Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex-mediated stimulation on INSR/IGF1R and ERK1/2. HEK293 cells were pretreated with dimethyl sulfoxide (linsitinib [−]) or with linsitinib (linsitinib [+]) and subsequently stimulated with Pas2r12, Pas2r12–EGFP, Pas2r12–IgG, or insulin for 2 min ( A – C ) or 10 min ( D – F ). Panels A and D show representative Western blot images. Panels B and E display the phosphorylation levels of INSR/IGF1R (pINSR/pIGF1R); panels C and F show the phosphorylation levels of ERK1/2 (pERK1/2); all values were normalized to GAPDH. Phosphorylation levels were analyzed and compared with the solvent control (DMEM only, linsitinib [−]) using Student’s t -test. For each treatment condition, linsitinib (+) was compared with the corresponding linsitinib (−) condition. Statistically significant differences compared with the control are indicated on the bar graphs; significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Statistical comparisons were performed against control cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05 and ** p < 0.01. N = 4.

Article Snippet: The cells were incubated for 24 h; fixed with 4% paraformaldehyde; permeabilized with phosphate-buffered saline containing 0.2% Triton X-100; blocked with Blocking One at room temperature; incubated overnight at 4 °C with primary antibodies against IGF1R (3B7, sc-462; Santa Cruz Biotechnology) or INSR (CT3, sc-57342; Santa Cruz Biotechnology) diluted 1:200 in Immuno Shot 1 solution; washed; and incubated at room temperature for 45 min with secondary antibody Alexa Fluor 488-labeled goat antimouse IgG and Hoechst 33342 diluted 1:500 in Immuno Shot 2 solution.

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

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R and INSR knockdown on the Pas2r12-mediated cytosolic delivery of EGFP. Western blot analyses ( A – C ) and confocal laser scanning microscopy images ( D , E ). Graphs B and C show IGF1R and INSR expression levels, respectively, normalized to GAPDH. Cells analyzed with confocal laser scanning microscopy in panel A were analyzed by Western blot ( A – C ). ( D ) Pas2r12-mediated cytosolic delivery of EGFP in knockdown cells, with EGFP fluorescence shown in green, and ( E ) percentage of cells exhibiting cytosolic EGFP delivery. Scale bars represent 20 μm. Statistical comparisons were performed against siNC cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 4.

Article Snippet: The cells were incubated for 24 h; fixed with 4% paraformaldehyde; permeabilized with phosphate-buffered saline containing 0.2% Triton X-100; blocked with Blocking One at room temperature; incubated overnight at 4 °C with primary antibodies against IGF1R (3B7, sc-462; Santa Cruz Biotechnology) or INSR (CT3, sc-57342; Santa Cruz Biotechnology) diluted 1:200 in Immuno Shot 1 solution; washed; and incubated at room temperature for 45 min with secondary antibody Alexa Fluor 488-labeled goat antimouse IgG and Hoechst 33342 diluted 1:500 in Immuno Shot 2 solution.

Techniques: Knockdown, Western Blot, Confocal Laser Scanning Microscopy, Expressing, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Assessment of IGF1R overexpression. ( A ) Verification of IGF1R expression levels in HEKI cells. ( B ) Relative levels of IGF1R expression normalized to GAPDH based on the data in panel A. Values for IGF1R/GAPDH are expressed compared with parental HEK293 cells (set to 1). Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3. ( C ) Subcellular localization of IGF1R is shown in green; nuclei were counterstained with Hoechst 33342 (blue). Scale bars represent 20 μm.

Article Snippet: The cells were incubated for 24 h; fixed with 4% paraformaldehyde; permeabilized with phosphate-buffered saline containing 0.2% Triton X-100; blocked with Blocking One at room temperature; incubated overnight at 4 °C with primary antibodies against IGF1R (3B7, sc-462; Santa Cruz Biotechnology) or INSR (CT3, sc-57342; Santa Cruz Biotechnology) diluted 1:200 in Immuno Shot 1 solution; washed; and incubated at room temperature for 45 min with secondary antibody Alexa Fluor 488-labeled goat antimouse IgG and Hoechst 33342 diluted 1:500 in Immuno Shot 2 solution.

Techniques: Over Expression, Expressing

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in IGF1R-overexpressing cells. ( A ) HEKI#66 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Level of phosphorylated INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R, normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated HEKI#66 control (linsitinib [−]), and, for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3.

Article Snippet: The cells were incubated for 24 h; fixed with 4% paraformaldehyde; permeabilized with phosphate-buffered saline containing 0.2% Triton X-100; blocked with Blocking One at room temperature; incubated overnight at 4 °C with primary antibodies against IGF1R (3B7, sc-462; Santa Cruz Biotechnology) or INSR (CT3, sc-57342; Santa Cruz Biotechnology) diluted 1:200 in Immuno Shot 1 solution; washed; and incubated at room temperature for 45 min with secondary antibody Alexa Fluor 488-labeled goat antimouse IgG and Hoechst 33342 diluted 1:500 in Immuno Shot 2 solution.

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

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R overexpression on the Pas2r12-mediated cytosolic delivery of EGFP. ( A ) Representative confocal images showing the cellular uptake of Pas2r12–EGFP in IGF1R-overexpressing (HEKI) cells. Scale bars represent 20 μm. ( B ) Relative levels of the cytosolic delivery efficiency of EGFP in HEKI cells compared with parental HEK293 cells (set to 100%). Merged images show EGFP fluorescence (green) and differential interference contrast. Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). ** p < 0.01. N = 3.

Article Snippet: The cells were incubated for 24 h; fixed with 4% paraformaldehyde; permeabilized with phosphate-buffered saline containing 0.2% Triton X-100; blocked with Blocking One at room temperature; incubated overnight at 4 °C with primary antibodies against IGF1R (3B7, sc-462; Santa Cruz Biotechnology) or INSR (CT3, sc-57342; Santa Cruz Biotechnology) diluted 1:200 in Immuno Shot 1 solution; washed; and incubated at room temperature for 45 min with secondary antibody Alexa Fluor 488-labeled goat antimouse IgG and Hoechst 33342 diluted 1:500 in Immuno Shot 2 solution.

Techniques: Over Expression, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in INSR-overexpressing cells. ( A ) IN#1 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Phosphorylation of INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R were normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated IN#1 control (linsitinib [−]), and for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001. N = 3.

Article Snippet: The cells were incubated for 24 h; fixed with 4% paraformaldehyde; permeabilized with phosphate-buffered saline containing 0.2% Triton X-100; blocked with Blocking One at room temperature; incubated overnight at 4 °C with primary antibodies against IGF1R (3B7, sc-462; Santa Cruz Biotechnology) or INSR (CT3, sc-57342; Santa Cruz Biotechnology) diluted 1:200 in Immuno Shot 1 solution; washed; and incubated at room temperature for 45 min with secondary antibody Alexa Fluor 488-labeled goat antimouse IgG and Hoechst 33342 diluted 1:500 in Immuno Shot 2 solution.

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