Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: The hypothetical pathway of this study. All the important molecular and pathways were indicated. Briefly, AMPK is a preferential substrate of Compound C than Akt in normal conditions. When TiO₂ NZs were added to cells, they inhibited the expression of AMPK and mTOR proteins, then with extra addition of Compound C or phenformin turned to inhibit the candidate target of Akt, and exerted contrary effects on regulating autophagy

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Expressing

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Contrasted effects of Compound C and phenformin on TiO₂ NZs–induced fetal growth impairment and placental cell energy/autophagy responses. ( A ) Representative fetal images and corresponding average fetal length and average fetal weight in the control, TiO₂ NZs, TiO₂ NZs + phenformin, and TiO₂ NZs + Compound C groups. ( B ) Immunofluorescence analysis of autophagy levels in HTR cells following exposure to 100 µg/mL TiO₂ NZs or 100 µg/mL bulk-TiO₂. ( C ) Corresponding cellular ATP levels measured after exposure to TiO₂ NZs and bulk-TiO₂. ( D - E ) Immunofluorescence analysis of autophagy levels following exposure to 100 µg/mL TiO₂ NZs or 100 µg/mL b-TiO₂, either alone or in combination with Compound C/phenformin. Cell nuclei were stained blue with DAPI, and autophagosomes were labeled red using CY3-conjugated secondary antibodies.Scale bar = 20 μm. ( F ) Uptake and location of TiO₂ NZs in HTR-8/Svneo (HTR) cells observed by TEM after cells were treated with 100 µg/mL of TiO₂ NZs. The TiO₂ NZs were indicated by red arrows. ( G ) The morphology of mitochondria observed by TEM after cells were exposed to 100 µg/mL of TiO₂ NZs. Normal or swollen mitochondria were indicated by red arrows, respectively. Data are expressed as mean ± SD. A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( A ). One-way ANOVA was used for ( C ). ** P < 0.01, *** P < 0.001 vs. control group; $ P < 0.05 TiO₂ NZs + phenformin vs. TiO₂ NZs group; # P < 0.05 TiO₂ NZs + Compound C vs. TiO₂ NZs group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Control, Immunofluorescence, Staining, Labeling, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Autophagosome accumulation and its modulation by Compound C and phenformin in placental cells. ( A - C ) Cell autophagy levels were assessed by immunofluorescence and observed using a confocal microscope after cells received corresponding treatments. ( D , E ) Cell autophagy levels were assessed by immunofluorescence after HTR cells were treated with Compound C and phenformin in the absence of TiO₂ NZs. ( F ) Autophagy levels were examined after Compound C was added to the starvation-induced autophagy group. Cell nuclei were stained with DAPI (blue), and autophagosomes were labeled with CY3-conjugated secondary antibodies (red). Scale bar = 100 μm

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Immunofluorescence, Microscopy, Staining, Labeling

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Compound C and phenformin differentially regulate AMPK/mTOR signaling and exhibit distinct binding modes to AKT1.( A ) Western blot analysis of AMPK, p-AMPK, mTOR, and p-mTOR expression levels in cells treated with 100 µg/mL TiO₂ NZs. ( B ) Western blot analysis of AMPK and p-AMPK expression levels in cells treated with TiO₂ NZs, either alone or in combination with Compound C or phenformin. ( C , D ) Western blot analysis of AMPK and p-AMPK expression levels in cells treated with Compound C ( C ) and phenformin ( D ). GAPDH served as a loading control. Molecular weights (kDa) are indicated beside the bands.Quantitative densitometric analysis corresponding to the western blots in ( A – D ) was performed using ImageJ software. All bands were normalized to GAPDH expression, and the tests were repeated three times. ( E ) Chemical structures of ATP-competitive AKT1 inhibitors. ( F ) Chemical structures of allosteric (auto-inhibitory) AKT1 inhibitors. ( G ) Docking conformation of Compound C at the interface of the AKT1 dimer, with AKT1a and AKT1b colored red and blue, respectively. ( H ) Docking conformation of the AKT inhibitor (4EJN) in AKT1. ( I ) Docking conformation of phenformin within the AKT1 binding site. ( J ) Superimposed binding modes of Compound C and phenformin in the AKT1 dimer. ( K, L ) Molecular docking results showing the overlapping binding region (blue frame) of Compound C and phenformin, with the upper interaction site specific to Compound C. ( M ) Docking scores (kcal/mol) of phenformin and Compound C bound to AKT1, where more negative values indicate stronger binding affinity.The data are presented as mean ± SD. An unpaired two-tailed t -test was used for ( A , C and D ). A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for (B). ** P < 0.01, *** P < 0.001 vs. control group; ### P < 0.001 vs. TiO₂ NZs group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Binding Assay, Western Blot, Expressing, Control, Software, Two Tailed Test, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Akt-targeted autophagy modulation emerges as AMPK declines in TiO₂ NZs-treated cells. ( A ) Western blot analysis of Akt and p-Akt protein levels in cells treated with 100 µg/mL TiO₂ NZs for 24 h. ( B, C ) Protein levels of Akt and p-Akt after treatment with Compound C or phenformin. ( D, E )Western blot analysis of Akt, p-Akt, mTOR, and p-mTOR in cells treated with TiO₂ NZs alone or in combination with Compound C or phenformin for 24 h. GAPDH served as a loading control. Molecular weights (kDa) are indicated beside the bands.Quantitative densitometric analysis corresponding to the western blots in ( A – E ) was performed using ImageJ software. All bands were normalized to GAPDH expression, and the tests were repeated three times. ( F ) AMPK mRNA and protein expression levels in cells transfected with AMPK siRNA or negative control. ( G ) Immunofluorescence analysis of autophagy levels in cells transfected with AMPK siRNA alone or in combination with Compound C or phenformin. ( H ) AMPK mRNA and protein expression levels in cells transfected with AMPK overexpression vector (pcDNA3.1-AMPK) or negative control vector (pcDNA3.1). All data are presented as the mean ± SD from three independent experiments. An unpaired two-tailed t-test was used for ( A - C ). A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( D , E , F , H ). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; ## P < 0.01, ### P < 0.001 vs. TiO₂ NZs group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Western Blot, Control, Software, Expressing, Transfection, Negative Control, Immunofluorescence, Over Expression, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Autophagy reversal via AMPK overexpression and Akt knockdown highlights dual-target mechanism. ( A , B ) Immunofluorescence analysis of autophagy levels in HTR cells exposed to the following treatments: ( A ) Control cells, TiO₂ NZs, TiO₂ NZs + AMPK overexpression vector (pcDNA3.1-AMPK), TiO₂ NZs + AMPK overexpression vector + Compound C, or TiO₂ NZs + Compound C; ( B ) Control cells, TiO₂ NZs, TiO₂ NZs + AMPK overexpression vector, TiO₂ NZs + AMPK overexpression vector + phenformin, or TiO₂ NZs + phenformin. Cell nuclei were stained blue with DAPI, and autophagosomes were labeled red using CY3-conjugated secondary antibodies. Scale bar = 100 μm. ( C ) AKT mRNA and protein levels examined by RT-PCR and western blotting after cells were transfected with the Akt siRNA and the negative control. ( D ) Immunofluorescence analysis of autophagy levels in HTR cells exposed to the following treatments: TiO₂ NZs, TiO₂ NZs + Akt siRNA, TiO₂ NZs + Akt siRNA + Compound C, TiO₂ NZs + Compound C, TiO₂ NZs + Akt siRNA + phenformin, or TiO₂ NZs + phenformin for 24 h. Cell nuclei were stained blue with DAPI, and autophagosomes were labeled red using CY3-conjugated secondary antibodies. All data are presented as the mean ± SD. A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( C ). **** P < 0.0001 vs. NC siRNA group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Over Expression, Knockdown, Immunofluorescence, Control, Plasmid Preparation, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Negative Control, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Phenformin and Compound C rewire AMPK/mTOR pathway in HTR cells post-TiO₂ exposure. ( A, B ) Western blot analysis of Akt, AMPK, mTOR, and their phosphorylated forms after cells were treated with 100 µg/mL TiO₂ NZs, TiO₂ NZs + AMPK overexpression vector (pcDNA3.1-AMPK), or TiO₂ NZs + AMPK overexpression vector + Compound C/phenformin for 24 h. ( C ) Protein levels of AKT, p-AKT, AMPK, p-AMPK, mTOR, p-mTOR, and LC3 in the control group, TiO₂ NZs exposure group, TiO₂ NZs + phenformin and TiO₂ NZs + Compound C groups, as determined by western blotting. GAPDH served as a loading control. Molecular weights (kDa) are indicated beside the bands. Quantitative densitometric analysis corresponding to the western blots in ( A – C ) was performed using ImageJ software. All bands were normalized to GAPDH expression, and the tests were repeated three times.Data were collected from three independent experiments and presented as mean ± SD. An unpaired two-tailed t -test was used for (C left panel). A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( A , B , C right panel). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001 TiO₂ NZs + AMPK-vector + Compound C vs. TiO₂ NZs group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 TiO₂ NZs + AMPK-vector vs. TiO₂ NZs group; † P < 0.05, †† P < 0.01, ††† P < 0.001 TiO₂ NZs + AMPK-vector + Compound C vs. TiO₂ NZs + AMPK-vector group (A-B). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; # P < 0.05, ### P < 0.001 TiO₂ NZs + Compound C vs. TiO₂ NZs group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 TiO₂ NZs + phenformin vs. TiO₂ NZs group ( C )

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Western Blot, Over Expression, Plasmid Preparation, Control, Software, Expressing, Two Tailed Test, Comparison

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Structures of netarsudil-family ROCKi test compounds. Color code used throughout all figures.

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques:

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Experimental design. HTM hydrogels were treated with vehicle control (=baseline) or TGFβ2 for 5 d to induce a glaucoma-like cell phenotype (=induction) before adding netarsudil-family ROCKi test compounds for the next 5 d in absence of TGFβ2 (=induction + rescue), with fresh media supplied every 2-3 days.

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Control

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Dose response effects of netarsudil-family ROCKi treatment following TGFβ2-induction on HTM hydrogel contraction. Representative brightfield micrographs of HTM hydrogels encapsulated with HTM07 subjected to (A) netarsudil, (C) compound A, (E) compound B, (G) compound C, or (I) compound D over a broad dose range for 10 d, with Y27632 serving as refence control (dashed lines outline original construct size at 0 d). Scale bars, 1 mm. Construct size quantification of HTM hydrogels subjected to (B) netarsudil, (D) compound A, (F) compound B, (H) compound C, or (J) compound D (N = 3-8 replicates per group). (K) ROCKi dose response curves with calculated EC 50 values. Data shown as Mean ± SD with individual data points. Significance was determined by one-way ANOVA using multiple comparisons tests (*p<0.05, ***p<0.001, ****p<0.0001; ns, not significant).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Control, Construct

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Effects of netarsudil-family ROCKi treatment at EC 50 following TGFβ2-induction on HTM hydrogel contraction. Representative brightfield micrographs of HTM hydrogels encapsulated with (A) HTM05, (C) HTM07, or (E) HTM36 subjected to the different treatments for 10 d, with max level netarsudil serving as refence control (dashed lines outline original construct size at 0 d). Scale bars, 1 mm. Construct size quantification of HTM hydrogels encapsulated with (B) HTM05, (D) HTM07, or (F) HTM36 (N = 3-8 replicates per group). (G) Construct size quantification of HTM hydrogels encapsulated with HTM05 (purple), HTM07 (maroon), or HTM36 (gray). Data shown as Mean ± SD with individual data points. Significance was determined by one- or two-way ANOVA using multiple comparisons tests (*p<0.05, ***p<0.001, ****p<0.0001; ns, not significant; # p<0.0001 vs. all other groups).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Control, Construct

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Effects of netarsudil-family ROCKi treatment at EC 50 following TGFβ2-induction on HTM cell F-actin stress fibers within ECM hydrogels. Representative confocal fluorescence micrographs of F-actin in HTM hydrogels encapsulated with (A) HTM05, (C) HTM07, or (E) HTM36 subjected to the different treatments for 10 d (F-actin = green). Scale bar, 200 μm. Quantification of relative F-actin signal intensity in HTM hydrogels encapsulated with (B) HTM05, (D) HTM07, or (F) HTM36 (N = 4 replicates per group). (G) Pooled quantification of relative F-actin signal intensity in HTM hydrogels encapsulated with HTM05 (purple), HTM07 (maroon), or HTM36 (gray) (N = 4 replicates per group and HTM cell strain). Data shown as Mean ± SD with individual data points. Significance was determined by one- or two-way ANOVA using multiple comparisons tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant; # p<0.01 vs. Ctrl, TGFβ2, netarsudil, and compounds A-C).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Fluorescence

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Effects of netarsudil-family ROCKi treatment at uniform netarsudil-EC 50 following TGFβ2-induction on HTM hydrogel contraction. Representative brightfield micrographs of HTM hydrogels encapsulated with (A) HTM07 or (C) HTM36 subjected to the different treatments for 10 d (dashed lines outline original construct size at 0 d). Scale bars, 1 mm. Construct size quantification of HTM hydrogels encapsulated with (B) HTM07 or (D) HTM36 (N = 3-4 replicates per group). (E) Construct size quantification of HTM hydrogels encapsulated with HTM07 (maroon) or HTM36 (gray). Data shown as Mean ± SD with individual data points. Significance was determined by one- or two-way ANOVA using multiple comparisons tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Construct

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 1. ETP/immature subtype pediatric and young adult patients with T-ALL highly express MEF2C and other stem cell factors HHEX, LMO2, BCL2, and LYL1. (A) Cluster analysis of 264 pediatric and young adult patients with T-ALL from the St. Jude study by Liu et al. (6) based on 371 available genes out of the 416-gene set (435 probe sets) as originally defined in the unsupervised cluster analysis of 117 pediatric patients with T-ALL by Homminga et al. (5). Specific clusters have been indicated as TLX1/NKX2.1 (formerly denoted as proliferative subtype), HOX, TLX3 (both formerly included in the TLX cluster), TALLMO, and ETP/immature. Specific oncogenic rearrangements are indicated for driving oncogenes, and patient characteristics include immunopheno- typic ETP, near ETP, and non-ETP patients. The absence of biallelic TRG deletions (ABD) are indicated as reported before (6). (B and C) Distribution of ETP, near ETP, and non-ETP cases or ABD and non-ABD patients with T-ALL over the ETP/immature cluster versus other T-ALL clusters.

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques:

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 2. MEF2C antagonizes Notch signaling in early progenitor T cells and induces B cell gene expression. (A) TCRγδ/CD3ε surface expression on LOUCY cells that were cultured in media or cocultured for 3 days on OP9-DL1 stromal cells, in the absence or presence of 10 μM of γ-secretase inhibitor DAPT visualized by flow cytometry (n = 3). (B) Transduced LOUCY cells containing a dox-inducible MEF2C-BFP overexpression construct (middle panels) or an IPTG-inducible MEF2C shRNA knockdown construct (bottom panels) were generated and compared with parental LOUCY cells (top panels). Blue fluorescence (BFP, blue-filled histograms), intracellular MEF2C levels (green-filled histograms), and surface CD3ε levels (red-filled histograms) were analyzed using flow cytometry in the absence (open histograms) or presence of dox or IPTG, respectively (filled histograms), as indicated. “Endog. MEF2C” refers to endogenous MEF2C expression levels in the MEF2C shRNA knockdown line. CD3ε expression after coculture on OP9 control or OP9-DL1 stromal cells (right columns). (C) Significantly enriched up- (red) or downregulated (blue) GO terms in MEF2C-induced LOUCY cells for 24 hours compared with noninduced LOUCY cells. (D) Heatmaps of significantly up- or downregulated probe sets (log2 fold change >0.6, P < 0.05, FDR < 0.1) in triplicate gene expression analysis (Affymetrix GeneChip Human Genome U133 Plus 2.0) per condition for LOUCY_MEF2C-BFP cells with/without MEF2C induction for 24 hours (± dox). Downregulated or upregulated probe sets following MEF2C-induction are shown in blue or red, respectively. Three of the 4 MEF2C probe sets, designated with asterisks, lie outside the cloned MEF2C cDNA sequence and thus represent endogenous MEF2C levels, whereas the other MEF2C probe set (indicated by the arrow) also covers the cloned cDNA construct.

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques: Gene Expression, Expressing, Cell Culture, Flow Cytometry, Over Expression, Construct, shRNA, Knockdown, Generated, Fluorescence, Control, Clone Assay, Sequencing

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 3. MEF2C binds to promoters and enhancers, which is reduced by Notch signaling. (A) Color-coded centered heatmaps indicating ChIP-Seq peak binding intensities (number of reads in a –1.0 kb to +1.0 kb region relative to the summit of the peak) for MEF2C, Bromodomain 4 (BRD4), and histone H3 with epigenetic trimethylation (H3K4me3) or acetylation marks (H3K27ac) for LOUCY parental cells (blue) or LOUCY parental cells cultured on OP9-DL1 stromal cells for 24 hours (green). Centered heatmaps for 5 kb regions upstream of transcription start site (uTSS), GeneHancer-defined enhancers (Enh), 5 kb regions upstream of long noncoding areas (defined by deepbase and lncipedia [uLnc]), or other regions (Supplemental Figure 3). (B) The number of upregulated genes (from collapsed probe sets; red circle) and downregulated genes (blue circle) with a log2 fold change > 0.6 (with P < 0.05 and q < 0.1) 24 hours after induction of MEF2C expression are displayed (see also Figure 2, C and D). The smaller black circles indicate the number of genes having MEF2C binding sites within the gene body or within 10 kb flanking regions. (C) Motifs as identified by MEME-ChIP found within 50 bp upstream or downstream regions relative to the MEF2C ChIP-Seq peak summits (101 bp window) are indicated including their expectancy values (E value), the number of sites identified, and their relative locations in these windows.

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques: ChIP-sequencing, Binding Assay, Cell Culture, Expressing

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 4. MEF2C induces BCL2 and provides a survival advantage under limiting serum levels. (A) LOUCY_MEF2C-BFP cells were cultured in triplicate without (dashed lines) or following induction of MEF2C (+dox, solid lines) in media containing 0%, 1%, or 10% fetal calf serum (FCS; mean values ± SD are shown). (B) Per- centage of cells expressing the MEF2C-BFP construct in the derivative LOUCY_MEF2C-BFP bulk line. Triplicate experiments grown for 22 days in the presence of 1% (left) or 10% (right) FCS. Each column displays the fraction of MEF2C-BFP expressing cells in blue. (C) LOUCY_MEF2C-BFP cells were grown in medium containing 1% FCS without (–dox, gray line) or with MEF2C-BFP induction (+dox, blue line). Dox was washed out at day 17 (dashed blue line), and cells were further cultured in 1% FCS media without dox. Mean values ± SD from an experiment in triplicate are shown; shown are representative examples of 3 independent experiments performed. (D) Western blot for MEF2C and BCL2 in LOUCY_MEF2C-BFP cells that were grown in 1% or 10% FCS media for 3 days without (–dox) or with MEF2C induction (+dox). Relative band intensities for BCL2 normalized to β-actin are indicated, with the –dox condition ratio set at 1. (E) Western blot of phospho-STAT5 (Tyr694), total STAT5, and BCL2 in LOUCY_MEF2C-BFP cells that were grown in 10% FCS media without (–dox) or with MEF2C induction (+dox) for 24 hours and subsequently incubated without and with 100 ng/mL IL-7 for 15 minutes (top) or 2 days (bottom). Ruxolitinib (2 μM) was added for 1 hour before IL-7 addition and remained present throughout the experiment. β-Actin was used as a loading control. Relative band intensity ratios for phospho-STAT5 (normalized to total STAT5 level) and for BCL2 (normalized to β-actin) are shown as explained in D.

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques: Cell Culture, Expressing, Construct, Western Blot, Incubation, Control

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 5. MEF2C blocks T cell development. (A) MEF2C-eGFP transgenic mice: loxP-flanked stop cassette followed by MEF2C cDNA, IRES, and eGFP following exon 1 of the Rosa26 locus (top). Cre recombinase activity removes the stop cassette and activates expression of MEF2C and eGFP from the Rosa26 promoter (bottom). SA, splice acceptor site; arrows, genotyping primers. (B) Genotype of spleens of mice with WT or MEF2C-eGFP transgene (tg) alleles, in the absence or the presence of Lck-Cre. (C) Western blot of endogenous or induced MEF2C and α-tubulin protein levels in total thymocytes from mice of different genotypes. (D) Flow cytometry histograms of eGFP expression in lymph node or thymus cells from duplicate MEF2C-eGFP hetero- or homozygous mice with or without Lck-Cre as indicated by the gray, light green, and dark green histograms. (E) Absolute cell numbers (mean ± SD) for cells from MEF2C-eGFP control (no Cre, open circles) and MEF2C-eGFP/Lck-Cre mice (blue filled circles) at 10–16 and 20–34 weeks of age. Populations are CD4/CD8 DN, DP, CD4 SP, and CD8 SP. DN are also gated on lineage– (CD3, B220, CD11b, Ly6G/C, Ter-119) cells to further divide the immature thymocyte populations into DN1-DN4. DN1, CD44+CD25–; DN2, CD44+CD25+; DN3, CD44–CD25+; DN4, CD44–CD25–. (F) Total CD45+ cell numbers (mean ± SD) as in E in the thymus (left panel) or inguinal lymph nodes (right panel). (G) Total CD19+ cell numbers (mean ± SD) as in E in the BM (left panel) or thymus (right panel). *P < 0.05, **P < 0.01 by Mann-Whitney U test.

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques: Transgenic Assay, Activity Assay, Expressing, Western Blot, Flow Cytometry, Control, MANN-WHITNEY

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 6. MEF2C blocks T cell lineage differentiation in murine and human cocultures. (A) Mouse differentiation assay: lineage-depleted (CD3, B220, CD11b, Ly6G/C, 7-4, Ter-119) BM cells from MEF2C-eGFPtg/tg mice were exposed to Tat-Cre before a coculture experiment (day 0) on OP9-DL1 or OP9 stromal cells to monitor T and B cell differentiation, respectively. (B) Human differentiation assay: human CD34+ cells from healthy umbilical cord blood donors transduced with BFP control (left) or MEF2C-t2a-BFP (right) lentiviruses and cocultured on OP9-DL1 stromal cells for 2 or 5 days. (C) Transduction as in B, but at day 12 after splitting the coculture. CD45+ cell numbers of a representative example of 3 independent coculture experiments are displayed. For transduced cells, the BFP+ percentage is indicated.

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques: Differentiation Assay, Cell Differentiation, Transduction, Control

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 7. MEF2C induces tumors in mice. (A) Flow cytometry analysis of CD3+ and/or CD19+ cell fractions of CD45+ thymus or BM cells from MEF2C-eGFP/Lck-Cre mouse #3 (with tumor) and mouse #5 (no tumor). (B) Summary of CD3+ and/or CD19+ cell fractions of GFP+CD45+ cells from 3 MEF2C-eGFP/Lck-Cre mice with tumors (mice #1–#3) and 3 mice without evidence of tumor growth (mice #4–#6). Percentages of different cell populations are indi- cated: CD3+CD19+ (red), CD3+ (green), CD19+ (blue), and CD3–CD19– (gray) cells. (C) Summary of CD3+ and/or CD19+ cell fractions of GFP+CD45+ cells as in B in NSG mice that were transplanted with tumors from indicated tissues of MEF2C-eGFP mice #1–#3 (arrows). Nine of 14 transplanted NSG mice are shown. (D) Survival curves of 14 NSG mice transplanted with MEF2C-eGFP/Lck-Cre tumors (red line) and 2 control NSG mice transplanted with splenocytes from 2-year-old MEF2C-eGFP/no-Cre control mice (black line).

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques: Flow Cytometry, Control

Journal: JCI insight

Article Title: MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

doi: 10.1172/jci.insight.150363

Figure Lengend Snippet: Figure 8. SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibi- tors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates. As controls, media, non-DLL4–stimulated cells or MEF2C-overexpressing (LOUCY_MEF2C-BFP) cells (+dox) have been indicated in blue. Normalized CD3ε fluorescence intensities have been plotted for duplicate experiments. (B) SIK kinase inhibitors YKL-06-062 and HG-9-91-01; dasatinib, which has SIK off-target activity (12 nM, 48 nM, and 180 nM for SIK1, SIK2, or SIK3, respectively) (MedChemExpress or Proteomicsdb.org), and imatinib were tested to induce a CD3ε+ phenotype in LOUCY_MEF2C- BFP cells when cultured on DLL4-coated plates in the absence of dox (except when indicated). (C) Western blot of MEF2C, P-MEF2C (Ser222), and BCL2 in noninduced (no dox) or MEF2C-induced (+dox) LOUCY_MEF2C-BFP cells that were incubated in the presence of increasing concentrations of inhibitors HG-9-91-01 for 4 days. Band intensity ratios for various proteins relatively to β-actin control levels is indicated, with the –dox condition set at the value = 1. (D) Cell viability of noninduced (no dox) or MEF2C-induced (+dox) LOUCY_MEF2C-BFP after a 4-day exposure to a serial dilution of prednisolone combined with a serial dilution of HG-9-91-01 at the concentrations indicated. ZIP synergy scores are calculated from an n = 3 per condition. ZIP scores < –10 are indicated with a dash-dot line and a blue color. ZIP scores > 10 are indicated by a dashed line.

Article Snippet: SIK inhibitors block MEF2C function. (A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 μM) were screened for their potential to induce a CD3ε+ phenotype in LOUCY cells on DLL4-coated plates.

Techniques: Blocking Assay, Drug discovery, Fluorescence, Activity Assay, Cell Culture, Western Blot, Incubation, Control, Serial Dilution