Journal: Frontiers in Pharmacology

Article Title: Ling-gui-zhu-gan promotes adipocytes browning via targeting the miR-27b/PRDM16 pathway in 3T3-L1 cells

doi: 10.3389/fphar.2024.1386794

Figure Lengend Snippet: The effect of LGZG-containing serum on the expression of miR-27b and browning-related markers in 3T3-L1 adipocytes. (A–G) Relative mRNA levels of UCP1, miR-27b, PRDM16, PGC-1α, PPARγ, CTBP1, and CTBP2 using RT-qPCR analysis. (H–M) The immunoblotting images and relative protein levels of UCP1, PRDM16, PGC-1α, PPARγ, CTBP1, and CTBP2 using Western blot analysis. All data were presented as mean ± SEM, with n = 6 for each group in (A–G) , n = 3 for each group in (H–M) . * p < 0.05 versus the non-treated 3T3-L1 adipocytes.

Article Snippet: Immediately after, the membrane was incubated with corresponding primary antibodies against UCP1 (23673-1-AP), PRDM16 (PA5-20872), PGC-1α (66369-1-Ig), PPARγ (16643-1-AP), CTBP1 (10972-1-AP), and CTBP2 (10346-1-AP) at 1:1,000 dilution, and a secondary antibody against β-Tubulin (66240-1-Ig) at 1:10,000 (all from Proteintech Group, Chicago, IL, United States).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Ling-gui-zhu-gan promotes adipocytes browning via targeting the miR-27b/PRDM16 pathway in 3T3-L1 cells

doi: 10.3389/fphar.2024.1386794

Figure Lengend Snippet: The effect of LGZG-containing serum on miR-27b/PRDM16 signaling pathway in 3T3-L1 adipocytes. (A–G) Relative mRNA levels of miR-27b, UCP1, PRDM16, PGC-1α, PPARγ, CTBP1, and CTBP2 after transfection with miR-27b mimic or miR-NC mimic for 48 h using RT-qPCR analysis. (H–N) The immunoblotting images and relative protein levels of UCP1, PRDM16, PGC-1α, PPARγ, CTBP1, and CTBP2 after transfection with miR-27b mimic or miR-NC mimic for 48 h using Western blot analysis. All data were presented as mean ± SEM, with n = 3 for each group in (A–N) . # p < 0.05 versus the negative control adipocytes; * p < 0.05 versus the miR-27b overexpressed adipocytes.

Article Snippet: Immediately after, the membrane was incubated with corresponding primary antibodies against UCP1 (23673-1-AP), PRDM16 (PA5-20872), PGC-1α (66369-1-Ig), PPARγ (16643-1-AP), CTBP1 (10972-1-AP), and CTBP2 (10346-1-AP) at 1:1,000 dilution, and a secondary antibody against β-Tubulin (66240-1-Ig) at 1:10,000 (all from Proteintech Group, Chicago, IL, United States).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Scientific Reports

Article Title: The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6b rd1/rd1 mouse model of retinal dystrophy

doi: 10.1038/s41598-024-52391-y

Figure Lengend Snippet: Antibodies utilized for immunohistochemical and immunofluorescent staining.

Article Snippet: CtBP2 , Novus , 1:50/1:200 , Rabbit , NBP3-15268.

Techniques: Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6b rd1/rd1 mouse model of retinal dystrophy

doi: 10.1038/s41598-024-52391-y

Figure Lengend Snippet: PKCα staining of rod bipolar cells ( A ), Calbindin staining of horizontal cell bodies ( B ), CtBP2 staining of presynaptic ribbon ( C ), hematoxylin staining of the nuclei ( D ) in WT and rd1 mouse retinal sections, and Quantification of immunohistochemical staining ( E ). No noticeable differences were observed in the staining of rd1 and WT retinas at P7. At P14, immunoreactivity for PKCα and CtBP2 in the OPL of the rd1 retina appeared reduced, and a reduction in processes originating from horizontal cell bodies was also observed. Image scale bar: 50 μm (N = 3 eyes from different mice, n = 5 images from each eye). ** P < 0.01, * P < 0.05 compared with rd1 mice by an independent two-sample t -test.

Article Snippet: CtBP2 , Novus , 1:50/1:200 , Rabbit , NBP3-15268.

Techniques: Staining, Immunohistochemical staining

Journal: Scientific Reports

Article Title: The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6b rd1/rd1 mouse model of retinal dystrophy

doi: 10.1038/s41598-024-52391-y

Figure Lengend Snippet: ( A ) Retina double labeling with PKCα (rod bipolar cells, red) and CtBP2 (presynaptic ribbon, green) antibodies revealed that CtBP2-positive puncta were sparse and appeared flattened in rd1 retina from P10, before photoreceptor degeneration. ( B ) In WT and rd1 retinas with both RBCs (labeled with PKCα in red) and HCs (marked with Calbindin in green) identified, the terminal dendrites of horizontal cells in the rd1 retina experienced a reduction since P10, and horizontal cell bodies decreased at P14. Image scale bar: 50 μm. OPL, outer plexiform layer; ONL, outer nuclear layer. ( C ) Quantification of immunofluorescence staining indicated the reduction of CtBP2 and PKCα positive area during retina degeneration and the significant loss of horizontal cell bodies per field at P14. (N = 3 eyes from different mice, n = 5 images from each eye). ** P < 0.01, * P < 0.05 compared with rd1 mice by an independent two-sample t -test.

Article Snippet: CtBP2 , Novus , 1:50/1:200 , Rabbit , NBP3-15268.

Techniques: Labeling, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6b rd1/rd1 mouse model of retinal dystrophy

doi: 10.1038/s41598-024-52391-y

Figure Lengend Snippet: Idebenone supplementation delayed photoreceptor cell death in rd1 retinas. ( A ) Quantification of ERG indicated that b-wave amplitudes of ERG showed no significant visual rescue in mice treated with idebenone, although there was a trend toward an increased response (black square box). WT mice exhibited more distinct waveforms than rd1 mice, irrespective of treatment. However, the amplitude of F-VEP showed significant visual rescue in mice treated with idebenone at P14, which was not significant at P21. (N = 4–10 eyes from different mice). ( B ) Histological examination of untreated or idebenone-treated rd1 retinas revealed that idebenone therapy promoted neuronal cell survival in the ONL at P14, though the effect was not significant at P21. Morphometric analysis of ONL thickness extending from the ONH also indicated that idebenone treatment notably preserved photoreceptor cells at P14. (N = 4–10 eyes from different mice, with multiple counts per eye). Image scale bar: 50 μm. ( C ) Retinal double staining using antibodies against PKCα (red) and CtBP2 (green) showed an increase in CtBP2-positive puncta following enhanced photoreceptor cell survival in rd1 mice treated with idebenone for 14 days, while the numbers remained unaltered and scarce in the treated groups at P21. In WT mice, the characteristic horseshoe-shaped CtBP2-positive puncta were observed, particularly at P21. ( D ) Rod bipolar cell (labeled with PKCα, red) and horizontal cell (marked by Calbindin, green) double-labeling revealed a substantial decrease in the terminal dendrites of horizontal cells in rd1 mice as opposed to their WT counterparts, while the untreated and idebenone-treated mice at the ages of P14 and P21 showed no differences. INL, inner nuclear layer; ONH, optic nerve head. Image scale bar: 50 μm. ( E ) Quantification of immunofluorescence staining indicated only CtBP2-positive area were increased following the photoreceptor cell survival in the rd1 mice after idebenone treatment for 14 days. (N = 3–6 eyes from different mice, n = 5 images from each eye). ** P < 0.01, * P < 0.05 compared with untreated rd1 and idebenone-treated rd1 mice by one-way ANOVA with LSD’s multiple comparisons.

Article Snippet: CtBP2 , Novus , 1:50/1:200 , Rabbit , NBP3-15268.

Techniques: Double Staining, Labeling, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Hearing impairment due to Mir183/96/182 mutations suggests both loss and gain of function effects

doi: 10.1101/579003

Figure Lengend Snippet: Colocalised pre- and postsynaptic densities in Mir183/96 dko and Mir182 ko mice. A. Synapses below inner hair cells (IHCs) in Mir183/96 dko (top) and Mir182 ko (bottom) wildtype (left), heterozygous (middle) and homozygous (right) knockout mice. Presynaptic ribbons are labelled with anti-Ribeye antibody (red) and postsynaptic densities with anti-GluR2 antibody (green); where they colocalise, the resulting colour is yellow. DAPI (blue) labelled the nuclei. Scale bar = 10µm. B. Mean counts of colocalised pre- and postsynaptic markers in wildtype (green), heterozygous (blue) and homozygous (red) Mir183/96 dko (top) and Mir182 ko (bottom) mice. There are significantly fewer colocalised synapses in Mir183/96 dko homozygotes (n=3) compared to wildtypes (n=5, p = 0.016, one way ANOVA, Bonferroni-corrected p=0.02**) and also compared to heterozygotes (n=7, Bonferroni-corrected p=0.035*), but no significant differences in heterozygotes compared to wildtypes (Bonferroni-corrected p=1.0). No significant difference was seen between synapse counts in Mir182 ko knockout heterozygotes (n=6), homozygotes (n=7) and wildtypes (n=4,p=0.818, one way ANOVA). Error bars show standard deviation.

Article Snippet: The primary antibodies used were anti-NFH (Abcam, cat. no: ab4680, diluted 1:800), anti-GluR2 (Millipore, cat. no: MAB397, diluted 1:200) and anti-Ribeye (Synaptic Systems, cat. no: 192 103, diluted 1:500), and the secondary antibodies were Alexa Fluor 488 goat anti-chicken (Invitrogen, cat. no: A11039, diluted 1:300), Alexa Fluor 546 goat anti-rabbit (Invitrogen, cat. no: A11035, diluted 1:300) and Alexa Fluor 488 goat anti-mouse (Invitrogen, cat. no: A21131, diluted 1:300).

Techniques: Knock-Out, Standard Deviation

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A Expression pattern of CtBP2 mRNA in K7M2 cell lines modified for CYR61, as assessed by RT-qPCR. GAPDH was used as internal reference gene. The relative mRNA level was calculated using the 2 −ΔΔCT method and expressed as box plot (n=3). An asterisk (*) indicates a statistically significant difference (P-value < 0.05). B Expression pattern of CtBP2 and CYR61 protein in K7M2 cell lines modified for CYR61, as assessed by western blot. Actin was used as loading control. C Spearman correlation between the CtBP2 and CYR61 mRNA level, evaluated by RT-qPCR in a panel of eight human osteosarcoma cell lines. The black dotted line shows the regression line.

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Modification, Quantitative RT-PCR, Western Blot, Control

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A Interaction network diagram of CtBP2 and CYR61 proteins. Network nodes represent proteins; the edges indicate both functional and physical protein associations; line thickness indicates the strength of data support: the thicker the gray connecting line, the stronger is the predicted interaction between the two proteins. B Spearman correlation between the indicated mRNA levels in K7M2 and U2OS modified cell lines, evaluated by RT-qPCR. GAPDH was used as internal reference gene. The relative mRNA level was calculated using the 2 −ΔΔCT method and expressed as box plot (n=3 independent experiments). The dotted lines show the regression lines. C Representative images from clonogenic assays after staining with crystal violet solution. K7M2 and U2OS cell lines were plated at 100 cells/cm², and incubated for 10 days. D Average size and number of colonies formed. Results are expressed as box plot (n=4). An asterisk (*) indicates a statistically significant difference (P-value < 0.05 vs. Control).

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Functional Assay, Modification, Quantitative RT-PCR, Staining, Incubation, Control

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A Expression pattern of CtBP2 mRNA in Control and stably modified cell lines, as assessed by RT-qPCR. GAPDH was used as internal reference gene. The relative mRNA level was calculated using the 2 −ΔΔCT method and expressed as box plot (n=3 independent experiments). An asterisk (*) indicates a statistically significant difference (P-value < 0.05 vs. Control). B Expression pattern of CtBP2 protein in K7M2 and U2OS modified cell lines, as assessed by western blot. Actin was used as loading control. C Relative cell metabolic activity, as assessed by the MTT assay. Results are expressed as mean ± standard deviation (n=30). D Relative DNA replication rate, as assessed by the BrdU incorporation assay. Results are expressed as mean ± standard deviation (n=17). E Representative images of Control and stably modified cells. F Distribution of cell shape parameters (cell surface, cell perimeter, and circularity index) for the Control and stably modified K7M2 and U2OS cells. Results are expressed as box plot.

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Control, Stable Transfection, Modification, Quantitative RT-PCR, Western Blot, Activity Assay, MTT Assay, Standard Deviation, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A Representative images of K7M2 cell migration after 6 h incubation, as assessed by Boyden chamber assay. CtBP2-silenced K7M2 cells were stained in green, and CtBP2-overexpressing cells were stained in red before seeding. B Estimation plot representing the relative number of migrating cells. The difference between the group means is represented on the right. C Representative images of cell migration (wound healing assay) taken at time 18 h after the wound. Control and CtBP2-silenced K7M2 cells were incubated in the presence or absence of recombinant CYR61 (1 µg/ml). The dotted rectangles outline the initial wound surface. D Quantitative evaluation of the cell migration rate. Results are expressed as box plot (n=6). An asterisk (*) indicates a statistically significant difference (P-value < 0.05 vs. control).

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, Incubation, Boyden Chamber Assay, Staining, Wound Healing Assay, Control, Recombinant

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A Representative images of U2OS cell migration after 18 h incubation, as assessed by Boyden chamber assay. CtBP2-silenced U2OS cells were stained in green, and CtBP2-overexpressing cells were stained in red. B Estimation plot representing the relative number of migrating cells as compared to Control. The difference between the group means is represented on the right. C Representative images of cell migration (wound healing assay) taken at time 18 h after the wound. Control and CtBP2-silenced U2OS cells were incubated in the presence or absence of recombinant CYR61 (1 µg/ml). The dotted rectangles outline the initial wound surface. D Quantitative evaluation of the cell migration rate. Results are expressed as box plot (n=6). An asterisk (*) indicates a statistically significant difference (P-value < 0.05 vs. control).

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, Incubation, Boyden Chamber Assay, Staining, Control, Wound Healing Assay, Recombinant

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A Relative Matrix Metalloproteinase-2 (MMP2) activity in K7M2 and U2OS modified cell lines, as assessed by colorimetric assay. Results are expressed as box plot (n=10). An asterisk (*) indicates a statistically significant difference (P-value < 0.05 vs. Control). B Representative images of spatial spheroid invasion assays. 3D cultured spheroid made of mix (1:1) of CtBP2-silenced and CtBP2-overexpressing K7M2 were embedded in Matrigel basement membrane, and incubated for 24 h (bar = 200 µm). CtBP2-silenced K7M2 cells were stained in green, and CtBP2-overexpressing cells were stained in red. C Quantitative evaluation of the cells invading the Matrigel basement membrane at different time points. Results are expressed as mean ± standard error of the mean (n=23). D Estimation plot representing the relative number of invading cells as compared to Control. The difference between the group means is represented on the right.

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Modification, Colorimetric Assay, Control, Cell Culture, Membrane, Incubation, Staining

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A CtBP2 immunohistochemical staining of FFPE tissue sections of Control, CYR61-silenced or CYR61-overexpressing cell line derived xenografts. Tumor (T) and surrounding muscle (M) as well as the tumor core area (C) and invasive front (F) are indicated. The scale bars represent 200 µm. B Estimation plots representing the relative CtBP2 signal intensity determined for Control, CYR61-silenced or CYR61-overexpressing cell line derived xenografts. Results are expressed as paired sample comparison between the tumor core and invasive front. The difference between the means are represented on the right of each plot. C Spearman correlation between the average CtBP2 signal intensity and the number of metastatic foci in the lungs. The dotted line shows the regression line. D Spearman correlation between the average CtBP2 signal intensity and the lung metastatic surface. The dotted line shows the regression line.

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Staining, Control, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: Representative images with higher magnification of CtBP2 immunohistochemical staining of FFPE tissue sections of CYR61-silenced or CYR61-overexpressing cell line derived xenografts. Tumor (T) and surrounding muscle (M) as well as the tumor core area (C) and invasive front (F) are indicated. The scale bars represent 200 µm.

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Staining, Derivative Assay

Journal: bioRxiv

Article Title: CtBP2 triggers CCN1-induced metastatic dissemination of osteosarcoma cells in a non-hypoxic microenvironment

doi: 10.1101/2023.12.07.570584

Figure Lengend Snippet: A Immunohistochemical staining for CtBP2 and GLUT1, and H&E staining of FFPE section of K7M2 cultured as 3D spheroid. The scale bars represent 100 µm or 50 µm for below insert. B Estimation plots representing the relative CtBP2 and GLUT1 signal intensity. Results are expressed as paired sample comparison between the spheroid central and front area. The difference between the means are represented on the right of each plot. C Spearman correlation between CtBP2 and GLUT1 signal intensities. The dotted line shows the regression line. D Expression pattern of CtBP2 and HIF-1α protein in K7M2 cells cultured as 2D monolayer for 6 h or 72 h in normoxic or hypoxic condition, as assessed by western blot. Actin was used as loading control.

Article Snippet: Ready-to-use lentiviral particles containing a vector encoding human or murine CtBP2 shRNA sequences or non-targeting (control) shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Staining, Cell Culture, Comparison, Expressing, Western Blot, Control