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