Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Protein S variants generated by site-directed mutagenesis

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Generated

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of APC and protein S on thrombin generation. Thrombin generation was performed in protein S–deficient plasma with 100nM inhibitory antibodies against TFPI. Up to 10nM APC had no effect on thrombin generation in the absence of protein S. All concentrations generate lines that are superimposable (A). After addition of 120nM protein S (at 0-10nM APC), an APC dose-dependent effect was observed (B). The top single line represents 0 to 10nM APC in the absence of protein S. Protein S in the presence of no or 2.5nM APC generated lines that were superimposable. Conditions used are noted adjacent to the peaks to which they refer. The anticoagulant effect of 10nM APC and 120nM protein S was inhibited by polyclonal antibodies against protein S (C) or against protein C (D). PS indicates protein S; PC, protein C. Representative experiments are shown (n = 3).

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Generated

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Screening of protein S variants for APC cofactor activity. The APC cofactor activity of protein S was evaluated at 16nM APC and 100nM protein S by CAT. The peak height in the absence of protein S was set to 100%. A high concentration of APC, leading to almost complete inhibition of thrombin generation with 100nM WT protein S, was chosen specifically for screening purposes as this allows widening of the assay window at which mutants with reduced APC cofactor activity are visualized. Results were confirmed by evaluating protein S (at 60 and 90nM) cofactor activity toward 4 or 9nM APC.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Activity Assay, Concentration Assay, Inhibition

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of WT protein S, D95A, D95N, D78A, and Q79A variants on thrombin generation. Thrombin generation was measured in protein S–deficient plasma supplemented with 9nM APC, 100nM antibodies against TFPI, and 0 to 120nM WT protein S (A), protein S D95A (B), protein S D95N (C), or 90nM purified WT (dashed line) or purified protein S D95A (dotted line; D). Protein S concentrations are positioned adjacent to the peaks to which they refer. The cofactor activity of 60nM WT protein S and protein S variants D95A, D78A, and Q79A was compared at 9nM APC (E). Typical experiments are shown (n = 3). Whereas the cofactor activity of WT protein S is highly dependent on the APC concentration used (Figure 1B), that of protein S D95A is not, explaining the difference in fold activity between WT protein S and protein S D95A in Figures 2 and ​and3.3. Dose-response data from titrations with WT protein S, protein S D95A, and protein S D95N in the presence of 9nM APC are shown in panel F (data are expressed as mean ± SD of 2 independent experiments performed in duplicate). Inset in panel B shows recognition of WT protein S and protein S D95A in media by polyclonal antibodies and a monoclonal antibody recognizing only γ-carboxylated Gla domains. Inset in panel D shows the SeeBlue-prestained marker, plasma-purified protein S from Enzyme Research Laboratories Ltd (lane 1), purified recombinant WT protein S (lane 2), and purified protein S D95A (lane 3) visualized with silver staining.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Purification, Activity Assay, Concentration Assay, Marker, Recombinant, Silver Staining

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Binding of protein S to phospholipid surfaces. Protein S (0-120nM) was incubated in a plate coated with 25 μg/mL phospholipids. Bound protein S was detected with an HRP-conjugated polyclonal antibody against protein S. A representative experiment is shown. The apparent Kd values, 5.69 ± 1.24 and 9.54 ± 2.26nM for WT protein S and protein S D95A, respectively, were obtained by calculating the mean ± SD of 3 independent experiments performed in duplicate. PL indicates phospholipids.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Binding Assay, Incubation

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Binding of protein S to phospholipids and domain-specific monoclonal antibodies

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Binding Assay, Mutagenesis

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Protein S enhancement of APC-mediated cleavage of FVa in Arg306. Protein S (0-120nM) in the presence of 0.5nM APC was incubated with 0.8nM FVa R506Q/R679Q in the presence of phospholipids for 10 minutes. The remaining FVa actvity was measured with a prothrombinase assay. Results are plotted as mean ± SD from 3 independent experiments performed in duplicate (A). A time course experiment was performed to calculate the apparent pseudo–first-order rate constants of WT protein S and protein S D95A. It is observed that approximately 6-fold more APC is needed in the presence of protein S D95A to obtain a similar amount of APC-mediated FVa R506Q/R679Q inactivation as with WT protein S (B).

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Incubation

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Location of Asp78, Gln79, and Asp95 within the protein S Gla-TSR-EGF1 model. Domains are labeled on the right side of the model. Residues mutated in the GLA2 variant, Asp78, Gln79, and Asp95, are in light gray on the left side surface model. Residues Asp78, Gln79, and Asp95 are highlighted by the box to show their proximal spatial location. The model is taken from Villoutreix et al.35

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Labeling, Variant Assay

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 2. Effect of the TRPC5 activator AM237 on acetylcholine (ACh)-induced relaxation and contraction in the normal-fat diet (NFD) mouse aorta. (a) TRPC5 protein expression in mouse aortic endothelial cells (MAoECs) from NFD ( n = 10) and high-fat diet (HFD)-induced obese ( n = 11) mice. (b) Co-immunostaining of TRPC5 and CD31 in mouse aortic rings (scale bar, 50 𝜇m). (c–e) Representative traces (c) and summary data (d, e) of phenylephrine (Phe)-precontracted ACh-induced relaxation followed by contraction at high concentrations in NFD ( n = 7) and AM237 (100 nmol/L)-pretreated NFD ( n = 6) aortas. (f–h) Original recordings (f) and data summary (g, h) showing the effect of AM237 (100 nmol/L) on ACh-induced contraction in the NFD aorta with or without endothelium ( + endo CTL, n = 5; + endo AM237, n = 5; –endo CTL, n = 4; –endo AM237, n = 4). Mean ± SEM; A, ∗ P < 0.05 vs NFD, Student’s unpaired two-tailed t test; d, ∗ P < 0.05, NS, no significant difference vs NFD, two-way ANOVA followed by Bonferroni test; e, ∗ P < 0.05, NS, no significant difference vs CTL, Student’s unpaired two-tailed t test; g, ∗ P < 0.05 vs + endo CTL, two-way ANOVA followed by Bonferroni test; h, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237, one-way ANOVA followed by Turkey’s multiple comparisons test.

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Expressing, Immunostaining, Two Tailed Test

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 3. Effect of TRPC5 inhibition on acetylcholine (ACh)-induced vaso- constriction in the high-fat diet (HFD)-induced obese mouse aorta. (a, b) Representative traces (a) and data summary (b) showing ACh-induced contrac- tion is attenuated by the TRPC5 inhibitor clemizole (20 𝜇mol/L), knockout of TRPC5, and the removal of endothelium (CTL, n = 5; clemizole, n = 6; TRPC5 − / − , n = 5; CTL(–Endo), n = 6; mean ± SEM; b, left, ∗ P < 0.05 vs CTL, two-way ANOVA followed by Bonferroni test; right, ∗ P < 0.05 vs CTL, one-way ANOVA followed by Dunnett’s multiple comparisons test).

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Inhibition, Knock-Out

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 4. TRPC5 regulates contractions via cytosolic phospholipase A 2 (cPLA 2 ) in the high-fat diet (HFD)-induced obese mouse aorta. (a) Western blots and analysis of cPLA 2 and phosphorylated cPLA 2 (p-cPLA 2 ) expression in normal-fat diet (NFD, n = 15), AM237 (100 nmol/L)-treated NFD ( n = 9), HFD-induced obese ( n = 14), clemizole (20 𝜇mol/L)-treated HFD ( n = 7), and TRPC5 − / − -HFD ( n = 5) mouse aortic endothelial cells (MAoECs). (b) Dose-dependent effect of AM237 on p-cPLA 2 levels in NFD MAoECs. AM237 (nmol/L), 0, n = 16; 50, n = 11; 100, n = 16; 200, n = 10. (c) Dose-dependent effect of clemizole on p-cPLA 2 levels in HFD MAoECs ( n = 5). (d) Representative fluorescence images of the Bis-BODIPY TM FL C 11 -PC stained en-face aorta and analysis of PLA 2 activity in endothelial cells of the NFD ( n = 9), AM237 (100 nmol/L)-pretreated NFD ( n = 7), HFD ( n = 17), clemizole (20 𝜇mol/L)-treated HFD ( n = 9), and TRPC5 − / − HFD ( n = 12) mouse aorta (scale bars, 10 𝜇m). (e) Acetylcholine (ACh)-induced contraction in HFD ( n = 4) and MAFP (10 𝜇mol/L)-treated HFD ( n = 6) mouse aortic rings. (f) ACh-induced contraction in the NFD ( n = 5), AM237 (100 nmol/L)-pretreated NFD ( n = 5), and AM237 (100 nmol/L) + MAFP (30 𝜇mol/L)-pretreated NFD ( n = 6) mouse aorta. Mean ± SEM; a, ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, NS, no significant difference, Kruskal-Wallis and Dunn’s post hoc non-parametric test (p-cPLA 2 ) and one-way ANOVA followed by Turkey’s multiple comparisons test (cPLA 2 ); b, ∗ P < 0.05, NS, no significant difference vs no AM237, one-way ANOVA followed by Dunnett’s multiple comparisons test; c, ∗ P < 0.05, NS, no significant difference vs no clemizole, one-way ANOVA followed by Dunnett’s multiple comparisons test; d, ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, one-way ANOVA followed by Turkey’s multiple comparisons test; e, ∗ P < 0.05 vs CTL, two-way ANOVA followed by Bonferroni test (left) and Student’s unpaired two-tailed t test (right); f, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Turkey’s multiple comparisons test (right).

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Western Blot, Expressing, Staining, Activity Assay, Two Tailed Test

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 6. Role of COX-2 in TRPC5-regulated vasoconstriction in the mouse aorta. (a) Western blots and analysis of COX-1 and COX-2 expression in normal-fat diet (NFD, n = 5), AM237 (100 nmol/L) pre-treated NFD ( n = 5), high-fat diet (HFD, n = 5), clemizole (20 𝜇mol/L) pre-treated HFD ( n = 5), and TRPC5 − / − HFD ( n = 5) mouse aortic endothelial cells (MAoECs). (b) Effect of the COX inhibitors NS-398 (3 𝜇mol/L) ( n = 5), VAS-2870 (30 𝜇mol/L) ( n = 3), and indomethacin (indo, 1 𝜇mol/L, n = 3) on acetylcholine (ACh)-induced contraction in the HFD mouse aorta (CTL, n = 3). (c) Effect of the COX-2 inhibitor NS-398 (3 𝜇mol/L) on ACh-induced contraction in the AM237 (100 nmol/L)-pretreated NFD mouse aorta (CTL, n = 5; AM237, n = 7; AM237 + NS-398, n = 7). Mean ± SEM; a, ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, NS, no significant difference, Kruskal-Wallis and Dunn’s post hoc non-parametric test (COX-1) and one-way ANOVA followed by Turkey’s multiple comparisons test (COX-2); b, ∗ P < 0.05, NS, no significant difference vs CTL, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Dunnett’s multiple comparisons test (right); c, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Turkey’s multiple comparisons test (right) .

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Western Blot, Expressing

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 5. TRPC5 contributes to acetylcholine (ACh)-induced Ca 2+ entry into endothelial cells of high-fat diet (HFD)-induced obese mouse aortas. (a, b) Representative traces (a) and data summary (b) showing an ACh (10 𝜇mol/L)- induced [Ca 2 + ] i rise in aortic endothelial cells from normal-fat diet (NFD) and HFD mice (NFD, n = 7; NFD-AM237 (100 nmol/L), n = 5; HFD, n = 5; HFD- clemizole (20 𝜇mol/L), n = 6; HFD-TRPC5 − / − , n = 6; mean ± SEM; ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, one-way ANOVA followed by Turkey’s multiple com- parisons test).

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques:

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 7. PGF 2 𝜶and PGE 2 are involved in TRPC5-related contraction in the high-fat diet (HFD)-induced obese mouse aorta. (a) Enzyme immunoassay (EIA) showing the PGF 2 𝛼( n = 7), PGE 2 ( n = 7), PGD 2 ( n = 5), PGI 2 ( n = 5), and 8-isoprostanes ( n = 6) levels in normal-fat diet (NFD) and HFD mouse aortic rings after exposure to acetylcholine (ACh, 10 𝜇mol/L). (b) Effect of clemizole (20 𝜇mol/L), MAFP (10 𝜇mol/L), NS-398 (3 𝜇mol/L), –endo, and knockout of TRPC5 − / − on ACh-induced PGF 2 𝛼and PGE 2 release in the ACh-stimulated HFD mouse aorta (PGF 2 𝛼, CTL, n = 7; clemizole, n = 5; TRPC5 − / − , n = 5; MAFP, n = 6; NS-398, n = 6; –endo, n = 6; PGE 2 , CTL, n = 7; clemizole, n = 5; TRPC5 − / − , n = 5; MAFP, n = 5; NS-398, n = 5; –endo, n = 5; (c) Effects of AM237 (100 nmol/L), MAFP (10 𝜇mol/L), and NS-398 (3 𝜇mol/L) treatment and the removal of endothelium (–Endo) on EIA for PGF 2 𝛼and PGE 2 in the NFD mouse aorta stimulated with ACh (10 𝜇mol/L) (PGF 2 𝛼, CTL, n = 7; AM237, n = 4; MAFP, n = 6; NS-398, n = 4; –endo, n = 4; PGE 2 , CTL, n = 10; AM237, n = 5; MAFP, n = 5; NS-398, n = 5; –endo, n = 7). (d) Schematic of the mechanism of TRPC5 regulation of endothelium-dependent contraction in the DIO mouse aorta. Mean ± SEM; a, ∗ P < 0.05, NS, no significant difference vs NFD, Student’s unpaired two-tailed t test; b, ∗ P < 0.05 vs CTL, one-way ANOVA followed by Dunnett’s multiple comparisons test; c, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237 group, one-way ANOVA followed by Turkey’s multiple comparisons test.

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Enzyme-linked Immunosorbent Assay, Knock-Out, Two Tailed Test

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 3. Ferroptosis-inducing and immunostimulatory capabilities of the biomimetic hydrogel. (a) Schematic diagram for the tumor cell/immune cell co- incubation system in transwell plates. The tumor cells (B16F10, 4T1) or immune cells were inoculated in the bottom chamber of the 24-well transwell culture plate, while the hydrogel soaking solution was placed in the upper chamber. (b) Changes of GPX4 activity in B16F10 cells after different treatments. (I) Control, (II) Gel, (III) RSL3, (IV) Gel@RSL3 (n = 4). (c) Flow cytometric analysis on the lipid ROS levels in B16F10 cells after different treatments. (d) CLSM imaging of lipid ROS generation in B16F10 cells after different treatments. Higher green fluorescence intensity indicates greater lipid ROS production. (e) Quantitative fluorescence analysis of lipid ROS levels in panel D (n = 4). (f) Flow cytometric analysis on the hydrogel-mediated ferroptosis levels of B16F10 cells after different treatments. (g) ATP levels in the supernatants of cell culture after different treatments. (I) Control, (II) Gel, (III) RSL3, (IV) Gel@RSL3 (n = 4). (h) CLSM imaging of CRT expression in B16F10 cells after different treatments. Stronger red fluorescence indicates higher expression levels. (i) Quantitative fluorescence analysis of CRT expression levels in panel H (n = 4). (j) CLSM imaging of cellular HMGB1 abundance after different treatments. Lower red fluorescence indicates greater HMGB1 release into the extracellular compartment. (k) Quantitative fluorescence analysis of HMGB1 release in panel J (n = 4). (l) Flow cytometric analysis on the treatment-induced maturation of BMDCs. (m) Flow cytometric analysis on the activation status of macrophages by monitoring the CD80 expression levels. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: Incubation, Activity Assay, Control, Imaging, Cell Culture, Expressing, Activation Assay

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 4. Gel@RSL3 + GM-CSF + aPD-L1 activates immune response in vitro. (a-d) Flow cytometric analysis of the activation status of DCs (CD11c + /MHC II + ), M1 macrophages (F4/80 + /CD80 + ) and T cells (CD8 + /CD3 + and CD8a + /IFN- 𝛾+ ) in the co-incubation system of splenic immune cells and B16F10 cells after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3 and (V) Gel@RSL3 + GM-CSF (n = 4). (e) Secretion levels of immunostimulatory cytokines including IFN- 𝛾, TNF- 𝛼and antitumor effector molecule GzmB in the supernatant from the co-culture system after different treatments (n = 4). (f) PD-L1 expression in tumor cells with after the hydrogel-mediated ferroptosis-immunotherapy. Group set-up for panel e-f: (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3 and (V) Gel@RSL3 + GM- CSF). (g-h) Flow cytometric analysis of the expression levels of effector T cell marker CD4 + /CD8 + and CD8a + /IFN- 𝛾+ in T cells co-incubated with B16F10 cells after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD-L1. (i) Secretion levels of immunostimulatory cytokines including IFN- 𝛾, TNF- 𝛼and antitumor effector molecule GzmB in the supernatant from the co-culture system after different treatments (n = 4). (j) Evaluation on the GSH levels in B16F10 cells after different treatments (n = 4). (k) Western blot analysis of the expression level of CRT, HMGB1 and SLC7A11 in different groups. (l) Flow cytometric analysis on the lipid ROS levels in B16F10 cells after different treatments. (m) MDA levels in B16F10 cells after different treatments (n = 4). Group set-up for panel I-M: (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD- L1). (n) Flow cytometric analysis on the death rate of B16F10 cells after different treatments, including (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD-L1. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01, ∗ ∗ ∗ indicates significance at P < 0.001, ∗ ∗ ∗ ∗ indicates significance at P < 0.0001.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: In Vitro, Activation Assay, Incubation, Control, Co-Culture Assay, Expressing, Marker, Western Blot

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 5. Antitumor effect of biomimetic hydrogel in vivo. (a) Schematic illustration of the treatment scheme of the B16F10-luc tumor-bearing mice (n = 7). (b) Treatment procedures on the tumor-bearing mice. (c) In vivo bioluminescence images of B16F10-luc tumor-bearing mice throughout the treatment period with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. (d) Tumor size changes during the incubation period after different treatments. (e) Survival analysis of mice after different treatments. (f) Body weight changes after treatment with different samples. (g) Evaluation on the GPX4 activity in tumor tissues after different treatments (n = 4). (h) Western blot analysis on the expression of CRT, HMGB1 and SLC7A11 in B16F10-luc tumors after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. (i) MDA levels in tumor tissue after different treatments (n = 4). (j) H&E and TUNEL staining of tumor tissue samples after treatment (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01, ∗ ∗ ∗ indicates significance at P < 0.001.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: In Vivo, Control, Incubation, Activity Assay, Western Blot, Expressing, TUNEL Assay, Staining

Journal: Fundamental Research

Article Title: O-GlcNAcylation of YTHDF2 antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma

doi: 10.1016/j.fmre.2024.07.003

Figure Lengend Snippet: Fig. 2 O-GlcNAcylation of YTHDF2 at Thr-49 antagonizes ERK-dependent

Article Snippet: The following primary antibodies were used for immunoblot: anti-Myc (PTM Bio, #PTM-5390, 1:3000), anti-c-Myc (ProteinTech, #10828-1-AP, 1:1000), anti-YTHDF2 (proteinTech, #24744-1-AP, 1:1000), anti-Flag (Sigma, #F1084, 1:1000), RL2 (Abcam, #AB2739, 1:1000), anti-GST (Gene Script, #A00865, 1:1000), anti-HA (Bethyl, #A190-108A, 1:1000), anti-OGT (Santa Cruz Biotechnology, #sc-74546, 1:1000), anti-β-actin (Sigma, #A5441, 1:1000), anti-Ubiquitin (PTM Bio, #PTM-1106RM, 1:1000) , anti-AXIN (ProteinTech,#16541-1-AP, 1:1000) and anti-YTHDF2-pS39 antibodies were generated using the sequence PYLpSPQAR by Dia-An Biotech, Inc. Peroxidase-conjugated secondary antibodies were from JacksonImmuno Research.

Techniques:

Journal: Fundamental Research

Article Title: O-GlcNAcylation of YTHDF2 antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma

doi: 10.1016/j.fmre.2024.07.003

Figure Lengend Snippet: Fig. 3 YTHDF2 O-GlcNAcylation promotes ubiquitination. (a) 293T cells were

Article Snippet: The following primary antibodies were used for immunoblot: anti-Myc (PTM Bio, #PTM-5390, 1:3000), anti-c-Myc (ProteinTech, #10828-1-AP, 1:1000), anti-YTHDF2 (proteinTech, #24744-1-AP, 1:1000), anti-Flag (Sigma, #F1084, 1:1000), RL2 (Abcam, #AB2739, 1:1000), anti-GST (Gene Script, #A00865, 1:1000), anti-HA (Bethyl, #A190-108A, 1:1000), anti-OGT (Santa Cruz Biotechnology, #sc-74546, 1:1000), anti-β-actin (Sigma, #A5441, 1:1000), anti-Ubiquitin (PTM Bio, #PTM-1106RM, 1:1000) , anti-AXIN (ProteinTech,#16541-1-AP, 1:1000) and anti-YTHDF2-pS39 antibodies were generated using the sequence PYLpSPQAR by Dia-An Biotech, Inc. Peroxidase-conjugated secondary antibodies were from JacksonImmuno Research.

Techniques: Ubiquitin Proteomics

Journal: Fundamental Research

Article Title: O-GlcNAcylation of YTHDF2 antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma

doi: 10.1016/j.fmre.2024.07.003

Figure Lengend Snippet: Fig. 4 O-GlcNAcylation of YTHDF2 downregulates c-Myc in H1299 and A549 lung

Article Snippet: The following primary antibodies were used for immunoblot: anti-Myc (PTM Bio, #PTM-5390, 1:3000), anti-c-Myc (ProteinTech, #10828-1-AP, 1:1000), anti-YTHDF2 (proteinTech, #24744-1-AP, 1:1000), anti-Flag (Sigma, #F1084, 1:1000), RL2 (Abcam, #AB2739, 1:1000), anti-GST (Gene Script, #A00865, 1:1000), anti-HA (Bethyl, #A190-108A, 1:1000), anti-OGT (Santa Cruz Biotechnology, #sc-74546, 1:1000), anti-β-actin (Sigma, #A5441, 1:1000), anti-Ubiquitin (PTM Bio, #PTM-1106RM, 1:1000) , anti-AXIN (ProteinTech,#16541-1-AP, 1:1000) and anti-YTHDF2-pS39 antibodies were generated using the sequence PYLpSPQAR by Dia-An Biotech, Inc. Peroxidase-conjugated secondary antibodies were from JacksonImmuno Research.

Techniques:

Journal: Fundamental Research

Article Title: O-GlcNAcylation of YTHDF2 antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma

doi: 10.1016/j.fmre.2024.07.003

Figure Lengend Snippet: Fig. 5 O-GlcNAcylation of YTHDF2 regulates the metastatic capacity of H1299 and

Article Snippet: The following primary antibodies were used for immunoblot: anti-Myc (PTM Bio, #PTM-5390, 1:3000), anti-c-Myc (ProteinTech, #10828-1-AP, 1:1000), anti-YTHDF2 (proteinTech, #24744-1-AP, 1:1000), anti-Flag (Sigma, #F1084, 1:1000), RL2 (Abcam, #AB2739, 1:1000), anti-GST (Gene Script, #A00865, 1:1000), anti-HA (Bethyl, #A190-108A, 1:1000), anti-OGT (Santa Cruz Biotechnology, #sc-74546, 1:1000), anti-β-actin (Sigma, #A5441, 1:1000), anti-Ubiquitin (PTM Bio, #PTM-1106RM, 1:1000) , anti-AXIN (ProteinTech,#16541-1-AP, 1:1000) and anti-YTHDF2-pS39 antibodies were generated using the sequence PYLpSPQAR by Dia-An Biotech, Inc. Peroxidase-conjugated secondary antibodies were from JacksonImmuno Research.

Techniques:

Journal: Fundamental Research

Article Title: O-GlcNAcylation of YTHDF2 antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma

doi: 10.1016/j.fmre.2024.07.003

Figure Lengend Snippet: Fig. 6 YTHDF2 O-GlcNAcylation inhibits lung cancer. (a-c) Xenografts in nude mice.

Article Snippet: The following primary antibodies were used for immunoblot: anti-Myc (PTM Bio, #PTM-5390, 1:3000), anti-c-Myc (ProteinTech, #10828-1-AP, 1:1000), anti-YTHDF2 (proteinTech, #24744-1-AP, 1:1000), anti-Flag (Sigma, #F1084, 1:1000), RL2 (Abcam, #AB2739, 1:1000), anti-GST (Gene Script, #A00865, 1:1000), anti-HA (Bethyl, #A190-108A, 1:1000), anti-OGT (Santa Cruz Biotechnology, #sc-74546, 1:1000), anti-β-actin (Sigma, #A5441, 1:1000), anti-Ubiquitin (PTM Bio, #PTM-1106RM, 1:1000) , anti-AXIN (ProteinTech,#16541-1-AP, 1:1000) and anti-YTHDF2-pS39 antibodies were generated using the sequence PYLpSPQAR by Dia-An Biotech, Inc. Peroxidase-conjugated secondary antibodies were from JacksonImmuno Research.

Techniques: