rabbit anti-mouse ki67 ab  (Thermo Fisher)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Loss of full-length pumilio 1 abrogates miRNA-221-induced gene p27 silencing-mediated cell proliferation in the heart

doi: 10.1016/j.omtn.2021.12.012

Figure Lengend Snippet: miR-221 downregulates P27 and increases cellular proliferation in vitro Rat ventricular myoblast H9c2 cells were transfected with 25 nM miR-221 mimics (miR-221) or control (MC) for 24 h and subjected to 20 h hypoxia/2 h re-oxygenation (H/R). The normoxia groups were incubated under normal culture conditions for an equal duration. p27 was measured by RT-qPCR (A) and western blot (B). (C) Cell proliferation was measured by ki67 staining (i) and cell count (ii). (D) p27 cDNA plasmid was co-transfected with mimics for phenotype rescue. The effects of P27 overexpression were validated by western blot (i), ki67 staining (ii), and cell count (iii). Data are expressed as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001 versus MC, #p < 0.05 versus miR-221 by one-way ANOVA with Bonferroni post hoc analysis. Experiments performed three times in triplicate.

Article Snippet: Briefly, cells were trypsinized, fixed, and permeabilized (eBioscience Intracellular Fixation & Permeabilization Buffer Set, Thermo Fisher Scientific, Singapore) for anti-ki67 Ab staining (clone 8D5, Cell Signaling Technology, Research Biolabs, Singapore) and anti-mouse IgG-PE (Invitrogen, Thermo Fisher Scientific, Singapore), followed by flow cytometry on MUSE cell analyzer (Merck, Singapore).

Techniques: In Vitro, Transfection, Control, Incubation, Quantitative RT-PCR, Western Blot, Staining, Cell Counting, Plasmid Preparation, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: Loss of full-length pumilio 1 abrogates miRNA-221-induced gene p27 silencing-mediated cell proliferation in the heart

doi: 10.1016/j.omtn.2021.12.012

Figure Lengend Snippet: Stress and repeated miR-221 treatment do not induce FL PUM1 expression or p27 regulation in rat MI heart (A) Rat hearts with MI were harvested at 48 h after surgery (MId2) and dissected by the infarct, border, and remote zones (Inf., Bor., Rem., respectively). PUM1 protein expression was assessed with western blot. HeLa cell lysate was used as a positive control. (B) Mimics of miR-221 were i.v. injected at 1 mg/kg immediately after surgery and tissue was harvested at MId2. A second injection was given at day 3 and tissue was harvested at MId7. miR-221 overexpression was measured by stem-loop qPCR. Results are presented as fold change over MI control. P27 expression in MI control and miR-221 mimic-treated rat hearts was measured by (C) RT-qPCR and (D) western blot. (E) Immunofluorescence staining of the heart sections (MId7) with three colors: cell proliferation marker ki67 (red), fibrosis marker α-SMA (green), nuclei stain DAPI (blue) (i). Scale bar, 200 μm. Results were analyzed as ki67% positive nuclei and normalized to MI control (ii). n = 5 animals for each group.

Article Snippet: Briefly, cells were trypsinized, fixed, and permeabilized (eBioscience Intracellular Fixation & Permeabilization Buffer Set, Thermo Fisher Scientific, Singapore) for anti-ki67 Ab staining (clone 8D5, Cell Signaling Technology, Research Biolabs, Singapore) and anti-mouse IgG-PE (Invitrogen, Thermo Fisher Scientific, Singapore), followed by flow cytometry on MUSE cell analyzer (Merck, Singapore).

Techniques: Expressing, Western Blot, Positive Control, Injection, Over Expression, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Marker

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: ( a ) Schematic representation of primers for genotyping and targeting strategy. ( b ) miR-31 genotyping using P1/P2, 1,064 bp band for miR-31 fl/fl and 235 bp band for miR-31 fl/fl /K5-Cre (cKO). DNA samples were prepared from total skin. ( c ) Cre-mediated tissue-specific deletion of miR-31 in epidermis. DNA samples were prepared from either epidermis or dermis. ( d ) miR-31 expression in epidermis derived from miR-31 fl/fl and cKO mice. ( e ) Phenotypic presentation of mouse back skin for miR-31 fl/fl or cKO mice treated with IMQ or vehicle for 7 days. ( f ) Splenomegaly and lymphadenopathy in miR-31 fl/fl or cKO mice treated with IMQ or vehicle for 7 days. Data are representative of more than five mice. ( g ) Skin thickness was measured on the days indicated. Symbols represent mean skin thickness±s.e.m. for five to six mice per group. ( h ) H&E staining of the back skin of miR-31 fl/fl or cKO mice treated with IMQ or vehicle. Dotted line indicates the border between the epidermis and the dermis. Scale bar, 100 μm. ( i ) Acanthosis of miR-31 fl/fl or cKO mice treated with IMQ or vehicle. ( j ) Dermal cellular infiltrates of miR-31 fl/fl or cKO mice treated with IMQ. ( k ) Immunostaining of Ki67 in lesional skin derived from miR-31 fl/fl (left panel) or cKO (right panel) mice treated with IMQ. Scale bar, 100 μm. ( l ) Quantitation of Ki67 + cells in epidermis. For all measurements, numbers of specifically stained Ki67 + cells in epidermis counted in three high-power fields for each section were used. ( m ) qPCR analysis of epidermal differentiation markers (Keratin 10, Loricrin and Filaggrin) in miR-31 fl/fl or cKO mice treated with IMQ or vehicle. Results ( d , m ) are presented as the ratio of miRNA to the small nuclear RNA U6 or of mRNA to the β-actin, relative to that in miR-31 fl/fl mice. * P <0.05, ** P <0.01, *** P <0.001, two-tailed Student's t -test for d , i , j , l , m , one-way ANOVA for g . Data ( g , i , j , l , m ) are representative of two independent experiments with four to six mice per group in each (mean and s.e.m.).

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: Expressing, Derivative Assay, Staining, Immunostaining, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: ( a , b ) Western blotting of ppp6c expression in the epidermis of normal skin derived from four healthy individuals (Ctr-1 to 4) or in psoriatic lesions derived from four patients with psoriasis (Pso-1 to 4). ( c ) Immunohistochemical staining of ppp6c in skin sections derived from healthy or psoriatic skin. Scale bar, 100 μm. ( d ) Primary mouse keratinocytes were transfected with scramble siRNA (Ctr) or with ppp6c siRNA (siRNA). Cell lysates were immunoblotted with anti-ppp6c or anti-actin. ( e ) Cell cycle analysis of mouse primary keratinocytes transfected with scrambled siRNA or ppp6c siRNA. ( f ) Western blotting of ppp6c expression in epidermis derived from lentiviral shRNA-control (LV-Ctr) or lentiviral shRNA-ppp6c (LV-shRNA) treated mice. ( g ) H&E staining of the back skin injected with LV-Ctr or LV-shRNA in mice applied with IMQ. Dotted line indicates the border between the epidermis and dermis. Scale bar, 100 μm. ( h ) Acanthosis of the back skin injected with LV-Ctr or LV-shRNA in mice applied with IMQ. ( i ) Immunohistochemical staining of Ki67 in skin sections derived from mice injected with LV-Ctr or LV-shRNA prior to IMQ painting. Dotted line indicates the border between the epidermis and dermis. Scale bar, 50 μm. Values ( a , d , f ) were expressed as fold changes relative to Ctr-1 ( a ), to scramble siRNA ( d ), or to lentiviral shRNA-control ( f ), and normalized to β-actin. * P <0.05, ** P <0.01, two-tailed Student's t -test. Data ( c – i ) are representative of two independent experiments with three to five samples per group.

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: Western Blot, Expressing, Derivative Assay, Immunohistochemical staining, Staining, Transfection, Cell Cycle Assay, shRNA, Control, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: ( a ) Undifferentiated NHEK proliferation induced by various concentrations of IL-6 for 24 h, and analysed by BrdU incorporation assay. ( b ) NHEK were transfected with scramble siRNA (Ctr) or with p65 siRNA (p65 siRNA). Cell cycle analysis of NHEK or transfected NHEK treated without or with IL-6 for 24 h. ( c ) In vitro wound healing rate of NHEK treated without or with IL-6 for 16 h. ( d , e ) Three-dimensional organotypic culture of HaCaT keratinocytes treated without or with IL-6. Scale bar, 100 μm. ( f ) 1 μg recombinant mouse IL-6 (in 25 μl PBS) or PBS was injected i.d. in ears of C57BL/6J mice. Ear sections were prepared for Ki67 staining 3 days after IL-6 administration. Scale bar, 100 μm. ( g ) NHEK were transfected with scramble siRNA (Ctr) or with p65 siRNA (p65 siRNA). Western blotting of ppp6c expression in NHEK with or without IL-6 treatment for 24 h. ( h ) Cell cycle analysis of primary mouse keratinocytes derived from miR-31 fl/fl or cKO mice in absence or presence of IL-6. Values were expressed as fold changes relative to non-stimulated HaCaT keratinocytes ( e ) or to non-stimulated NHEK ( g ) and normalized to β-actin. IL-6 was used at the concentration of 50 ng ml −1 ( b – e , g , h ). ** P <0.01, *** P <0.001, two-tailed Student's t -test. Data are representative of at least two independent experiments.

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: BrdU Incorporation Assay, Transfection, Cell Cycle Assay, In Vitro, Recombinant, Injection, Staining, Western Blot, Expressing, Derivative Assay, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis

doi: 10.1038/ncomms8652

Figure Lengend Snippet: Mice were injected subcutaneously with an irrelevant antagomir (NC) or an antagomir to miR-31 (anti-miR-31). The first injection was administered 3 days before the application of IMQ and thereafter was performed every other day until the end of the experiment. ( a ) H&E staining of the back skin derived from mice injected with NC (upper panel) or anti-miR-31 (lower panel). Scale bar, 100 μm. ( b , c ) Acanthosis and dermal cellular infiltrates were quantitated for mice treated with NC or anti-miR-31. ( d , e ) Ppp6c mRNA and protein levels in NC- or anti-miR-31-treated mice. ( f , g ) Immunohistochemical staining of ppp6c or Ki67 in skin sections derived from NC- or anti-miR-31-treated mice after induction of skin phenotype by IMQ ( n =8–9). Scale bar, 50 μm ( f ) or 100 μm ( g ). For all measurements ( c ), the median number of specifically stained dermal nucleated cells was counted in three high-power fields per section. Results ( d ) are presented as the ratio of mRNA to the β-actin, relative to that in NC-treated mice. * P <0.05, *** P <0.001, two-tailed Student's t -test. Data ( a – g ) are representative of at least two independent experiments with four to nine samples per group in each (mean and s.e.m.).

Article Snippet: Mouse anti-actin Ab (1:3,000; Cell Signaling Technology, #3700S), rabbit anti-ppp6c Ab (1:1,000; Abcam, #EPR8764), rat anti-p65 Ab (Cell Signaling Technology, #8242S) and rabbit anti-mouse Ki67 Ab (eBioscience, #42–5698) were used.

Techniques: Injection, Staining, Derivative Assay, Immunohistochemical staining, Two Tailed Test