643 alone, cycloheximide alone (CHX) or NS1643 and CHX at different time points (0, 30, 60, 90 min) on c-Myc protein density in A549 lung cancer cells. Intensities were normalized for the loading control histone H3. B Quantification of the events in ( A ) for the 0, 30, 60 min time points. C Western blot showing the effect of NS1643 (50 mM; 24 h) alone or with the proteasome inhibitor MG132 in A549 cells. The bar graph indicates quantification of ( C ). D Western blot showing the effect of NS1643 (50 mM; 24 h) on phosphorylation of Serine 62 or Threonine 58 of c-Myc in A549 cell line. E Western blot showing the effect of NS1643 (50 mM; 24 h) on the doxycycline-induced c-Myc T58A in A549 cells. F Western blot showing the effect of NS1643 (50 mM; 24 h) on c-Myc in A549 cells transiently transfected with either OTUD6B1 or OTUD6B2 in pCDNA3. Similar results were obtained in H1299. *, **statistically significant ( P < 0.05 or P < 0.01, respectively).

Article Snippet: MYC T58A (human) was PCR-amplified from MSCV Myc T58A puro, which was a gift from Scott Lowe (Addgene plasmid # 18773; http://n2t.net/addgene:18773 ; RRID:Addgene_18773).

Techniques: Western Blot, Control, Phospho-proteomics, Transfection

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Schematic of the protocol used to generate pulmonary neuroendocrine cells (PNECs) by stepwise differentiation of human pluripotent stem cells (hPSCs) to form: definitive endoderm (DE), day 3; anterior foregut endoderm (AFE), day 6; and lung progenitor cells (LPs) days 15–25. LPs were further differentiated into the types of lung cells (LCs) found in mature human lung parenchyma and airway epithelium, days 25–55. DAPT (10 μM) encourages the formation of PNECs, and addition of doxycycline (1 μM; DOX) induces expression of shRNAs against RB1 and TP53 mRNAs, as well as expression of MYC or MYC (T58A), as described in the text. ( B ) Western blot of extracts of RUES2 LCs at day 25 of differentiation protocol treated with DOX (1 μM for 72 hr); cells unexposed to DOX served as negative expression controls. Apparent differences in MYC protein levels may be attributable to the HA-tagged version of MYC (T58A), which migrates slightly slower than wild-type MYC protein. ( C ) Schematic representation of tumorigenesis experiments comparing injection sites (renal capsule or subcutaneous), DOX treatment (+/-DOX diet), and genotypes (see Materials and methods for additional details). Total numbers of animals are six to seven per experimental arm with two injection sites per mouse (right and left flank). Renal capsule injections were performed on a single kidney. Transgenic lines of RUES2 hESCs were differentiated and grown in DAPT (10 μM) from days 25 to 55. At day 55, PNECs were separated from other LCs by sorting for PE+ CGRP-expressing cells (see Materials and methods). PNECs were then injected either subcutaneously or into the renal capsular space in NOG mice, half of which then received DOX in their feed as described in Materials and methods. ( D ) Table summary of experiments with xenografted mice, indicating the number of animals that developed visible tumors (≥250 mm 3 in volume) at the site of injection or the number of visible metastases in the liver or lung. *, p<0.05; **, p<0.01 by Fisher’s test to denote significant differences between mice that did not receive DOX diet. As before, abbreviations for cell lines are: RP = shRB1+shTP53; RPM = shRB1+shTP53+WT MYC; RPM (T58A)=shRB1+shTP53+MYC (T58A). Figure 1—source data 1. Raw data of western blot bands in . Figure 1—source data 2. Raw data of .

Article Snippet: Recombinant DNA reagent , pLV-tetO-myc T58A (Konrad Hochedlinger) , Addgene , RRID:Addgene_19763 , T58A mutant MYC with 3X-HA tag.

Techniques: Expressing, Western Blot, Injection, Transgenic Assay

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Subcutaneous engraftment of 10 6 viable cells at ~day 50 of RUES2 lung differentiation protocol from indicated genotypes. Cells were cultured in the presence of media containing 1 μM doxycycline (DOX) and 10 μM DAPT from days 25 to 55. All immunocompromised mice were maintained on the DOX diet throughout this study; n=5/arm. ( B ) Left: subcutaneous engraftment of 10 6 viable RPM tumor cells from the first passage (mouse-to-mouse passage) into immunocompromised mice (NSG) maintained on DOX or normal chow; n=5 animals per arm with single flank engraftments, ± standard error of the mean (SEM). **p<0.05. Right: after 1 month on normal chow, mice were placed on DOX chow (DOX ‘add-back’); tumor volumes were measured 1 month later (2 months on study). Paired t-test p-values are shown for each comparison. ( C ) Representative H&E tumor histology from RPM or RPM (T58A) engraftments; 2× (scalebar 1 mm) and 20× (scalebar 100 µm). Representative whole lung sagittal sections showed no evidence of gross metastasis from subcutaneous injections inthe flank at study endpoints; n=3 analyzed per genotype. Dashed lines shown for boundaries of lung lobes. Figure 2—source data 1. High resolution source data of .

Article Snippet: Recombinant DNA reagent , pLV-tetO-myc T58A (Konrad Hochedlinger) , Addgene , RRID:Addgene_19763 , T58A mutant MYC with 3X-HA tag.

Techniques: Cell Culture, Comparison

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Subcutaneous xenografts formed with human embryonic stem cell (hESC)-derived pulmonary neuroendocrine cells (PNECs) with RP, RPM, or RPM (T58A) genotypes. Photographs of representative tumors formed with cells of the indicated genotypes are shown for RP and RPM; indicated scale of 1 cm. ( B ) Quantification of the tumor sizes and paired comparisons for a secondary in vivo experiment; n=5 animals per arm with single subcutaneous engraftments; **p<0.01. ( C ) H&E staining of the indicated tumors from panel B. ( D ) Gross and histologic pathology of renal capsular xenografts and liver metastases formed with the RPM or RPM (T58A) cells. Left panels, gross appearance of representative tumors within the liver (metastasis) or kidney (primary); right panels, H&E staining of primary and metastatic tumors ( T ) formed in kidney ( K ) and liver ( L ) in the RPM (left) or RPM (T58A) model. ( E ) Immunostaining of tumor samples from D. Samples of primary peri-renal tumors and hepatic metastases in mice injected with hESC-derived PNECs programmed to reduce levels of TP53 and RB1 mRNA and to express wild-type MYC were stained with antisera for Ki67 (left) or MYC (right). ( F ) Immunofluorescence staining for neuroendocrine markers ASCL1, NEUROD1, and CD56 from sections in E of the RPM tumors. ( G ) Immunofluorescence staining for neuroendocrine markers, CGRP, ASCL1, NEUROD1, and CD56 in the RP tumors. H&E staining serves as the bright-field comparison of the indicated tumors. Figure 3—source data 1. High resolution source data of .

Article Snippet: Recombinant DNA reagent , pLV-tetO-myc T58A (Konrad Hochedlinger) , Addgene , RRID:Addgene_19763 , T58A mutant MYC with 3X-HA tag.

Techniques: Derivative Assay, In Vivo, Staining, Immunostaining, Injection, Immunofluorescence, Comparison

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Schematic of the protocol used to generate pulmonary neuroendocrine cells (PNECs) by stepwise differentiation of human pluripotent stem cells (hPSCs) to form: definitive endoderm (DE), day 3; anterior foregut endoderm (AFE), day 6; and lung progenitor cells (LPs) days 15–25. LPs were further differentiated into the types of lung cells (LCs) found in mature human lung parenchyma and airway epithelium, days 25–55. DAPT (10 μM) encourages the formation of PNECs, and addition of doxycycline (1 μM; DOX) induces expression of shRNAs against RB1 and TP53 mRNAs, as well as expression of MYC or MYC (T58A), as described in the text. ( B ) Western blot of extracts of RUES2 LCs at day 25 of differentiation protocol treated with DOX (1 μM for 72 hr); cells unexposed to DOX served as negative expression controls. Apparent differences in MYC protein levels may be attributable to the HA-tagged version of MYC (T58A), which migrates slightly slower than wild-type MYC protein. ( C ) Schematic representation of tumorigenesis experiments comparing injection sites (renal capsule or subcutaneous), DOX treatment (+/-DOX diet), and genotypes (see Materials and methods for additional details). Total numbers of animals are six to seven per experimental arm with two injection sites per mouse (right and left flank). Renal capsule injections were performed on a single kidney. Transgenic lines of RUES2 hESCs were differentiated and grown in DAPT (10 μM) from days 25 to 55. At day 55, PNECs were separated from other LCs by sorting for PE+ CGRP-expressing cells (see Materials and methods). PNECs were then injected either subcutaneously or into the renal capsular space in NOG mice, half of which then received DOX in their feed as described in Materials and methods. ( D ) Table summary of experiments with xenografted mice, indicating the number of animals that developed visible tumors (≥250 mm 3 in volume) at the site of injection or the number of visible metastases in the liver or lung. *, p<0.05; **, p<0.01 by Fisher’s test to denote significant differences between mice that did not receive DOX diet. As before, abbreviations for cell lines are: RP = shRB1+shTP53; RPM = shRB1+shTP53+WT MYC; RPM (T58A)=shRB1+shTP53+MYC (T58A). Figure 1—source data 1. Raw data of western blot bands in . Figure 1—source data 2. Raw data of .

Article Snippet: Recombinant DNA reagent , pLV-tetO-myc T58A (Konrad Hochedlinger) , Addgene , RRID:Addgene_19763 , T58A mutant MYC with 3X-HA tag.

Techniques: Expressing, Western Blot, Injection, Transgenic Assay

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Subcutaneous engraftment of 10 6 viable cells at ~day 50 of RUES2 lung differentiation protocol from indicated genotypes. Cells were cultured in the presence of media containing 1 μM doxycycline (DOX) and 10 μM DAPT from days 25 to 55. All immunocompromised mice were maintained on the DOX diet throughout this study; n=5/arm. ( B ) Left: subcutaneous engraftment of 10 6 viable RPM tumor cells from the first passage (mouse-to-mouse passage) into immunocompromised mice (NSG) maintained on DOX or normal chow; n=5 animals per arm with single flank engraftments, ± standard error of the mean (SEM). **p<0.05. Right: after 1 month on normal chow, mice were placed on DOX chow (DOX ‘add-back’); tumor volumes were measured 1 month later (2 months on study). Paired t-test p-values are shown for each comparison. ( C ) Representative H&E tumor histology from RPM or RPM (T58A) engraftments; 2× (scalebar 1 mm) and 20× (scalebar 100 µm). Representative whole lung sagittal sections showed no evidence of gross metastasis from subcutaneous injections inthe flank at study endpoints; n=3 analyzed per genotype. Dashed lines shown for boundaries of lung lobes. Figure 2—source data 1. High resolution source data of .

Article Snippet: Recombinant DNA reagent , pLV-tetO-myc T58A (Konrad Hochedlinger) , Addgene , RRID:Addgene_19763 , T58A mutant MYC with 3X-HA tag.

Techniques: Cell Culture, Comparison

Journal: eLife

Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs

doi: 10.7554/eLife.93170

Figure Lengend Snippet: ( A ) Subcutaneous xenografts formed with human embryonic stem cell (hESC)-derived pulmonary neuroendocrine cells (PNECs) with RP, RPM, or RPM (T58A) genotypes. Photographs of representative tumors formed with cells of the indicated genotypes are shown for RP and RPM; indicated scale of 1 cm. ( B ) Quantification of the tumor sizes and paired comparisons for a secondary in vivo experiment; n=5 animals per arm with single subcutaneous engraftments; **p<0.01. ( C ) H&E staining of the indicated tumors from panel B. ( D ) Gross and histologic pathology of renal capsular xenografts and liver metastases formed with the RPM or RPM (T58A) cells. Left panels, gross appearance of representative tumors within the liver (metastasis) or kidney (primary); right panels, H&E staining of primary and metastatic tumors ( T ) formed in kidney ( K ) and liver ( L ) in the RPM (left) or RPM (T58A) model. ( E ) Immunostaining of tumor samples from D. Samples of primary peri-renal tumors and hepatic metastases in mice injected with hESC-derived PNECs programmed to reduce levels of TP53 and RB1 mRNA and to express wild-type MYC were stained with antisera for Ki67 (left) or MYC (right). ( F ) Immunofluorescence staining for neuroendocrine markers ASCL1, NEUROD1, and CD56 from sections in E of the RPM tumors. ( G ) Immunofluorescence staining for neuroendocrine markers, CGRP, ASCL1, NEUROD1, and CD56 in the RP tumors. H&E staining serves as the bright-field comparison of the indicated tumors. Figure 3—source data 1. High resolution source data of .

Article Snippet: Recombinant DNA reagent , pLV-tetO-myc T58A (Konrad Hochedlinger) , Addgene , RRID:Addgene_19763 , T58A mutant MYC with 3X-HA tag.

Techniques: Derivative Assay, In Vivo, Staining, Immunostaining, Injection, Immunofluorescence, Comparison