vectors carrying full-length mtor with an n-terminal flag tag  (Addgene inc)

Journal: Bioscience Reports

Article Title: Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-InsP7

doi: 10.1042/bsr20240792

Figure Lengend Snippet: Figure 2. IP6K1 binds pyrophosphorylation substrate proteins and CK2 (A–C) Co-precipitation of endogenous NOLC1 (A), TCOF (B) or UBF1 (C) with IP6K1. N-terminally SFB-tagged IP6K1 was expressed in HEK293T cells, pulled down with streptavidin-sepharose beads, and probed to detect endogenous NOLC1, TCOF or UBF1. (D)

Article Snippet: Human IP6K1 cDNA (GenBank ID NM 001242829.2) and catalytically inactive IP6K1 (K226A) mutant were subcloned in pCDNA3.1 as previously described [22]. cDNA encoding human IP6K1 domains, i.e., FL, N-lobe, C-lobe, IDR-1, IDR-2, PDKG motif with IP helices, and IDR-1 with C-lobe, were cloned into the N-terminal SFB (S protein, Flag, streptavidin binding peptide)-tag destination vector, using the Gateway cloning strategy (Thermo Fisher Scientific).

Techniques:

Journal: Heliyon

Article Title: Interplay of p62-mTORC1 and EGFR signaling promotes cisplatin resistance in oral cancer

doi: 10.1016/j.heliyon.2024.e28406

Figure Lengend Snippet: p62-mTOR modulates CDDP tolerance in SAS cells. (A) p62 levels in SAS and OECM-1 cells shown by Western blot, under the treatment of insulin, IGF-1, CDDP, or Taxol. Numbers represent average of normalized band intensities, based on at least duplicate analyses. (B) Dose-dependent cell viability curves of CDDP in five cancer cell lines (n = 6). (C) The effects of CDDP on p62 and mTOR pathway activation in SAS and OECM-1 cells. Numbers below immunoblotting bands represent means ± SE of normalized intensities (n = 3). (D) Time series of p62 mRNA levels upon CDDP treatment. Error bars indicate standard error of the mean (n = 3). P values were determined by two-tailed unpaired t -test with equal variance (** p < 0.01, *** p < 0.001). (E) Transient inhibition of p62 using sip62 and its effect on SAS cell survival. siCtrl, non-targeting siRNA control. P values were determined by two-way ANOVA test with equal variance (*** p < 0.001; n = 3). (F) Effect of p62 inhibition on SAS colony formation upon CDDP treatment (n = 2). The top diagram depicts the experimental timeline. (G) p62 inhibition by sip62 abolishes the activating effect of CDDP on the p62-mTORC1 pathway. Numbers below each band represent means ± SE of normalized intensities (n = 3).

Article Snippet: For exogenous expression, vectors carrying full-length EGFR (referred to as “EGFR”), full-length Akt with an N-terminal myristoylation signal tag (designated as “myr-Akt”, a constitutively active form of Akt), and full-length mTOR with an N-terminal Flag tag (labeled as “Flag-mTOR”) were all purchased from Addgene (USA).

Techniques: Western Blot, Activation Assay, Two Tailed Test, Inhibition, Control

Journal: Heliyon

Article Title: Interplay of p62-mTORC1 and EGFR signaling promotes cisplatin resistance in oral cancer

doi: 10.1016/j.heliyon.2024.e28406

Figure Lengend Snippet: CDDP-induced drug resistance depends on p62-mTOR activity. (A) Schematics illustrating the process of inducing drug-resistant cells using CDDP. (B) Dose-dependent cell viability curves of SAS and SAScis (n = 6). (C) Downregulation of p62 and activation of the mTOR pathway in SAScis cells compared to SAS. Numbers below each band represent means ± SE of normalized intensities (n = 3). (D) Stable knockdown of p62 using shp62 and its effect on SAScis survival, in 5 or 10 μM of CDDP. shLuc, luciferase targeting shRNA control (n = 3). (E) Effect of p62 inhibition on SAScis colony formation in the presence of 5 or 10 μM of CDDP treatment. The top diagram depicts the experimental timeline. (F) CDDP induced p62 in SAScis cells. P values in (D, E) were determined using two-way ANOVA test or two-tailed unpaired t -test with equal variance (* p < 0.05, *** p < 0.001).

Article Snippet: For exogenous expression, vectors carrying full-length EGFR (referred to as “EGFR”), full-length Akt with an N-terminal myristoylation signal tag (designated as “myr-Akt”, a constitutively active form of Akt), and full-length mTOR with an N-terminal Flag tag (labeled as “Flag-mTOR”) were all purchased from Addgene (USA).

Techniques: Activity Assay, Activation Assay, Knockdown, Luciferase, shRNA, Control, Inhibition, Two Tailed Test

Journal: Heliyon

Article Title: Interplay of p62-mTORC1 and EGFR signaling promotes cisplatin resistance in oral cancer

doi: 10.1016/j.heliyon.2024.e28406

Figure Lengend Snippet: High mTOR activity correlates with poor prognosis in HNSCC patients. (A) Immunohistochemistry of OSCC tumor specimens displaying p -mTOR expression. Three representative images for high, moderate, and low levels of p -mTOR were shown. (B) Significantly higher levels of p -mTOR in OSCC tumor specimens relative to paired NCMT tissues among 34 patients. Statistical analysis was based on two-tailed paired t -test. (C) Patient survival curves comparing moderate/high (+/++) to low (−) expression levels of p -mTOR. P values were determined by log-rank test. (D) Recurrence of OSCC was associated with p -mTOR expression. P values were determined by Fisher's exact test.

Article Snippet: For exogenous expression, vectors carrying full-length EGFR (referred to as “EGFR”), full-length Akt with an N-terminal myristoylation signal tag (designated as “myr-Akt”, a constitutively active form of Akt), and full-length mTOR with an N-terminal Flag tag (labeled as “Flag-mTOR”) were all purchased from Addgene (USA).

Techniques: Activity Assay, Immunohistochemistry, Expressing, Two Tailed Test

Journal: Heliyon

Article Title: Interplay of p62-mTORC1 and EGFR signaling promotes cisplatin resistance in oral cancer

doi: 10.1016/j.heliyon.2024.e28406

Figure Lengend Snippet: mTOR activity influences stemness in OSCC cells. (A, B) Influence of mTOR inhibition using Rapamycin and Metformin on the proliferative capacity of SAS and OECM-1 cells: (A) MTT assay (n = 6); (B) trypan-blue exclusion assay (n = 3). (C) ALDH + cell populations quantified post-mTOR inhibition, using Aldefluor assay and flow cytometry. DEAB was used as an ALDH-specific inhibitor for negative control. IGF-1 was used as a positive control in (A–C). (D) Western blot analysis showing Oct4 and Nanog expression after mTOR inhibition. (E, F) Effects of mTOR overexpression on stemness markers (Oct4, Nanog, and Sox2) at mRNA (E) and protein (F) levels. Error bars indicate standard error of the mean (n = 3). P values were determined by two-tailed unpaired t -test with equal variance (* p < 0.05, *** p < 0.001). Band intensities in (D, F) were normalized and then averaged, based on at least duplicate analyses.

Article Snippet: For exogenous expression, vectors carrying full-length EGFR (referred to as “EGFR”), full-length Akt with an N-terminal myristoylation signal tag (designated as “myr-Akt”, a constitutively active form of Akt), and full-length mTOR with an N-terminal Flag tag (labeled as “Flag-mTOR”) were all purchased from Addgene (USA).

Techniques: Activity Assay, Inhibition, MTT Assay, Trypan Blue Exclusion Assay, Flow Cytometry, Negative Control, Positive Control, Western Blot, Expressing, Over Expression, Two Tailed Test

Journal: Heliyon

Article Title: Interplay of p62-mTORC1 and EGFR signaling promotes cisplatin resistance in oral cancer

doi: 10.1016/j.heliyon.2024.e28406

Figure Lengend Snippet: Inhibition of mTOR activity sensitizes SAS, SAScis, and OECM-1 cells to CDDP. Combinatorial treatment of Rapamycin or Metformin with CDDP on the viability of SAS, SAScis, and OECM-1 cells: (A) trypan-blue exclusion assay (n = 4); (B) MTT assay (n = 6).

Article Snippet: For exogenous expression, vectors carrying full-length EGFR (referred to as “EGFR”), full-length Akt with an N-terminal myristoylation signal tag (designated as “myr-Akt”, a constitutively active form of Akt), and full-length mTOR with an N-terminal Flag tag (labeled as “Flag-mTOR”) were all purchased from Addgene (USA).

Techniques: Inhibition, Activity Assay, Trypan Blue Exclusion Assay, MTT Assay