Journal: Frontiers in Pharmacology

Article Title: Matrine promotes mitochondrial biosynthesis and reduces oxidative stress in experimental optic neuritis

doi: 10.3389/fphar.2022.936632

Figure Lengend Snippet: MAT promoted SIRT1 expression in RGCs. Eyeballs were harvested from naive rats, MAT- and vehicle-treated EAE rats. (A) Immunofluorescence double staining suggested that SIRT1 (FITC, green) were colocalized with Brn3a (Cy3, red) in the eyeball retina of EAE rats (200×). Scale bars, 10 μm. (B) Quantitative analysis of the number of positive cells. (C) Quantitative analysis of the rate of positive cells. Data represent mean ± SD; n = 10 rats per group. ** p < 0.01, *** p < 0.001, comparison between naive and vehicle-treated EAE groups. ## p < 0.01, comparison between vehicle- and MAT-treated EAE groups.

Article Snippet: The sections were then incubated in blocking solution at 4°C overnight with primary antibodies specific for rabbit anti-Nrf2 (1:100), rabbit anti-SIRT1 (1:100), rabbit anti-PGC-1α (1:300), and rabbit anti-TOMM20 (1:250) (all from Abcam, Cambridge, United Kingdom) and then incubated with secondary antibody donkey anti-rabbit FITC (1:200; IgG; Proteintech, Wuhan, China) at room temperature (RT) for 2 h. After being permeabilized with 1% BSA-PBS for 3 × 5 min, the sections were incubated with rabbit anti-Brn3a (1:100; IgG; Bioss, Beijing, China) specific primary antibody in blocking solution overnight at 4°C, then incubated with secondary antibody donkey anti-rabbit Cy3 (1:200; IgG; Proteintech) at RT for 2 h, mounted with 4’,6-diamidino-2-phenylindole (DAPI, 1:1,000; Roche, Basel, Switzerland), washed with PBS, cover-slipped, and examined under a fluorescence microscope (Leica Microsystem AG, Switzerland).

Techniques: Expressing, Immunofluorescence, Double Staining

Journal: Frontiers in Pharmacology

Article Title: Matrine promotes mitochondrial biosynthesis and reduces oxidative stress in experimental optic neuritis

doi: 10.3389/fphar.2022.936632

Figure Lengend Snippet: MAT elevated the expression of Nrf2 and PGC-1α. Eyeballs were harvested from naive rats, MAT- and vehicle-treated rats. (A) Immunofluorescence double staining suggested that Nrf2 (FITC, green) were colocalized with Brn3a (Cy3, red) (200×). Scale bars, 10 μm. (B) Quantitative analysis of the number of positive cells. (C) Quantitative analysis of the rate of positive cells. (D) Immunofluorescence double staining suggested that PGC-1α (FITC, green) were colocalized with Brn3a (Cy3, red) in the eyeball retina (200×). Scale bars, 10 μm. (E) Quantitative analysis of the number of positive cells. (F) Quantitative analysis of the rate of positive cells. Symbols represent mean ± SD; n = 10 rats per group. ** p < 0.01, *** p < 0.001, comparison between naive and vehicle-treated groups. ## p < 0.01, comparison between vehicle- and MAT-treated EAE groups.

Article Snippet: The sections were then incubated in blocking solution at 4°C overnight with primary antibodies specific for rabbit anti-Nrf2 (1:100), rabbit anti-SIRT1 (1:100), rabbit anti-PGC-1α (1:300), and rabbit anti-TOMM20 (1:250) (all from Abcam, Cambridge, United Kingdom) and then incubated with secondary antibody donkey anti-rabbit FITC (1:200; IgG; Proteintech, Wuhan, China) at room temperature (RT) for 2 h. After being permeabilized with 1% BSA-PBS for 3 × 5 min, the sections were incubated with rabbit anti-Brn3a (1:100; IgG; Bioss, Beijing, China) specific primary antibody in blocking solution overnight at 4°C, then incubated with secondary antibody donkey anti-rabbit Cy3 (1:200; IgG; Proteintech) at RT for 2 h, mounted with 4’,6-diamidino-2-phenylindole (DAPI, 1:1,000; Roche, Basel, Switzerland), washed with PBS, cover-slipped, and examined under a fluorescence microscope (Leica Microsystem AG, Switzerland).

Techniques: Expressing, Immunofluorescence, Double Staining

Journal: Frontiers in Pharmacology

Article Title: Matrine promotes mitochondrial biosynthesis and reduces oxidative stress in experimental optic neuritis

doi: 10.3389/fphar.2022.936632

Figure Lengend Snippet: MAT motivated mitochondrial biosynthesis. Eyeballs were harvested from naive rats, MAT- and vehicle-treated EAE rats. (A) Immunofluorescence double staining suggested that TOMM20 (FITC, green) were colocalized with Brn3a (Cy3, red) in the eyeball retina of EAE rats. Scale bars, 10 μm. (B) Quantitative analysis of the number of positive cells. (C) Quantitative analysis of the rate of positive cells. Symbols represent mean ± SD; n = 10 rats per group. ** p < 0.01, *** p < 0.001, comparison between naive and vehicle-treated EAE groups. ## p < 0.01, comparison between vehicle- and MAT-treated EAE groups. (D-G) TNF-α expose increase in loss of mitochondrial membrane potential (ΔѰM) in retinal cells. FACS analysis using JC-1 dye and Rhodamine-123 dye showing increase in mitochondrial membrane potential ((ΔѰM) with MAT treatmen in RGC-5 cells and this effect could be attenuated by EX527. Symbols represent mean ± SD; ** p < 0.01, comparison between control and TNF-α groups. && p < 0.01, comparison between TNF-α and MAT groups. ## p < 0.01 comparison between MAT and MAT+EX527 groups.

Article Snippet: The sections were then incubated in blocking solution at 4°C overnight with primary antibodies specific for rabbit anti-Nrf2 (1:100), rabbit anti-SIRT1 (1:100), rabbit anti-PGC-1α (1:300), and rabbit anti-TOMM20 (1:250) (all from Abcam, Cambridge, United Kingdom) and then incubated with secondary antibody donkey anti-rabbit FITC (1:200; IgG; Proteintech, Wuhan, China) at room temperature (RT) for 2 h. After being permeabilized with 1% BSA-PBS for 3 × 5 min, the sections were incubated with rabbit anti-Brn3a (1:100; IgG; Bioss, Beijing, China) specific primary antibody in blocking solution overnight at 4°C, then incubated with secondary antibody donkey anti-rabbit Cy3 (1:200; IgG; Proteintech) at RT for 2 h, mounted with 4’,6-diamidino-2-phenylindole (DAPI, 1:1,000; Roche, Basel, Switzerland), washed with PBS, cover-slipped, and examined under a fluorescence microscope (Leica Microsystem AG, Switzerland).

Techniques: Immunofluorescence, Double Staining

Journal: Stem Cell Research & Therapy

Article Title: Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

doi: 10.1186/s13287-015-0264-1

Figure Lengend Snippet: OPA1 +/− -iPSCs failed to differentiate into RGCs with culture medium supplemented with 10 % FBS and DAPT. Neurospheres were plated onto PLO/L-coated plates and cultured in hESC medium containing 10 μM DAPT and 10 % FBS for 7 days before fixation with 4 % PFA on day 24. Cells were stained with TUJ1 ( red ), BRN3a ( green , a ) and ISLET-1 ( green , b ) antibodies. Scale bars = 20 μM

Article Snippet: The following primary antibodies were used: rabbit anti-human TUJ1 (1:100, Fitzgerald Industrial Industries, Acton, MA, USA); mouse anti-human BRN3a (1:50, Santa Cruz Biotechnology, Dallas, Texas, USA); ISLET-1 (1:40, Developmental Studies Hybridoma Bank - University of Iowa, Iowa City, Iowa, USA); ZO-1 (1:200, Life Technologies, Carlsbad, CA, USA); and rabbit anti-human PAX6 (1:200, BioLegend, formerly Covance Antibody Products Covance, San Diego, CA, USA).

Techniques: Cell Culture, Staining

Journal: Stem Cell Research & Therapy

Article Title: Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

doi: 10.1186/s13287-015-0264-1

Figure Lengend Snippet: Neuron induction medium (NIM) promoted OPA1 +/− -RGC generation detected by IF. Neurospheres derived from the control and VO-iPSCs in the presence of NIM were plated onto PLO/L-coated plates and were cultured with hESC medium containing 10 μM DAPT, 10 % FBS, and 10 % NIM for 14 days. Cells were fixed with 4 % PFA on day 31. a TUJ1 ( green ) and BRN3a ( red ) staining are shown. b TUJ1 ( green ) and ISLET1 ( red ) staining are shown. Scale bars = 20 μM

Article Snippet: The following primary antibodies were used: rabbit anti-human TUJ1 (1:100, Fitzgerald Industrial Industries, Acton, MA, USA); mouse anti-human BRN3a (1:50, Santa Cruz Biotechnology, Dallas, Texas, USA); ISLET-1 (1:40, Developmental Studies Hybridoma Bank - University of Iowa, Iowa City, Iowa, USA); ZO-1 (1:200, Life Technologies, Carlsbad, CA, USA); and rabbit anti-human PAX6 (1:200, BioLegend, formerly Covance Antibody Products Covance, San Diego, CA, USA).

Techniques: Derivative Assay, Control, Cell Culture, Staining

Journal: Stem Cell Research & Therapy

Article Title: Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

doi: 10.1186/s13287-015-0264-1

Figure Lengend Snippet: Noggin rescued OPA1 +/− -iPSC differentiation into RGCs confirmed by IF. Mature EBs were cultured with 100 ng/mL Noggin to obtain putative RGCs. Cells were fixed with 4 % PFA on day 31. a TUJ-1 ( green ) and BRN3a ( red ) staining are shown. b TUJ-1 ( green ) and ISLET1 ( red ) staining are shown. Scale bars = 50 μM in panel ( a ) and 100 μM in panel ( b )

Article Snippet: The following primary antibodies were used: rabbit anti-human TUJ1 (1:100, Fitzgerald Industrial Industries, Acton, MA, USA); mouse anti-human BRN3a (1:50, Santa Cruz Biotechnology, Dallas, Texas, USA); ISLET-1 (1:40, Developmental Studies Hybridoma Bank - University of Iowa, Iowa City, Iowa, USA); ZO-1 (1:200, Life Technologies, Carlsbad, CA, USA); and rabbit anti-human PAX6 (1:200, BioLegend, formerly Covance Antibody Products Covance, San Diego, CA, USA).

Techniques: Cell Culture, Staining

Journal: Stem Cell Research & Therapy

Article Title: Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

doi: 10.1186/s13287-015-0264-1

Figure Lengend Snippet: Noggin treatment only during OPA1 +/− -iPSC differentiation into neural rosettes could rescue OPA1 +/− -RGC generation. Control and VO-iPSCs were incubated with 100 ng/mL Noggin in the cell culture medium only during the differentiation of neural rosettes and Noggin was removed in afterward differentation. Cells were fixed with 4 % PFA on day 31. Upper panel , TUJ1 ( green ) and BRN3a ( red ) staining are shown. Lower panel , TUJ1 ( green ) and ISLET-1 ( red ) staining are shown. Scale bars = 100 μM. Data from the control under the same treatment are not shown

Article Snippet: The following primary antibodies were used: rabbit anti-human TUJ1 (1:100, Fitzgerald Industrial Industries, Acton, MA, USA); mouse anti-human BRN3a (1:50, Santa Cruz Biotechnology, Dallas, Texas, USA); ISLET-1 (1:40, Developmental Studies Hybridoma Bank - University of Iowa, Iowa City, Iowa, USA); ZO-1 (1:200, Life Technologies, Carlsbad, CA, USA); and rabbit anti-human PAX6 (1:200, BioLegend, formerly Covance Antibody Products Covance, San Diego, CA, USA).

Techniques: Control, Incubation, Cell Culture, Staining

Journal: Stem Cell Research & Therapy

Article Title: Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

doi: 10.1186/s13287-015-0264-1

Figure Lengend Snippet: Addition of 17β-estradiol in RGC differentiation medium promoted generation of OPA1 +/− -RGCs. Control and VO RGCs were incubated with 100 nM 17β-estradiol in the cell culture medium during all of the differentiation stages, including iPSC culturing. Putative RGCs were then fixed with 4 % PFA on day 31. a TUJ1 ( green ) and BRN3a ( red ) staining are shown. b TUJ1 ( green ) and ISLET-1 ( red ) staining are shown. Scale bars = 50 μM in panel ( a ) and 100 μM in panel ( b )

Article Snippet: The following primary antibodies were used: rabbit anti-human TUJ1 (1:100, Fitzgerald Industrial Industries, Acton, MA, USA); mouse anti-human BRN3a (1:50, Santa Cruz Biotechnology, Dallas, Texas, USA); ISLET-1 (1:40, Developmental Studies Hybridoma Bank - University of Iowa, Iowa City, Iowa, USA); ZO-1 (1:200, Life Technologies, Carlsbad, CA, USA); and rabbit anti-human PAX6 (1:200, BioLegend, formerly Covance Antibody Products Covance, San Diego, CA, USA).

Techniques: Control, Incubation, Cell Culture, Staining