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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

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: 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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

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: Assessment of INSR overexpression. ( A ) Verification of INSR expression levels in INSR-overexpressing (IN) cells. ( B ) Relative levels of INSR expression normalized to GAPDH, based on the data in panel A. The value for INSR/GAPDH in parental HEK293 cells was set to 1.0 for comparison. 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 and ** p < 0.01. N = 3. ( C ) Subcellular localization of INSR in IN cells. Green indicates INSR, and blue indicates nuclei. Scale bars represent 20 μm.

Article Snippet: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

Techniques: Over Expression, Expressing, Comparison

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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

Techniques: Phospho-proteomics, Western Blot, 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 INSR overexpression on the Pas2r12-mediated cytosolic delivery of EGFP. ( A ) Representative confocal images showing cellular uptake of Pas2r12–EGFP in INSR-overexpressing (IN) cells. Merged images show EGFP fluorescence (green), Hoechst 33342 nuclear staining (blue), and differential interference contrast. Scale bars represent 20 μm. ( B ) Relative levels of the cytosolic delivery efficiency of EGFP in IN cells compared with parental HEK293 cells (set to 100%). 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. N = 3.

Article Snippet: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

Techniques: Over Expression, Fluorescence, Staining

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for GLUT4 and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Isolation, Immunoprecipitation, Phospho-proteomics, Western Blot, Two Tailed Test

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a. RT–PCR of Ins1 fold change in mRNA of isolated RPE from control and Ins2 KO mice. n = 8 mice used for each genotype. b. Insulin receptor was immunoprecipitated from various tissue lysates of control and Ins2 KO mice that were starved overnight. Phosphorylation status of the InsR was probed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. n = 2 mice for control vs Ins2 KO. Plots are presented as in Fig. 1.

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Immunoprecipitation, Phospho-proteomics, Western Blot

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a. Schematic of streptozotocin (STZ) treatment. b. ELISA measuring C-peptide levels in blood of control, control STZ-treated, and Ins2 KO STZ-treated mice starved mice. N = 6 mice for control and N = 4 mice for STZ-treated Control and STZ-treated Ins2 KO. *p < .05, one-way ANOVA with Dunnet’s multiple comparisons test. c. Insulin receptor (InsR) was immunoprecipitated from retina tissue lysates of control, control STZ-treated, and Ins2KO STZ-treated mice, starved overnight (left) or control and control STZ-treated mice fed ad libitum (right). Phosphorylation status of the InsR was probed using phospho-specific antibodies (Tyr1150/1151) via immunoblotting and quantified as the ratio of p-InsR to total InsR, normalized to untreated WT mice. N = 3 mice for all conditions. *p < 05, one-way ANOVA with Tukey’s multiple comparison’s test. Plots are presented as in Fig. 1.

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Immunoprecipitation, Phospho-proteomics, Western Blot

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a, Representative image of three independent experiments showing immunofluorescence analysis with anti-rhodopsin (magenta) on retina sections 2 h after light onset (peak phagocytosis time) from fed and starved (10–14 h) mice. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). RPE is outlined in white. b, Flow cytometry-based phagocytosis assay on isolated fixed and permeabilized RPE stained with rhodopsin, from fed or starved mice, obtained 2 h after light onset. Phagocytosis was measured as % of rhodopsin+ RPE within total RPE. n = 3 mice per condition. c, RT–PCR for Ins2 expression in RPE from fed mice at different times of the day. Lights on at 6:00 and lights off at 20:00. n = 8, 7, 7, 7, 3 and 3 mice used for times of 5:00, 6:00, 8:00, 15:00, 20:00 and 1:00, respectively. FC, fold change. d, InsR immunoprecipitated from retina lysates, at different times of day from overnight starved control and Ins2 KO mice, were probed using phospho-specific InsR antibodies. The same retina lysates were probed for GLUT4. Values were normalized to the average of control across all time points. n = 3 mice for each time point. e, Schematic of phagocytic receptors used in POS recognition (left). RT–PCR for Ins2 in RPE isolated from WT and MerTK KO or CD36 KO mice 2 h after light onset (right). n = 10 and 11 mice for WT versus MerTK KO and n = 6 and 5 mice for WT versus CD36 KO. f, Phagocytosis quantification using flow cytometry on isolated RPE from WT and MerTKCR mice (middle). n = 3 mice used per genotype. RT–PCR measuring Ins2 expression in isolated RPE from WT and MerTKCR (right). n = 14 and 10 mice for WT and MerTKCR, respectively. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA (c), two-way ANOVA with Tukey’s multiple comparisons test (d), unpaired two-tailed t-test (e,f RT–PCR) and paired two-tailed t-test (f phagocytosis quantification).

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Immunofluorescence, Flow Cytometry, Phagocytosis Assay, Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation, Control, Two Tailed Test

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a. Quantification of OS phagocytosis by the RPE 2 hours after light onset using immunohistochemistry. Quantification of phagocytosis was measured by the amount of Rhodopsin immunoreactivity in the RPE divided by pixels and presented as phagosomes per area on the y-axis (left). Quantification of the number of phagosomes in RPE was done by counting Rhodopsin puncta in the RPE (right). n = 3 mice used for each condition. *p ≤ .05 paired two-tailed t-test. b. Western blot against Rhodopsin on isolated RPE protein lysates from fed and starved mice at 8 am and 10 am (left). Right panel is quantification of the blot to evaluate POS degradation showing 10 am band intensity as a percent of 8 am (peak phagocytosis) band intensity (right). n = 2 mice used for each time point. c. Insulin receptor was immunoprecipitated from lysates of retina from control and MerTK KO mice that were starved overnight. The lysates were probed for InsR phosphorylation or GLUT4 levels by immunoblotting. C-peptide 2 levels were determined by ELISA. N = 6 mice for each condition. *p < .05 paired two-tailed t-test. d. Schematic of WT and cleavage-resistant ‘gain of function’ MerTKCR mice with altered cleavage sites indicated (left). e. Phagocytosis quantification of ingested photoreceptor outer segments using flow cytometry on isolated RPE stained with antibody against rhodopsin from Control and Ins2 KO mice two hours after light onset. n = 7 mice used for each genotype. Plots are presented as in Fig. 1.

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Immunohistochemistry, Two Tailed Test, Western Blot, Isolation, Immunoprecipitation, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining